Identification of anti-tumor components from toad venom.

Secretion of granular glands from the skin of amphibians is a fascinating resource of active substances, particularly for cancer therapy in clinical practice of Traditional Chinese Medicine. A variety of anti-tumor peptides have been isolated from different toads and frogs; however, no anti-tumor peptides are reported in toad venom of Bufo gargarizans. Firstly, soluble fraction from fresh toad venom (FTV) was compared with that from dried toad venom (DTV), using HPLC analysis. It was revealed that FTV has a different HPLC chromatography compared with DTV. Soluble fraction of FTV is more toxic to SH-SY5Y cells than that of DTV, as evaluated by MTT assay. Secondly, it was identified that protein components from soluble fractions of FTV and DTV possess different patterns by SDS-PAGE analysis, and proteins from FTV are also more toxic than that from DTV. Thirdly, an immobilized basic fibroblast growth factor (bFGF) affinity column was used to isolate bFGF-binding components from soluble fraction of FTV, and it was identified that bFGF-binding components prohibited bFGF-dependent neurite growth of SH-SY5Y cells. Finally, it was identified that bFGF-binding components activated apoptosis via upregulation of caspase-3 and caspase-8 expression in SH-SY5Y cells. These data suggest that FTV contains active components that interact with bFGF and activate apoptosis in SH-SY5Y cells.

The chaperone Grp78 in protein folding disorders of the nervous system.

Chaperones are essential for the proper folding of proteins, and their dysfunction or depletion may be a key factor in protein folding disorders in the central nervous system. In normal conditions the cell regulates the proper folding of proteins by endoplasmic reticulum chaperones, called heat shock proteins, the cellular machinery that correctly folds newly synthesized and partially folded proteins or initiates degradation of misfolded proteins. Maintaining protein homeostasis within the cell is vital for the cells to function and survive. However, under conditions of cellular stress, proteastatic mechanisms must be activated to recycle, refold, or initiate degradation of misfolded or unfolded proteins. In this commentary, we will discuss the importance of chaperones, more specifically the 78 kd glucose regulated protein Grp78 (also known as BiP and HSP5a), in Parkinson's, Alzheimer's, Huntington's, and prion diseases, and the role that metals may play in exacerbating neurodegenerative diseases.

HSPA5 forms specific complexes with copper.

Our previous study indicated that Hspa5 directly interacts with copper (Cu) to maintain Cu homeostasis in astrocytes. In this study, we explored the possibility that Cu forms a specific complex with Hspa5 by assaying stoichiometric binding of Cu and other metals to recombinant human HSPA5 (rh-HSPA5) in silico. Spectrophotometric analysis showed that incubation of rh-HSPA5 with Cu but not with Fe, Mn, Zn, or Pb in the presence of ascorbic acid produced an absorbance peak at 470 nm. Furthermore, the absorbance peak was absent when bovine serum albumin was incubated with Cu and when another recombinant protein YWHAZ-14-3-3-Zeta carrying a 6× histidine tag identical to the tag in the rh-HSPA5 was incubated with Cu. The absorbance peak produced by Cu and rh-HSPA5 was abolished by EDTA treatment and was stabilized at pH levels above 6.5. Assay of the stoichiometry of metal binding to the purified rh-HSPA5 showed that one molecule of the rh-HSPA5 could chelate 1 or 2 Cu, 13 iron (Fe), 5 zinc (Zn) and 10 lead (Pb) ions but not manganese (Mn). These data further support our previous finding that HSPA5 specifically forms a complex with Cu to help maintain Cu homeostasis.

Involvement of the molecular chaperone Hspa5 in copper homeostasis in astrocytes.

