[Show abstract][Hide abstract] ABSTRACT: Paraoxonase 2 (PON2), a member of a gene family that also includes PON1 and PON3, is expressed in most tissues, including the brain. In mouse brain, PON2 levels are highest in dopaminergic areas (e.g. striatum), and are higher in astrocytes than in neurons. PON2 is primarily located in mitochondria and exerts a potent antioxidant effect, protecting mouse CNS cells against oxidative stress. The aim of this study was to characterize PON2 expression and functions in the brains of male and female mice. Levels of PON2 (protein, mRNA, and lactonase activity) were higher in brain regions and cells of female mice. Astrocytes and neurons from male mice were significantly more sensitive (by 3-4-fold) to oxidative stress-induced toxicity than the same cells from female mice. Glutathione levels did not differ between genders. Importantly, no significant gender differences in susceptibility to the same oxidants were seen in cells from PON2(-/-) mice. Treatment with estradiol induced a time- and concentration-dependent increase in the levels of PON2 protein and mRNA in male (4.5-fold) and female (1.8-fold) astrocytes, which was dependent on activation of estrogen receptor alpha. In ovariectomized mice, PON2 protein and mRNA were decreased to male levels in brain regions and in liver. Estradiol protected astrocytes from wild-type mice against oxidative stress-induced neurotoxicity, but did not protect cells from PON2(-/-) mice. These results suggest that PON2 is a novel major intracellular factor that protects CNS cells against oxidative stress, and confers gender-dependent susceptibility to such stress. The lower expression of PON2 in males may have broad ramifications for susceptibility to diseases involving oxidative stress, including neurodegenerative diseases.
No preview · Article · Jan 2013 · Free Radical Biology and Medicine
[Show abstract][Hide abstract] ABSTRACT: Neurite outgrowth in CNS neurons is primarily dependent on extracellular matrix proteins that are produced and released by glial cells. One of these proteins, fibronectin, together with laminin, plays a pivotal role in neuronal differentiation mediated by astrocytes. When co-cultured together, astrocytes interact with neurons and promote their differentiation; interference with this interaction can indirectly affect neuronal development. Cholinergic agonists, through activation of muscarinic M3 receptors in astrocytes increase their ability to promote neuritogenesis, largely by increasing the expression and release of fibronectin and laminin. This effect is mediated by activation of multiple signal transduction pathways in astrocytes. Ethanol, a known developmental neurotoxicant, inhibits muscarinic receptor signal transduction at the level of phosphor-lipase D, and antagonizes the astrocyte-mediated neuritogenic effect on hippocampal neurons. Similar effects of muscarinic agonists and of ethanol have been also seen in the in vitro hippocampal slice. Thyroid hormones (T3) can increase fibronectin expression in astrocytes, and hence increase neuritogenesis in cerebellar neurons. Conversely, astrocytes from hypothyroid animals, a condition know to cause profound developmental neurotoxicity, have decreased levels of fibronectin and decreased ability to promote neuritogenesis. The metal manganese, also increasingly recognized as a developmental neurotoxicant, accumulates in astrocytes, where it causes oxidative stress. Upon exposure to manganese, astrocytes have a decreased expression /release of fibronectin, and a decreased ability to foster neuritogenesis in hippocampal neurons. These effects are due to manganese-induced oxidative stress and are antagonized by antioxidants. Other oxidants, such as hydrogen peroxide or 2,3-dimethoxy-1,4-naphtoquinone, cause similar effects on astrocytic fibronectin and on neuritogenesis. Additionally, certain organophosphorus insecticides, such as diazinon and its oxygen analog diazoxon, which can also cause oxidative stress, are also capable of altering fibronectin expression and neuritogenesis. These studies indicate that interference with fibronectin expression/release in astrocytes greatly affects their ability to exert neuritogenic effects on developing neurons.
[Show abstract][Hide abstract] ABSTRACT: Signal transduction is a key process to transmit information from the extracellular milieu, and to elicit changes in the biological activity of target cells. Several cell signaling pathways can be targeted by neurotoxicants and developmental neurotoxicants. This chapter focuses on the interactions of ethanol, a known human developmental neurotoxicant, with signal transduction pathways stimulated by acetylcholine through activation of muscarinic receptors. It shows how initial observations in vivo, upon developmental exposure to ethanol, have been followed-up by a series of studies in cell culture systems which have allowed the discoveries that ethanol, by interfering with muscarinic signaling in astroglial cells, inhibits their proliferation and their ability to foster neuronal differentiation. Such effects of alcohol may be related to microencephaly and abnormal neuronal development, two hallmarks of the fetal alcohol syndrome.
