2015 Volume 38 Issue 8 Pages 1109-1112
Environmental influences, such as chemical exposure, have long been considered potential risk factors for neurodegenerative disorders, including neuromuscular diseases. However, no definitive links between environmental chemical exposure and a pathogenic mechanism of neurodegenerative disease has yet been established. In this study, we describe that exposure to arsenic, an environmental pollutant naturally found in drinking water, induces neuronal cell death and alteration of morphology, particularly neurite outgrowth and in the cytoskeleton of neurons. Since progressive cell loss accompanied by the alteration of neuronal structures and cytoskeleton is considered the major pathologic feature of neurodegenerative disorders, arsenic-induced neurotoxicity might contribute to an etiologic mechanism of some neurodegenerative diseases. Further, we discuss the importance of in vitro assay, particularly an embryonic toxicity test, for assessing the neurotoxicity of chemicals, because most of chemicals found in our environment remain to be evaluated regarding their neurotoxicity risk for neurodegenerative diseases.
Spinal muscular atrophy (SMA) is the most common genetic disease causing infant death due to an extended loss of motor neurons. This neuromuscular disorder results from deletions and/or mutations within the Survival Motor Neuron 1 (SMN1) gene, leading to a pathological decreased expression of functional full-length SMN protein.1) Environmental factors also have long been considered potential risk factors in the development of neurodegenerative diseases.2) For example, amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder involving primarily upper and lower motor neurons in the cerebral cortex, brainstem, and spinal cord,3) and the underlying cause of ALS remains largely unknown,2,4) although 5–10% of the diagnosed cases are associated with a genetic basis. A number of epidemiologic studies have suggested that ALS patients have been exposed to environmental toxins,5–9) and accumulating evidence has demonstrated that exposure to environmental and occupational chemicals (lead, mercury, and pesticides) increases the risk of developing neuromuscular diseases such as ALS, Guan ALS, Parkinson’s disease (PD), and multiple system atrophy.4) These data are highly favorable to the possibility that exposure to the environmental chemicals may play an important role in neuromuscular diseases. Since progressive cell death in specific neuronal cell populations is the major pathological feature of neuromuscular diseases and other neurodegenerative diseases,10) it is important to clarify the role of environmental chemical exposure in neural cell death in order to prevent neurodegenerative disorders such as neuromuscular disease.
In addition, abnormal organization of the cytoskeleton contributes to the pathology of some neuromuscular diseases such as SMA. Previous studies suggest that the small guanosine triphosphatase (GTPase) RhoA and its major downstream effector, Rho kinase (ROCK), both of which play an instrumental role in cytoskeleton organization, may contribute to the pathology of motor neuron disease, including SMA.1) Neural axon and dendrite functions are important for neuronal physiology.11,12) Neurons regulate structural functions with three cytoskeletal components: microtubules, microfilaments, and neurofilaments. Dynamic changes in cytoskeletal components occur during neuronal cell proliferation, migration, neuritogenesis, spinogenesis and synaptogenesis.13) Thus, the failure of neural cytoskeletal formation leads to neural dysfunction. This means that it is important to clarify the role of environmental chemical exposure in neural cytoskeletal formation in order to prevent neurite degeneration.