Copper (Cu) ion availability in tissues and cells must be closely regulated within safe limits by Cu transporters and chaperones. Astrocytes play key roles in metal homeostasis and distribution in the brain that are only partially understood. The purpose of this study was to define the role that the protein chaperone Hspa5, also known as Grp78, plays in Cu homeostasis in astrocytes. First passage cultures of primary astrocytes from neonatal rats and cultures of the C6 rat glioma cells were used as models. We found that the level of Cu accumulation in astrocyte cultures increased with Cu concentrations in the medium, and Cu treatment significantly reduced cellular levels of iron (Fe), manganese (Mn) and zinc (Zn). Cu accumulation specifically induced protein expression of Hspa5 but not metallothioneins (MTs) in astrocytes. In C6 cells, Hspa5 was identified as one component of a Cu-binding complex and shown to directly bind Cu. However, the level of Hspa5 expression was not proportional to Cu accumulation in astrocytes and C6 cells: astrocytes expressed low protein levels of Hspa5 compared to C6 cells but accumulated significantly more Cu than did C6 cells. Consistent with this finding, astrocytes expressed a lower level of the Cu-extruding protein Atp7a than did C6 cells, and depletion of Hspa5 by RNA interference resulted in significantly increased Cu accumulation and induction of MT1/2 expression. These data demonstrate that Hspa5 is involved in Cu homeostasis in astrocytes but not as a Cu storage protein.

ER chaperone-metal interactions: links to protein folding disorders.

Chaperones in the endoplasmic reticulum play vital roles in the folding, assembly, and post-translational modification of secretory proteins and also recycle, refold, or initiate degradation of misfolded proteins. Chaperone deficiencies in either amount or function are implicated in the etiology or pathogenesis of Alzheimer's disease and other protein folding disorders of the central nervous system. In this review, we discuss evidence that chaperones become pathologic through deleterious interactions with metals and then promote protein folding disorders. The "master regulator" chaperone GRP78 in the endoplasmic reticulum is a compelling subject for investigation in this context because it is located at the hub of signaling pathways in a complex chaperone network. It has therefore been studied by several laboratories in conjunction with exposure to toxic metals. The key points of this review are that metals are implicated in the etiology or pathogenesis of Alzheimer's disease and other protein folding disorders, metals induce the expression GRP78, often associated with oxidative stress, some metals bind to GRP78, and lead (Pb) impairs GRP78 function when it binds to GRP78. If certain metals do indeed cause or promote the aggregation of toxic proteins in the central nervous system, as the available evidence indicates, the identification of the mechanisms by which they act would provide valuable leads for the development of therapies to prevent or reverse toxic protein aggregation.

Copper handling by astrocytes: insights into neurodegenerative diseases.

Copper (Cu) is an essential trace element in the brain that can be toxic at elevated levels. Cu accumulation is a suspected etiology in several neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and prion-induced disorders. Astrocytes are a proposed depot in the brain for Cu and other metals, including lead (Pb). This article describes the physiological roles of Cu in the central nervous system and in selected neurodegenerative diseases, and reviews evidence that astrocytes accumulate Cu and protect neurons from Cu toxicity. Findings from murine genetic models of Menkes disease and from cell culture models concerning the molecular mechanisms by which astrocytes take up, store, and buffer Cu intracellularly are discussed, as well as potential mechanistic linkages between astrocyte functions in Cu handling and neurodegenerative diseases.

Determination of metal interactions with the chaperone Hspa5 in human astrocytoma cells and rat astrocyte primary cultures.

Molecular chaperones assist the folding of nascent proteins during translation into their correct conformations. Neurotoxic metals such as copper (Cu) and lead (Pb) may produce a deficiency in chaperone function that compromises protein secretion and exacerbates protein aggregation, potentially promoting neurodegenerative diseases that exhibit protein aggregation. Because astrocytes function as depots in the brain for certain metals, including Cu and Pb, the interaction of metals with chaperones in these cells is of interest. Furthermore, Pb and Cu bind strongly to the molecular chaperone heat shock 70 kDa protein Hspa5, also known as glucose-regulated protein 78 (Grp78) or immunoglobulin-binding protein (BiP). This chapter describes methods for expressing fluorescent chimeric proteins in astrocytes and astrocytoma cells in order to examine the metal-induced cytosolic redistribution of Hspa5, as well as associated effects on the secretion of interleukin-6 (IL-6).

Defining and modeling known adverse outcome pathways: Domoic acid and neuronal signaling as a case study.

