Gennaro Giordano

Neurotoxicity of pesticides: A brief review

Pesticides are substances widely used to control unwanted pests such as insects, weeds, fungi and rodents. Most pesticides are not highly selective, and are also toxic to nontarget species, including humans. A number of pesticides can cause neurotoxicity. Insecticides, which kill insects by targeting their nervous system, have neurotoxic effect in mammals as well. This family of chemicals comprises the organophosphates, the carbamates, the pyrethroids, the organochlorines, and other compounds. Insecticides interfere with chemical neurotransmission or ion channels, and usually cause reversible neurotoxic effects, that could nevertheless be lethal. Some herbicides and fungicides have also been shown to possess neurotoxic properties. The effects of pesticides on the nervous system may be involved in their acute toxicity, as in case of most insecticides, or may contribute to chronic neurodegenerative disorders, most notably Parkinsons disease. This brief review highlights some of the main neurotoxic pesticides, their effects, and mechanisms of action.

Pharmacological and dietary modulators of paraoxonase 1 (PON1) activity and expression: The hunt goes on ☆

Paraoxonase 1 (PON1) is a high density lipoprotein (HDL)-associated enzyme displaying esterase and lactonase activity. PON1 hydrolyzes several organophosphorus (OP) insecticides and nerve agents, a number of exogenous and endogenous lactones, and metabolizes toxic oxidized lipids of low density lipoproteins (LDL) and HDL. As such, PON1 plays a relevant role in determining susceptibility to OP toxicity, cardiovascular diseases and several other diseases. Serum PON1 activity in a given population can vary by at least 40-fold. Most of this variation can be accounted for by genetic polymorphisms in the coding region (Q192R, L55M) and in the promoter region (T-108C). However, exogenous factors may also modulate PON1 activity and/or level of expression. This paper examines various factors that have been found to positively modulate PON1. Certain drugs (e.g. hypolipemic and anti-diabetic compounds), dietary factors (antioxidants, polyphenols), and life-style factors (moderate alcohol consumption) appear to increase PON1 activity. Given the relevance of PON1 in protecting from certain environmental exposure and from cardiovascular and other diseases, there is a need for further mechanistic, animal, and clinical research in this area, and for consideration of possible alternative strategies for increasing the levels and activity of PON1.

Is decabromodiphenyl ether (BDE-209) a developmental neurotoxicant?

Polybrominated diphenyl ether (PBDE) flame retardants have become ubiquitous environmental pollutants. The relatively higher body burden in toddlers and children has raised concern for their potential developmental neurotoxicity, which has been suggested by animal studies, in vitro experiments, and recent human epidemiological evidence. While lower brominated PBDEs have been banned in several countries, the fully brominated decaBDE (BDE-209) is still utilized, though manufacturers will discontinue production in the U.S.A. in 2013. The recent decision by the U.S. Environmental Protection Agency to base the reference dose (RfD) for BDE-209 on a developmental neurotoxicity study has generated some controversy. Because of its bulky configuration, BDE-209 is poorly absorbed and does not easily penetrate the cell wall. Its acute and chronic toxicities are relatively low, with the liver and the thyroid as the primary targets, though there is some evidence of carcinogenicity. A few animal studies have indicated that BDE-209 may cause developmental neurotoxicity, affecting motor and cognitive domains, as seen for other PBDEs. Limited in vivo and in vitro studies have also evidenced effects of BDE-209 on thyroid hormone homeostasis and direct effects on nervous cells, again similar to what found with other lower brominated PBDEs. In contrast, a recent developmental neurotoxicity study, carried out according to international guidelines, has provided no evidence of adverse effects on neurodevelopment, and this should be considered in a future re-evaluation of BDE-209. While estimated exposure to BDE-209 in children is believed to be several orders of magnitude below the most conservative RfD proposed by the USEPA, questions remain on the extent and relevance of BDE-209 metabolism to lower brominated PBDEs in the environment and in humans.

Neurotoxicity of domoic acid in cerebellar granule neurons in a genetic model of glutathione deficiency

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 α-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.