In this study, we describe neuronal cell death and the failure of neural cytoskeletal formation caused by exposure to an environmental pollutant, arsenic. Arsenic is a heavy metal and neurotoxic compound found naturally in drinking water throughout the world. Exposure to inorganic arsenic for a long period can cause peripheral neuropathy, which is similar to Guillain–Barre syndrome,14,15) and an early manifestation of neurodegenerative diseases such as Alzheimer’s disease.16) We also discuss the importance of in vitro assay for assessing neurotoxicity, particularly embryotoxicity, of environmental chemicals, since developmental exposure to environmental chemicals has been described as associated with late-life neurodegenerative processes.17,18)
Exposure to sodium arsenite, an inorganic arsenic, may lead to skin and lung cancer and various disorders such as vascular disease and peripheral neuropathy in humans. Patients intoxicated with arsenic show neurological symptoms in their feet and hands. These patients show significantly lower nerve conduction velocities in their peripheral nerves in comparison with controls. Animal studies have shown that arsenic can cross the blood–brain barrier, accumulate in different regions of the brain, including the striatum,19) alter neurotransmitter synthesis and release, and decrease locomotor activity.19,20) Rats and mice exposed to arsenic during gestation and early childhood exhibit behavioral deficits such as changes in locomotor activity, learning, memory, depression-like behavior and neuromotor reflex.21–23) The development of the central nervous system (CNS) in neonatal rats is also affected by arsenic, and exposure to arsenic has been shown to cause neuronal death in the adult rat brain.24)
Sodium arsenite at a concentration of 5 µM induces cytotoxicity at a cellular level via acting as a sulfhydryl reagent, which binds to the free thiol groups of numerous enzymes, inhibiting their functions and depleting levels of glutathione (GSH), thus destroying cell metabolism.25) Furthermore, sodium arsenite has been described to induce oxidative stress, mitochondrial damage and a broad range of pathological conditions by inciting the accumulation of reactive oxygen species. These effects of sodium arsenite ultimately lead to the induction of cell death.26–28) A previous study indicated that arsenic exposure induces free radical generation in rat neuronal cells, which diminishes the mitochondrial potential and enzyme activities of all the complexes of the electron transport chain. These early events, along with diminished ATP levels, could be co-related with the later events of the release of cytochrome c into cytosol, altered bax/bcl-2 ratio, and increased caspase-3 activity.29) In addition, Lu et al. recently reported that inorganic arsenic induces reactive oxygen species, causing neuronal cell death via both c-Jun N terminal kinase/extracellular signal-regulated kinase (JNK/ERK)-mediated mitochondria-dependent and GRP 78/CHOP-triggered apoptosis pathways.30) These results indicate that exposure to inorganic arsenic at least contributes to neuronal cell death via a mitochondrial dependent pathway.
Accumulating evidence demonstrates that arsenic-induced oxidative stress leads to neurite damage and cytoskeletal alteration.31,32) Cytoskeletal alteration has been considered a major histopathologic hallmark of neurodegenerative disease. For example, abnormal accumulations of neurofilaments in the cell body and proximal axons are major pathologic features of ALS.33) Neurofilaments are composed of light (NF-L), medium (NF-M), and heavy (NF-H) subunits, and organization of the neruofilament cytoskeleton is required for the formation of large-caliber axons.34) It has been reported that arsenic exposure alters the expression of NF in neuronal cells, decreases NF transport to axonal outgrowth, and increases perikaryal NF expression.31) Overexpression of NF-L causes an abnormal accumulation of neurofilaments in the perikaryal and proximal axons of neurons.33) In our study, a remarkable increase in the gene expression of NF-L and NF-M proteins was observed in neural cells exposed to sodium arsenite, in which the suppression of neurite outgrowth was also observed35) (Table 1). These data suggest that alteration to the NF dynamic contributes to arsenic-induced neurite suppression. We also observed that exposure to sodium arsenite significantly decreased the gene expression of microtubule (Tubulin) and microtubule-associated protein (Tau) in neural cells35) (Table 1). Tau is mainly expressed in axonal outgrowth and is essential to the microtubule assembly.36) Dysfunctional Tau impairs the proper regulation of neuronal microtubule dynamics, which is followed by neuronal death37); and low levels of Tau expression inhibit axonal elaboration,38) suggesting the important role of Tau expression in the formation of neurite outgrowth. Taken together, it is possible that an alteration in the expression of cytoskeletal components, particularly neurofilaments and microtubule proteins, contributes to arsenic-induced neurite outgrowth suppression.