An adverse outcome pathway (AOP) is a sequence of key events from a molecular-level initiating event and an ensuing cascade of steps to an adverse outcome with population-level significance. To implement a predictive strategy for ecotoxicology, the multiscale nature of an AOP requires computational models to link salient processes (e.g., in chemical uptake, toxicokinetics, toxicodynamics, and population dynamics). A case study with domoic acid was used to demonstrate strategies and enable generic recommendations for developing computational models in an effort to move toward a toxicity testing paradigm focused on toxicity pathway perturbations applicable to ecological risk assessment. Domoic acid, an algal toxin with adverse effects on both wildlife and humans, is a potent agonist for kainate receptors (ionotropic glutamate receptors whose activation leads to the influx of Na(+) and Ca²(+)). Increased Ca²(+) concentrations result in neuronal excitotoxicity and cell death, primarily in the hippocampus, which produces seizures, impairs learning and memory, and alters behavior in some species. Altered neuronal Ca²(+) is a key process in domoic acid toxicity, which can be evaluated in vitro. Furthermore, results of these assays would be amenable to mechanistic modeling for identifying domoic acid concentrations and Ca²(+) perturbations that are normal, adaptive, or clearly toxic. In vitro assays with outputs amenable to measurement in exposed populations can link in vitro to in vivo conditions, and toxicokinetic information will aid in linking in vitro results to the individual organism. Development of an AOP required an iterative process with three important outcomes: a critically reviewed, stressor-specific AOP; identification of key processes suitable for evaluation with in vitro assays; and strategies for model development.

Paraquat induces cyclooxygenase-2 (COX-2) implicated toxicity in human neuroblastoma SH-SY5Y cells.

Paraquat produces dopaminergic pathologies of Parkinson's disease, in which cyclooxygenase-2 (COX-2) is implicated. However, it is unclear whether paraquat induces toxicity within dopaminergic neurons through COX-2. To address this, human neuroblastoma SH-SY5Y cells were treated with paraquat and then the involving mechanism of COX-2 was investigated. We initially examined the involvement of COX-2 in paraquat-induced toxicity. Data suggest that COX-2 is implicated in paraquat-induced reduction of viability in SY5Y cells. Then, to confirm the presence of COX-2 in SY5Y cells, we examined COX-2 mRNA and protein levels, which are regulated by NF-κB. Data indicate that paraquat activates NF-κB and up-regulates COX-2. We then checked quinone-bound proteins as quinones produced by COX-2 bind to intracellular proteins. Paraquat obviously forms quinone-bound proteins, in particular, quinone-bound DJ-1 and this formation is attenuated by meloxicam. Finally, we investigated antioxidant system including nuclear factor erythroid-related factor 2 (Nrf2), gamma glutamylcysteine synthetase (γGCS), and glutathione (GSH) as DJ-1 is linked to Nrf2 and Nrf2 regulates γGCS expression and γGCS is a GSH synthesis enzyme. Paraquat decreases protein levels of Nrf2 and γGCS and intracellular GSH level and these decreases are alleviated by meloxicam. Therefore, collectively, our data indicate that paraquat induces COX-2 implicated toxicity in SY5Y cells. In conclusion, current findings support the idea that paraquat might produce toxicity in dopaminergic neurons through COX-2.

Image analysis of Ca2+ signals as a basis for neurotoxicity assays: promises and challenges.

Free intracellular calcium ([Ca(2+)](i)) controls a wide range of cellular functions such as contraction, neurotransmitter and hormone release, metabolism, cell division and differentiation. Cytosolic Ca(2+) levels are abnormal in cells exposed to toxicants and understanding how these levels become altered may improve our ability to design high-throughput methods for the sensitive detection of cellular responses to a toxic exposure. Because Ca(2+) is involved in multiple aspects of cellular function, its role in signaling is complex. It is therefore necessary to identify the individual pathways targeted during toxicant exposure in order to use them as a tool for predictive measurements of toxicity and as targets for prevention or reversal of injury. This review illustrates several methods available for analysis of Ca(2+) responses in vitro and their applicability for understanding mechanisms of toxicity at the molecular and cellular levels. The review will also consider the usefulness of Ca(2+) imaging for predicting a unique signature for classes of toxicants. Towards this end, two methodological approaches for assessment of Ca(2+) responses to toxicants are examined: steady state measurements and complex spatial and/or temporal measurements. Each of the methods described and appropriately used results in reliable and reproducible measurements which may be applied in a high-throughput fashion to individualize in vitro assessment of cellular responses caused by toxicants.

Paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in human neuroblastoma SH-SY5Y cells.

Epidemiologic and laboratory studies suggest that paraquat can be an environmental etiologic factor in Parkinson's disease (PD). One mechanism by which paraquat may mediate cell death of dopaminergic neurons is by inducing endoplasmic reticulum (ER) stress, as suggested in a recent report. In this study, we further investigated this linkage by examining ER stress cascades. To this aim, human neuroblastoma cells (SH-SY5Y cells) were treated with paraquat and the signaling cascades through which ER stress results in apoptosis were examined. Then, it was examined whether ER stress is produced by paraquat. Paraquat increased ER stress biomarker proteins, glucose-regulated protein 78 (GRP78), ER degradation-enhancing alpha-mannosidae-like protein (EDEM), and C/EBP homologous protein (CHOP). Then, it was investigated which ER stress cascades are affected by paraquat. Paraquat activated inositol-requiring enzyme 1 (IRE1), apoptosis signal regulating kinase 1 (ASK1), and c-jun kinase (JNK). Also, paraquat activated calpain and caspase 3, but did not affect the levels of intracellular calcium and the activity of caspase 12. Finally, apoptotic DNA damage by paraquat was investigated and this damage was attenuated by salubrinal (ER stress inhibitor), thioredoxin (ASK1 inhibitor) and SP600125 (JNK inhibitor). Therefore, current data indicate that paraquat activates the IRE1/ASK1/JNK cascade associated with apoptosis in SY5Y cells.

Valproate reversibly reduces neurite outgrowth by human SY5Y neuroblastoma cells.

Valproate (VPA) is a commonly prescribed mood stabilizer. However, emerging evidence indicates that VPA administration may cause reversible symptoms of Parkinsonism and cognitive decline (P/CD) in some manic patients. The mechanism of this phenomenon is unknown. In this study, we used human SY5Y neuroblastoma cells as a neuronal model to investigate the effects of VPA on neurite outgrowth and neurofilament expression. Data showed that the treatment with VPA at therapeutic plasma levels (0.5 mM) significantly reduced cell proliferation from day 4 through day 6, and neurite outgrowth length from day 1 through day 6. Conversely, VPA had no effect on cell proliferation of human CCF astrocytoma cells but stimulated nerve growth factor (NGF)-induced neurite outgrowth from rat PC12 pheochromocytoma cells. In parallel to these alterations in human SY5Y cells, both mRNA and protein levels of neurofilament 160 (NF160) were significantly reduced, starting at day 2 and day 3, respectively, by the treatment. The inhibition of neurite outgrowth by VPA was completely reversed 2 days after cessation of VPA exposure. Furthermore, NF160 protein levels also rebounded to control levels after VPA removal. NGF application significantly alleviated the inhibition of neurite outgrowth by VPA. These data suggest that VPA-modulated NF160 expression was involved in the inhibition and the reversal of neurite outgrowth in human neuronal cells.

Multiphoton spectral analysis of benzo[a]pyrene uptake and metabolism in breast epithelial cell lines.

Quantification of polycyclic aromatic hydrocarbons (PAH) and their metabolites within living cells and tissues in real time using fluorescence methods is complicated due to overlaping excitation and/or emission spectra of metabolites. In this study, simultaneous analysis of several metabolites of a prototype carcinogenic PAH, benzo[a]pyrene (BaP) in undifferentiated (MCF10A) and differentiated (MCF10CA1h) breast cancer cells was performed using single-cell multiphoton spectral analysis. The two cell types were selected for this study because they are known to have differences in BaP uptake and metabolism and induction of aryl hydrocarbon receptor-dependent ethoxyresorufin-O-deethylase (EROD) activity. Multiphoton microscopy spectral analysis performed in cells exposed to BaP for 24 hr identified 5 major peaks of fluorescence that were monitored within spectral bands. A comparison of the fluorescence peaks within these bands to those of BaP metabolite standards indicated that a peak in the spectral range of 393-415 nm matched benzo[a]pyrene-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-),(anti) (BPDE), the ultimate carcinogenic BaP metabolite. In addition, the 426-447 nm band matched the major metabolites 3-hydroxybenzo[a]pyrene (3-OH BaP) and 9-hydroxybenzo[a]pyrene (9-OH BaP); the 458-479 nm band corresponded to the secondary metabolite benzo[a]pyrene-3,6-dione (3,6 BPQ); and a peak at 490-530 nm matched the parent compound, BaP. Multiphoton spectral analysis also revealed differences in fluorescence intensities between MCF10A and MCF10CA1h cells within three spectral bands: 393-415 nm, 426-447 nm and 458-479 nm which were partially reversed with cyclosporine A suggesting differences in efflux mechanisms between cell lines. These results demonstrate the feasibility of analyzing BaP metabolism in situ by multiphoton spectral analysis and also identifying cell-type differences in BaP accumulation and metabolism.