Neurotoxicity of a polybrominated diphenyl ether mixture (DE-71) in mouse neurons and astrocytes is modulated by intracellular glutathione levels

Polybrominated diphenyl ether (PBDE) flame retardants have become widespread environmental contaminants. Body burden in the U.S. population has been shown to be higher than in other countries, and infants and toddlers have highest exposure through maternal breast milk and household dust. The primary concern for adverse health effects of PBDEs relates to their potential developmental neurotoxicity, which has been found in a number of animal studies. Information on the possible mechanisms of PBDE neurotoxicity is limited, though some studies have suggested that PBDEs may elicit oxidative stress. The present study examined the in vitro neurotoxicity of DE-71, a penta-BDE mixture, in primary neurons and astrocytes obtained from wild-type and Gclm knockout mice, which lack the modifier subunit of glutamate-cysteine ligase and, as a consequence, have very low levels of glutathione (GSH). These experiments show that neurotoxicity of DE-71 in these cells is modulated by cellular GSH levels. Cerebellar granule neurons (CGNs) from Gclm (-/-) mice displayed a higher sensitivity to DE-71 toxicity compared to CGNs from wild-type animals. DE-71 neurotoxicity in CGNs from Gclm (+/+) mice was exacerbated by GSH depletion, and in CGNs from both genotypes it was antagonized by increasing GSH levels and by antioxidants. DE-71 caused an increase in reactive oxygen species and in lipid peroxidation in CGNs, that was more pronounced in Gclm (-/-) mice. Toxicity of DE-71 was mostly due to the induction of apoptotic cell death. An analysis of DE-71-induced cytotoxicity and apoptosis in neurons and astrocytes from different brain areas (cerebellum, hippocampus, cerebral cortex) in both mouse genotypes showed a significant correlation with intracellular GSH levels. As an example, DE-71 caused cytotoxicity in hippocampal neurons with IC50s of 2.2 and 0.3 microM, depending on genotype, and apoptosis with IC50s of 2.3 and 0.4 microM, respectively. These findings suggest that the developmental neurotoxicity of PBDE may involve oxidative stress, and that individual with genetic polymorphisms leading to lower GSH levels may be more susceptible to their adverse effects.

Low concentrations of the brominated flame retardants BDE-47 and BDE-99 induce synergistic oxidative stress-mediated neurotoxicity in human neuroblastoma cells.

Polybrominated diphenyl ether (PBDE) flame retardants have become widespread environmental contaminants. The highest body burden has been found in toddlers and infants, due to their exposure through breast milk and house dust, and the current concern for potential adverse health effects of PBDEs relates to their developmental neurotoxicity. The mechanisms underlying the neurotoxicity of PBDEs are largely not understood, though there is evidence that PBDEs may elicit oxidative stress. In this study, two different mathematical models were used to evaluate the interaction between BDE-47 and BDE-99 on viability of neuronal cells. The combined exposure to these compounds induced synergistic effects at concentrations of BDE-47 below its threshold doses, and in a wide range of BDE-99 concentrations below its IC(50). In contrast, at concentrations of BDE-47 near its IC(50) value, and in a wide range of BDE-99 concentrations, antagonistic effects were observed. The interactions observed on cell viability were confirmed by an assessment of induction of oxidative stress. The finding that co-exposure to BDE-47 and BDE-99 could induce synergistic neurotoxic effects, in particular at low doses of BDE-47, is of much toxicological interest, as humans are exposed to mixtures of PBDEs, most notably tetra- and penta-BDE congeners.

DOMOIC ACID AS A DEVELOPMENTAL NEUROTOXIN

Domoic acid (DomA) is an excitatory amino acid which can accumulate in shellfish and finfish under certain environmental conditions. DomA is a potent neurotoxin. In humans and in non-human primates, oral exposure to a few mg/kg DomA elicits gastrointestinal effects, while slightly higher doses cause neurological symptoms, seizures, memory impairment, and limbic system degeneration. In rodents, which appear to be less sensitive than humans or non-human primates, oral doses cause behavioral abnormalities (e.g. hindlimb scratching), followed by seizures and hippocampal degeneration. Similar effects are also seen in other species (from sea lions to zebrafish), indicating that DomA exerts similar neurotoxic effects across species. The neurotoxicity of DomA is ascribed to its ability to interact and activate the AMPA/KA receptors, a subfamily of receptors for the neuroexcitatory neurotransmitter glutamate. Studies exploring the neurotoxic effects of DomA on the developing nervous system indicate that DomA elicits similar behavioral, biochemical and morphological effects as in adult animals. However, most importantly, developmental neurotoxicity is seen at doses of DomA that are one to two orders of magnitude lower than those exerting neurotoxicity in adults. This difference may be due to toxicokinetic and/or toxicodynamic differences. Estimated safe doses may be exceeded in adults by high consumption of shellfish contaminated with DomA at the current limit of 20 microg/g. Given the potential higher susceptibility of the young to DomA neurotoxicity, additional studies investigating exposure to, and effects of this neurotoxin during brain development are warranted.