[Show abstract][Hide abstract] ABSTRACT: Human paraoxonase 1 (PON1) is a high-density lipoprotein (HDL)-associated serum enzyme that exhibits a broad substrate specificity. In addition to protecting against exposure to some organophosphorus (OP) pesticides by hydrolyzing their toxic oxon metabolites, PON1 is important in protecting against vascular disease by metabolizing oxidized lipids. Recently, PON1 has also been shown to play a role in inactivating the quorum sensing factor N-(3-oxododecanoyl)-l-homoserine lactone (3OC12-HSL) of Pseudomonas aeruginosa. Native, untagged engineered recombinant human PON1 (rHuPON1) expressed in Escherichia coli and purified by conventional column chromatographic purification is stable, active, and capable of protecting PON1 knockout mice (PON1(-/-)) from exposure to high levels of the OP compound diazoxon. The bacterially derived rHuPON1 can be produced in large quantities and lacks the glycosylation of eukaryotic systems that can produce immunogenic complications when inappropriately glycosylated recombinant proteins are used as therapeutics. Previous studies have shown that the determination of PON1 status, which reveals both PON1(192) functional genotype and serum enzyme activity level, is required for a meaningful evaluation of PON1's role in risk of disease or exposure. We have developed a new two-substrate assay/analysis protocol that provides PON1 status without use of toxic OP substrates, allowing for use of this protocol in non-specialized laboratories. Factors were also determined for inter-converting rates of hydrolysis of different substrates. PON1 status also plays an important role in revealing changes in HDL-associated PON1 activities in male patients with Parkinson disease (PD). Immunolocalization studies of PONs 1, 2 and 3 in nearly all mouse tissues suggest that the functions of PONs 1 and 3 extend beyond the plasma and the HDL particle.
Preview · Article · Mar 2010 · Chemico-biological interactions
[Show abstract][Hide abstract] ABSTRACT: INTRODUCTION The human nervous system is one of the most complex organ systems in terms of both structure and function. It contains billions of neurons, each forming thousands of synapses leading to a very large number of connections. It also contains perhaps ten times more glial cells (astrocytes, oligodendrocytes, microglia) than neurons, which play important roles in the overall development and functioning of the nervous system. Anatomically, the nervous system is composed of a central (CNS) and a peripheral (PNS) component, whose basic functions are to detect and relay sensory information inside and outside the body, to direct motor functions, and to integrate thought processes, learning, and memory. Such functions and their complexity, together with some intrinsic characteristics (e.g., mature neurons do not divide, they are highly dependent upon oxygen and glucose) make the nervous system particularly vulnerable to toxic insults. Neurotoxicity can be defined as any adverse effect on the chemistry, structure, or function of the nervous system, during development or at maturity, induced by chemical or physical influences. A first issue is what constitutes an adverse effect. A proposed definition of an adverse effect is “any treatment related change which interferes with normal function and compromises adaptation to the environment.” Thus, most morphological changes such as neuronopathy (a loss of neurons), axonopathy (a degeneration of the neuronal axon), or myelinopathy (a loss of the glial cells surrounding the axon), or other gliopathies, would be considered adverse, even if structural and/or functional changes were mild or transitory.
[Show abstract][Hide abstract] ABSTRACT: Polybrominated diphenyl ethers (PBDEs) are an important group of flame retardants.Though several PBDEs have been banned or restricted in recent years, their past wide useand leach from the polymers, have made them worldwide environmental contaminants.Of special concern is the high body burden in infants and toddlers, as a result of exposurethrough maternal milk and house dust, particularly in North America. A number ofstudies have indicated the potential developmental neurotoxicity of PBDEs, however, thepossible underlying mechanisms are still unknown. Evidence accumulating in the pastfew years is suggesting that PBDEs may cause oxidative stress. Increases in reactiveoxygen species and/or lipid peroxidation have been observed in vitro in primary rodentneurons (cerebellum, hippocampus), in neuroblastoma and hepatoma cells, in neutrophilgranulocytes, and in fetal hematopoietic cells. Evidence of PBDE-induced oxidativestress has also been found upon in vivo administration in American kestrels anddeveloping mice. A series of studies in our own laboratory have shown that theantioxidant glutathione (GSH) is an important determinant of PBDE neurotoxicity.Genetically-determined reduced GSH levels increase PBDE-induced oxidative stress andensuing neurotoxicity which is primarily apoptotic in nature, both in vitro and in vivo.The developing brain is particularly sensitive to oxidative stress, due to its richness infree iron and a limited antioxidant capacity, and oxidative stress may contribute topathological apoptosis during brain development. Available evidence for oxidative stressin PBDE neurotoxicity, its possible relevance, and potential underlying mechanisms arediscussed in this commentary.