Paraquat-induced apoptosis in human neuroblastoma SH-SY5Y cells: involvement of p53 and mitochondria.

The herbicide paraquat is a suspected etiologic factor in the development of Parkinson's disease (PD). Paraquat was therefore used to reproduce Parkinsonian syndromes in lab animals, in which it produces dopaminergic pathogenesis. However, the factors or mechanisms by which paraquat kills dopaminergic neurons are not fully understood. Based on reported evidence that paraquat increases p53 protein levels and inhibits mitochondrial function, it was hypothesized that paraquat induces cell death in dopaminergic neurons through a mechanism in which p53 and mitochondrial apoptotic pathway are linked. To explore this possibility, dopaminergic SY5Y cells were treated with paraquat for 48 h and p53 responses were investigated, as well as biomarkers of the mitochondrial intrinsic pathway of apoptosis. Paraquat significantly increased protein levels of p53 and one of its target genes, Bax. By 24 h, paraquat decreased mitochondrial complex I activity and mitochondrial transmembrane potential and induced the release of cytochrome c from mitochondria. In addition, paraquat increased the activities of caspases 9 and 3. Finally, nuclear condensation and DNA fragmentation occurred 48 h after treatment. The decrease of mitochondrial functions, the release of cytochrome c, the increase of caspase 9 and 3 activities, and DNA damage that were produced by paraquat were inhibited by a specific p53 inhibitor, pifithrin-alpha. These findings support the conclusion that paraquat produced apoptosis in SY5Y cells through the mitochondrial intrinsic pathway associated with p53.

The bipyridyl herbicide paraquat induces proteasome dysfunction in human neuroblastoma SH-SY5Y cells.

Paraquat (PQ) is suspected to be an environmental risk factor for Parkinson's disease (PD). A strong correlation between exposure to paraquat and the occurrence of PD was reported in Canada, Taiwan, and the United States. This correlation is supported by in vivo work showing that paraquat produces dopaminergic pathogenesis. In particular, paraquat forms abnormal protein aggregates in dopaminergic neurons of mice. However, it is not clear how paraquat produces this pathology. Given that proteasome dysfunction induces aberrant protein aggregation, it was hypothesized that paraquat induces proteasome dysfunction. To explore this possibility, proteasome activity and some factors possibly contributing to proteasome dysfunction were investigated in dopaminergic SY5Y cells treated with paraquat. Furthermore, levels of alpha-synuclein and ubiquitin-conjugated proteins were measured to test whether paraquat induces protein accumulation in SY5Y cells. Results showed that at a concentration of paraquat that reduced viability by about 60% at 48 h (0.5 mM) loss of proteasome activity occurred. In addition, the cells showed decreased ATP levels and reduced mitochondrial complex V activity. These changes were significant 24 h after treatment with paraquat. Furthermore, paraquat-treated cells showed decreased protein levels of proteasome 19S subunits, but not 20S alpha or beta subunits, suggesting that the effects observed were not the result of general cytotoxicity. Paraquat also increased levels of alpha-synuclein and ubiquitinated proteins, suggesting that paraquat-induced proteasome dysfunction leads to aberrant protein accumulation. Taken together, these findings support the hypothesis that paraquat impairs proteasome function in SY5Y cells.

Activation profiles of HSPA5 during the glomerular mesangial cell stress response to chemical injury.