| Cytoskeletal components | Alteration of gene expression by NaAsO2 | Function | Effects of sodium arsenite exposure |
|---|---|---|---|
| Neurofilaments | |||
| ・Neurofilament-light (NF-L) | ↑ | ・Structural support for axon and dendrite | ・Decreased NF transport to axon and dendrite |
| ・Neurofilament-medium (NF-M) | ↑ | ・Accumulation of NF in perikaryal and proximal axon | |
| ・Neurofilament-heavy (NF-H) | → | ||
| Microtubules | |||
| ・Tau | ↓ | ・Formation of axon and dendrite | ・Impaired microtubules dynamics |
| ・Tubulin | ↓ | ・Axonal/dendritic extension and retraction | ・Neurite degeneration and cell death |
| ・Microtubule associated protein 2 (MAP2) | → | ||
| Microfilament | |||
| ・Actin | → | ・Formation of growth cone and neurite extension | ・No effects on actin organization |
Because environmental chemicals pose many potential risks for the development and progress of neurodegenerative disorders, the neurotoxic assessment of environmental chemicals, including during the developmental stage, is important to the prevention of neurodegenerative disorders caused by chemicals. Especially, in vitro assessment methods have promise in the neurotoxic assessment of environmental chemicals due to their cost performance and high throughput. In 1997, Spielmann et al. developed an in vitro model for screening embryotoxicity based on mouse embryonic stem (ES) cells.39) This is termed the “embryonic stem cell test” (EST). The EST is based on the assessment of three toxicological endpoints: (1) the morphological analysis of beating cardiomyocytes in embryoid body (EB) outgrowths compared to cytotoxic effects on (2) mouse ES cells as undifferentiated cells, and (3) mouse embryonic fibroblasts as differentiated cells. As an in vitro system, which mirrors both proliferation and differentiation, EST was proven in an international validation study by the European Centre for the Validation of Alternative Methods (ECVAM) to be a reliable assay for the prediction of embryotoxicity in vivo.40) Although EST has been demonstrated to be a reliable in vitro embryotoxicity test, it may not be a useful method to screen embryotoxic chemicals that affect the differentiation of noncardiac tissues, because the inhibition of cardiac differentiation alone is used as the endpoint of differentiation in the EST. Therefore, several suggestions have been made in regard to how to add additional endpoints.41–43) These include additional ES cell differentiation endpoints, as well as the development of new molecular markers for the detection of toxic effects on embryonic development.43) Baek et al. improved the EST and involved a neural endpoint (Tuj-1 expression) for the correct classification of developmental neurotoxic heavy metal, including arsenic compounds, and classified arsenic compounds as weakly or strongly neurotoxicant.44) Furthermore, a recent study showed that arsenite decreased muscle and neuronal cell differentiation.45) Arsenic exposure affected the sonic hedgehog signaling pathway, which plays an important role during the differentiation of both neurons and skeletal muscle, ultimately decreasing the expression of the Gli2 transcription factor in embryonic stem cells.45) These results suggest that arsenic exposure in fetal and/or infant stages also lead to neurodegenerative disorders, including neuromuscular diseases. Since most of the chemicals found in our environment have not yet been evaluated for their neurotoxicity, assessment of the toxic effects of environmental chemicals on the differentiation of neurons using in vitro assay, including EST, may provide valuable information regarding neurotoxicity risks for neurodegenerative diseases.
In this study, we determined that arsenic exposure induces neuronal cell death and alterations in the morphology and cytoskeletal components of neurons. Since cell death and abnormal alterations in the morphology and cytoskeletal components of neurons is the common feature of neurodegenerative diseases, arsenic-induced neurotoxicity serves as an important investigative link between environmental exposure and the pathogenic mechanisms of neurodegenerative diseases. Further studies are needed to assess the toxic effects of arsenic on the morphology of neurons and behavior in vivo, which may provide a new animal model of neurodegenerative disease induced by chemical exposure. We also discussed the importance of an in vitro assay to assess the neurotoxicity of chemicals. Although the influence of environmental chemicals has long been suspected to play an important role in neurodegenerative diseases, most chemicals found in our environment remain to be evaluated regarding their neurotoxicity risk for neurodegenerative diseases. Identifying such potential risk of chemicals, using in vitro and in vivo experimental models, is important for the prevention and reduction of the burden of these diseases on human health.
The authors declare no conflict of interest.