[Show abstract][Hide abstract] ABSTRACT: In mouse cerebellar granule neurons (CGNs) low concentrations of domoic acid (DomA) induce apoptotic cell death, which is mediated by oxidative stress; apoptosis is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate cysteine ligase (GCL) and have very low GSH levels. By activating M(3) muscarinic receptors, the cholinergic agonist carbachol inhibits DomA-induced apoptosis, and the anti-apoptotic action of carbachol is more pronounced in CGNs from Gclm (+/+) mice. Carbachol does not prevent DomA-induced increase in reactive oxygen species, suggesting that its anti-apoptotic effect is downstream of reactive oxygen species production. Carbachol inhibits DomA-induced activation of Jun N-terminal (JNK) and p38 kinases, increased translocation to mitochondria of the pro-apoptotic protein Bax, and activation of caspase-3. Carbachol activates extracellular signal-regulated kinases 1/2 (ERK1/2) MAPK and phospahtidylinositol-3 kinase (PI3K) in CGNs from both genotypes. However, while the protective effect of carbachol is mediated by ERK1/2 MAPK in CGNs from both mouse genotypes, inhibitors of PI3K are only effective at antagonizing the action of carbachol in CGNs from Gclm (+/+) mice. In CGNs from both Gclm (+/+) and (-/-) mice, carbachol induces a MAPK-dependent increase in the level of the anti-apoptotic protein Bcl-2. In contrast, carbachol causes a PI3K-dependent increase in GCL activity and of GSH levels only in CGNs from Gclm (+/+) mice. Such increase in GCL is not because of a transcriptionally-mediated increase in glutamate cysteine ligase catalytic subunit or glutamate cysteine ligase modifier subunit, but rather to an increase in the formation of the GCL holoenzyme. The results indicate that multiple pathways may contribute to the protective action of carbachol toward DomA-induced apoptosis. Compromised GCLM expression, which is also found in a common genetic polymorphism in humans, leads to lower GSH levels, which can exacerbate the neurotoxicity of DomA, and decreases the anti-apoptotic effectiveness of muscarinic agonists.
Full-text · Article · Mar 2009 · Journal of Neurochemistry
[Show abstract][Hide abstract] ABSTRACT: In mouse cerebellar granule neurons (CGNs) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase and phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs.
Full-text · Article · Jun 2008 · Neurochemistry International
[Show abstract][Hide abstract] ABSTRACT: Paraoxonase (PON1) is a high density lipoprotein-associated enzyme capable of hydrolyzing multiple substrates, including several
organophosphorus (OP) insecticides and nerve agents, oxidized lipids and a number of drugs or pro-drugs. Several polymorphisms
in the PON1 gene have been described, which have been shown to affect either the catalytic efficiency of hydrolysis or the
expression level of the enzyme. Animal studies have shown that PON1 is an important determinant of the toxicity of certain
OPs. Evidence for this was provided by cross-species comparisons, by administration of exogenous PON1 and by experiments in
PON1 knockout and transgenic mice. Low PON1 plays also a role in the higher susceptibility of the young to OP toxicity. Recent
findings also suggest that PON1 may modulate the toxicity resulting from exposure to mixtures of OP compounds
[Show abstract][Hide abstract] ABSTRACT: Early research on population distributions of plasma PON1 paraoxonase activity indicated a polymorphic distribution with high,
intermediate and low metabolizers. Cloning and characterization of the cDNA encoding human PON1 and follow-on experiments
demonstrated that the molecular basis of the activity polymorphism (PM) was a Q192R PM with PON1R192 specifying high paraoxonase activity. Further research demonstrated that the PON1192 polymorphism had little effect on the catalytic efficiencies of hydrolysis of phenylacetate and diazoxon (DZO), but did affect
the efficiencies of hydrolysis of chlorpyrifos oxon (CPO), soman and sarin, with PON1R192 having a higher efficiency of CPO hydrolysis and PON1Q192 having higher rates of hydrolysis of soman and sarin. Plots of rates of DZO hydrolysis (at a salt concentration that differentially
inhibited PON1R192) vs. paraoxon hydrolysis clearly separated the three PON1192 phenotypes (QQ, QR, RR) and also showed a wide range of activity among individuals with the same PON1192 genotype. The term PON1 status was introduced to include both PON1192 functional genotype and plasma PON1 level,both important in determining risk for either exposure to specific organophosphorus
compounds (OPs) or disease. Characterization of 5 promoter-region polymorphisms by several groups indicated that an Sp1 binding
site was responsible for significant(~30%) variation in plasma PON1 levels. Re-sequencing of the PON1 genes of 47 individuals
(24 African-American/23 European) revealed an additional 180 polymorphisms in 27 kb of the PON1 genomic DNA including 8 more
5' regulatory region PMs, 1 coding region polymorphism (W194X), 162 additional intronic PMs and 9 additional 3' UTR PMs. The generation of PON1 null mice and “PON1 humanized mice” expressing either tgHuPON1R192 or tgHuPON1Q192 at the same levels on the PON1−/− background allowed for a functional analysis of the Q192R PM under physiological conditions.