Environmental injury has been associated with endoplasmic reticulum (ER) stress, a response characterized by activation of the unfolded protein response, proteasomal degradation of proteins, and induction of HSPA5, also known as GRP78 or BiP. Although HSPA5 has been implicated in the stress response to environmental injury in several cell types, its role in the glomerular ER stress response is unknown. In this study, we evaluated HSPA5 activation profiles in rat glomerular mesangial cells (rGMCs) challenged with heavy metals (HgCl2 or Pb2+ acetate) or polycyclic aromatic hydrocarbons (PAHs, ie, benzo(a)pyrene [BaP]). Challenge of rGMCs with 1 or 10 microM HgCl2 or Pb2+ acetate increased HSPA5 mRNA and protein levels. The induction response was sensitive to transcriptional and translational inhibition by actinomycin D (AD) and cyclohexamide, respectively. HSPA5 mRNA was induced by 3 microM BaP in an AD-sensitive manner, but this response was unaffected by the presence of heavy metals. A promoter construct containing sequences that mediate thapsigargin (TH) inducibility of the HSPA5 promoter was refractory to both heavy metals and BaP. The HSPA5 induction response in rGMCs is conserved because it was reproduced with fidelity in immunolocalization experiments of HSPA5 protein in M15 and HEK293 cells in embryonic lines of murine and human origin, respectively. Collectively, these findings identify HSPA5 in the stress response of rGMCs and implicate regulatory mechanisms that are distinct from those involved in TH inducibility.

A 78-kDa glucose-regulated protein is involved in the decrease of interleukin-6 secretion by lead treatment from astrocytes.

Interleukin (IL)-6 is a cytokine produced mainly by microglia and astrocytes and plays a pleiotropic role in the central nervous system. In this study, we cloned rat IL-6 cDNA into an enhanced green fluorescent protein (EGFP) or a red fluorescent protein (DsRed2) vector and rat 78-kDa glucose-regulated protein (GRP78) cDNA into an EGFP vector to construct IL-6-EGFP, IL-6-DsRed2, and GRP78-EGFP chimeras for the investigation of the mechanism of IL-6 secretion from astrocytes. The data showed that constructed IL-6-EGFP and IL-6-DsRed2 chimeras retained the secretory property, and the secretion of IL-6-EGFP from astrocytes could be attenuated by GRP78 depletion with double-stranded RNA interference. Coexpression of IL-6-DsRed2 and dysfunctional GRP78-EGFP abolished IL-6-DsRed2 secretion, and two chimeric proteins colocalized inside living astrocytes. Coimmunoprecipitation analysis indicated that IL-6 and GRP78 resided in the same complex. The data further revealed that IL-6-EGFP secretion from astrocytes was blocked by the heavy metal lead (Pb) in a concentration-dependent manner. Analysis of the Pb interaction with protein on a Pb-affinity column demonstrated that Pb bound to GRP78 but failed to bind to IL-6. Therefore, these data suggest that IL-6-EGFP or IL-6-DsRed2 chimeras can be used as imaging probes to study IL-6 secretion from living cells, that GRP78 is involved in IL-6 secretion from astrocytes, and that Pb can block IL-6 secretion from astrocytes via targeting GRP78.

Effects of propofol on intracellular Ca2+ homeostasis in human astrocytoma cells.

The effects of propofol, a short-acting general anesthetic, upon cell growth and Ca(2+) signaling in a human astrocytic cell line were examined. Exposure of cells to graded concentrations of propofol resulted in a dose-dependent decrease in cell number with an inhibitory concentration of cell viability (IC50) of 31.7+/-1.2 microM. To evaluate the changes in intracellular Ca(2+) homeostasis induced by propofol, cytoplasmic and mitochondrial Ca(2+) were measured by fluorescence imaging. Mitochondrial Ca(2+) increased while cytoplasmic Ca(2+) decreased significantly at a propofol concentration lower than the IC50 (10 microM for 24 h, 1 microM for 72 h). In addition, propofol diminished the Ca(2+) response induced by fetal bovine serum (FBS). To determine the source of Ca(2+) alterations induced by propofol, pharmacologic agents targeting intracellular Ca(2+) homeostasis mechanisms were used. Nifedipine, an L-type Ca(2+) channel blocker, decreased FBS-induced Ca(2+) response of control cells to a level similar to propofol treated cells. However, diazoxide (a K(+)-ATP channel opener) administered 1 h before FBS addition restored the FBS response in propofol treated cells to a level similar to control. In addition, diazoxide increased mitochondrial Ca(2+) in control cells to a level comparable to propofol treated cells suggesting activation of these channels by propofol treatment. Addition of 1 muM RU-360 (a selective blocker of the mitochondrial Ca(2+) uniporter) for 30 min prior to propofol treatment restored mitochondrial and cytoplasmic Ca(2+) to control levels. These data suggest that voltage operated Ca(2+) channels, mitochondrial Ca(2+) and K(+)-ATP channels may be targets of propofol action in astrocytes.