Modulation of Neuritogenesis by Astrocyte Muscarinic Receptors

Astrocytes have been shown to release factors that have promoting or inhibiting effects on neuronal development. However, mechanisms controlling the release of such factors from astrocytes are not well established. Astrocytes express muscarinic receptors whose activation stimulates a robust intracellular signaling, although the role of these receptors in glial cells is not well understood. Acetylcholine and acetylcholine receptors are present in the brain before synaptogenesis occurs and are believed to be involved in neuronal maturation. The present study was undertaken to investigate whether stimulation of muscarinic receptors in astrocytes would modulate neurite outgrowth in hippocampal neurons. Rat hippocampal neurons, co-cultured with rat cortical astrocytes previously exposed to the cholinergic agonist carbachol, displayed longer neurites. The effect of carbachol in astrocytes was due to the activation of M3 muscarinic receptors. Exposure of astrocytes to carbachol increased the expression of the extracellular matrix proteins fibronectin and laminin-1 in these cells. This effect was mediated in part by an increase in laminin-1 and fibronectin mRNA levels and in part by the up-regulation of the production and release of plasminogen activator inhibitor-1, an inhibitor of the proteolytic degradation of the extracellular matrix. The inhibition of fibronectin activity strongly reduced the effect of carbachol on the elongation of all the neurites, whereas inhibition of laminin-1 activity reduced the elongation of minor neurites only. Plasminogen activator inhibitor-1 also induced neurite elongation through a direct effect on neurons. Taken together, these results demonstrate that cholinergic muscarinic stimulation of astrocytes induces the release of permissive factors that accelerate neuronal development.

Comparative Cytotoxicity and Intracellular Accumulation of Five Polybrominated Diphenyl Ether Congeners in Mouse Cerebellar Granule Neurons

Polybrominated diphenyl ethers (PBDEs), a group of flame retardants comprising 209 congeners, have become widespread environmental pollutants. High levels of PBDEs have been detected in human tissues, particularly in North America, and body burden is especially high in infants and toddlers because of exposure through breast milk and house dust. Increasing evidence, provided by animal studies, suggests that PBDEs are developmental neurotoxicants, although the underlying mechanisms are still unknown. Various PBDEs have been reported to cause oxidative stress and to induce apoptotic cell death in several cell types. In the present study, we investigated the comparative neurotoxicity in mouse cerebellar granule neurons of five brominated diphenyl ether (BDE) congeners, chosen among the most commonly found at the highest levels in human tissues. All BDE congener tested (BDE-47, BDE-99, BDE-100, BDE-153, and BDE-209) decreased cell viability and induced apoptotic cell death, which was prevented by antioxidants. They also caused oxidative stress, as indicated by an increase in reactive oxygen species and in lipid peroxidation. For all end points measured, the potency ranking of the congeners was BDE-100 > BDE-47 > BDE-99 > BDE-153 >> BDE-209. Measurement of BDE congener levels in neurons after exposure to different concentrations showed a significant accumulation in cells, which followed the same relative ranking. The findings suggest that all BDE congeners tested exhibit the same general mode of action (induction of oxidative stress-mediated apoptosis) and that the ability of each isomer to elicit such effects is dependent upon their accumulation in neurons, particularly in the microsomal fraction and the mitochondria.

Mouse cerebellar astrocytes protect cerebellar granule neurons against toxicity of the polybrominated diphenyl ether (PBDE) mixture DE-71

A large body of evidence indicates that polybrominated diphenyl ether (PBDE) flame retardants have become widespread environmental pollutants. Body burden is particularly high in infants and toddlers, due to exposure through maternal milk and house dust. Animal studies suggest that PBDEs may exert developmental neurotoxicity, via mechanisms that are still elusive. PBDEs have been reported to cause oxidative stress and apoptotic cell death in neurons in vitro, when tested in mono-cultures. Here we report the results of experiments in which mouse cerebellar granule neurons (CGNs) were co-cultured with cerebellar astrocytes. Astrocytes were found to protect neurons against the toxicity of the PBDE mixture DE-71. Astrocytes from Gclm (-/-) mice, which lack the modifier subunit of glutamate cysteine ligase and, as a consequence, have very low GSH levels, were much less effective at protecting CGNs from DE-71 toxicity. The protective effects were mostly due to the ability of Gclm (+/+) astrocytes to increase GSH levels in neurons. By increasing GSH, GSH ethylester provided a similar protective effect. In vivo, where both neurons and astrocytes would be either Gclm (+/+) or Gclm (-/-), the toxicity of DE-71 to CGNs is predicted to vary 16.8-fold, depending on genotype. Hence, in addition to being intrinsically more susceptible to DE-71 toxicity because of their low GSH content, CGNs in Gclm (-/-) mice would also lack the full protective effect provided by astrocytes. Since several polymorphisms, including some in the Gclm gene, cause very low levels of GSH, it may be speculated that such individuals might display a higher susceptibility to the neurotoxic effects of PBDEs.