Toxicology experiments with the PON1 humanized mice and the PON1 null mice injected with purified human PON1192 alloforms clearly demonstrated that the catalytic efficiency of substrate hydrolysis is important in determining whether
PON1 is able to protect against a given OP exposure. HuPON1R192 protects well against CPO and DZO exposure, but HuPON1Q192 does not protect well against CPO exposure and neither protects
against PO exposure. Studies on PON1 status and carotid artery disease show that low PON1 levels are a risk factor. The effects
of PON1192 alloforms on rates of hydrolysis of quorum sensing factors are not yet known. Taken together, these data along with those
of the leading researchers in the PON1 field indicate that it is important to measure PON1 levels/activities in any epidemiological
study. SNP analysis alone is inadequate for epidemiological studies, due to the wide variability of PON1 levels within the
three PON1192 genotypes Q/Q, Q/R R/R). Even the most comprehensive PON1 SNP analyses are unable to accurately predict PON1 levels. PON1
activity or level accurately predicts CHD risk, while genotype does not
[Show abstract][Hide abstract] ABSTRACT: Cholesterol is an essential component of cell membranes and plays an important role in signal transduction. This brief overview presents evidence from the literature that ethanol may affect cholesterol homeostasis and that, in the developing brain, this may be involved in its developmental neurotoxicity. The effects caused by inborn errors of cholesterol synthesis and by in utero ethanol exposure present several similarities in humans (eg, Smith-Lemli-Opitz syndrome and fetal alcohol syndrome), as well as in animal models. Ethanol has a cholesterol-reducing effect on the cardiovascular system, and a protective effect against Alzheimer's disease, whose pathogenesis has been linked to altered cholesterol homeostasis in the brain. In vitro, ethanol affects several functions that are mediated by cholesterol and important for brain development, such as glial cell proliferation, synaptogenesis, neuronal survival and neurite outgrowth. The brain contains high levels of cholesterol, mostly synthesized in situ. Astrocytes produce large amounts of cholesterol that can be released by these cells and utilized by neurons to form synapses. Ethanol up-regulates the cholesterol transporter ATP binding cassette A1 and cholesterol efflux from primary astrocyte cultures without affecting cholesterol synthesis.
No preview · Article · May 2007 · Human & Experimental Toxicology
[Show abstract][Hide abstract] ABSTRACT: This study investigated the role of cellular antioxidant defense mechanisms in modulating the neurotoxicity of domoic acid (DomA), by using cerebellar granule neurons (CGNs) from mice lacking the modifier subunit of glutamate-cysteine ligase (Gclm). Glutamate-cysteine ligase (Glc) catalyzes the first and rate-limiting step in glutathione (GSH) biosynthesis. CGNs from Gclm (-/-) mice have very low levels of GSH and are 10-fold more sensitive to DomA-induced toxicity than CGNs from Gclm (+/+) mice. GSH ethyl ester decreased, whereas the Gcl inhibitor buthionine sulfoximine increased DomA toxicity. Antagonists of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid/kainate receptors and of N-methyl-D-aspartate (NMDA) receptors blocked DomA toxicity, and NMDA receptors were activated by DomA-induced l-glutamate release. The differential susceptibility of CGNs to DomA toxicity was not due to a differential expression of ionotropic glutamate receptors, as evidenced by similar calcium responses and L-glutamate release in the two genotypes. A calcium chelator and several antioxidants antagonized DomA-induced toxicity. DomA caused a rapid decrease in cellular GSH, which preceded toxicity, and the decrease was primarily due to DomA-induced GSH efflux. DomA also caused an increase in oxidative stress as indicated by increases in reactive oxygen species and lipid peroxidation, which was subsequent to GSH efflux. Astrocytes from both genotypes were resistant to DomA toxicity and presented a diminished calcium response to DomA and a lack of DomA-induced L-glutamate release. Because polymorphisms in the GCLM gene in humans are associated with low GSH levels, such individuals, as well as others with genetic conditions or environmental exposures that lead to GSH deficiency, may be more susceptible to DomA-induced neurotoxicity.
Full-text · Article · Jan 2007 · Molecular Pharmacology