Comparative non-cholinergic neurotoxic effects of paraoxon and diisopropyl fluorophosphate (DFP) on human neuroblastoma and astrocytoma cell lines.

The objective of this study was to evaluate the comparative non-cholinergic neurotoxic effects of paraoxon, which is acutely neurotoxic, and diisopropyl fluorophosphate (DFP), which induces OPIDN, in the human neuroblastoma SY5Y and the human astrocytoma cell line CCF-STTG1. SY5Y cells have been studied extensively as a model for OP-induced neurotoxicity, but CCF cells have not previously been studied. We conducted a preliminary human gene array assay of OP-treated SY5Y cells in order to assess at the gene level whether these cells can distinguish between OP compounds that do and do not cause OPIDN. Paraoxon and DFP induced dramatically different profiles of gene expression. Two genes were upregulated and 13 downregulated by at least 2-fold in paraoxon-treated cells. In contrast, one gene was upregulated by DFP and none was downregulated at the 2-fold threshold. This finding is consistent with current and previous observations that SY5Y cells can distinguish between OPs that do or do not induce OPIDN. We also examined gene array results for possible novel target proteins or metabolic pathways for OP neurotoxicity. Protein levels of glucose regulated protein 78 (GRP78) revealed that paraoxon exposure at 3 microM for 24 h significantly reduced GRP78 levels by 30% in neuroblastoma cells, whereas DFP treatment had no effect. In comparison with SY5Y neuroblastoma cells, paraoxon and DFP (3 microM for 24 h) each significantly increased GRP78 levels by 23-24% in CCF astrocytoma cells. As we have previously evaluated intracellular changes in Ca(2+) levels in SY5Y cells, we investigated the effects of paraoxon and DFP on cellular Ca(2+) homeostasis in CCF by studying cytosolic and mitochondrial basal calcium levels. A significant decrease in the ratio of mitochondrial to cytosolic Ca(2+) fluorescence was detected in CCF cultures treated for either 1 or 3 days with 1, 3, 10, or 30 microM paraoxon. In contrast, treatment with DFP for 1 day had no significant effect on the ratio of mitochondrial to cytosolic Ca(2+) fluorescence; after 3 days treatment, only 30 microM decreased the ratio. These results are consistent with the finding that paraoxon induced a greater decrease than did DFP of intracellular esterase activity in CCF cells. The changes seen in the ratio of mitochondrial to cytosolic Ca(2+) represent a good indicator of the degree of injury induced by each chemical tested. This work further develops in vitro models that distinguish between compounds that cause OPIDN and those that induce acute neurotoxicity only. The study also exposes additional OP-induced toxicities that may be obscured in vivo.

In vitro models for assessing neurotoxicity of mixtures.

Rapid and inexpensive methods are needed to investigate the interactions of complex mixtures. This commentary addresses the use of cell cultures to detect neurotoxicity of simple binary mixtures, which is a first step in the development of such methods. A small number of recent studies from our laboratory are examined. Though such studies are few, they offer guidance for optimizing the value of cell cultures as systems for chemical toxicity screening and mechanistic research. The same issues that apply to in vitro neurotoxicity studies of single agents also apply to the study of mixtures, such as relevance of endpoints tested, biological usefulness and limitations of cell culture models, and relevance of exposures tested. In this commentary we will focus on several aspects of these issues.