Hispidulin, a naturally occurring flavonoid found in various medicinal plants, exhibits anti-proliferative effects in multiple cancer models; however, its role in regulating intracellular calcium (Ca2+) signaling in human breast cancer cells remains unclear. Here, we investigated its effects on intracellular Ca2+ dynamics, cytotoxicity, and Ca2+-associated signaling in T-47D human breast cancer cells. Hispidulin (40–120 μM) induced a concentration-dependent increase in intracellular Ca2+ levels ([Ca2+]i) accompanied by reduced cell viability, and pretreatment with the intracellular Ca2+ chelator BAPTA-AM further enhanced cytotoxicity, indicating that disruption of Ca2+ homeostasis potentiates cell death. Removal of extracellular Ca2+ partially attenuated the response, with additional inhibition by nifedipine, a dihydropyridine-sensitive Ca2+ channel blocker, suggesting involvement of voltage-dependent Ca2+ influx. The Ca2+ response was also reduced by 2-aminoethoxydiphenyl borate (2-APB), an inhibitor of store-operated Ca2+ entry (SOCE), and by the protein kinase C (PKC) inhibitor GF109203X, implicating SOCE and PKC signaling. Thapsigargin-induced depletion of endoplasmic reticulum (ER) Ca2+ stores indicated that hispidulin mobilizes ER Ca2+, while phospholipase C (PLC) inhibition by U73122 completely abolished the [Ca2+]i increase. Collectively, these findings demonstrate that hispidulin regulates intracellular Ca2+ homeostasis via PLC-dependent ER Ca2+ release and extracellular Ca2+ influx through SOCE and nifedipine-sensitive Ca2+ entry components, leading to PKC activation. Enhanced cytotoxicity following Ca2+ chelation further underscores the importance of Ca2+ homeostasis in determining cellular responses to hispidulin.

Alternariol (AOH), a mycotoxin produced by Alternaria species, has been associated with oxidative and inflammatory toxicity, although its effects on astrocytes remain poorly understood. This study investigated AOH-induced cellular responses and the protective effects of eicosapentaenoic acid (EPA) in human astrocytes. Exposure to AOH (25-125 μM) reduced cell viability, increased intracellular reactive oxygen species (ROS) production, depleted glutathione (GSH), and elevated the expression of pro-inflammatory cytokines, including TNF-α, IL-1β, and IL-6. AOH also induced apoptosis-related molecular alterations, characterized by increased Bax, cleaved caspase-9, and cleaved caspase-3 expression together with reduced Bcl-2 levels. In addition, AOH modulated the expression of Nrf2 and its downstream antioxidant targets, heme oxygenase-1 (HO-1) and NAD(P)H oxidoreductase 1 (NQO1), suggesting involvement of antioxidant defense-related signaling. Pretreatment with EPA (5 μM) significantly attenuated AOH-induced oxidative stress, inflammatory responses, and apoptosis-related molecular changes. These findings further identify astrocytes as a sensitive cellular target of Alternaria mycotoxins. Collectively, the findings indicate that AOH disrupts redox homeostasis and promotes cellular stress responses in human astrocytes, whereas EPA mitigates these effects and helps maintain cellular homeostasis.

Bupivacaine, a commonly used aminoamide local anaesthetic, exerts cellular effects beyond anaesthesia. However, its effects on calcium (Ca2+) signalling and the physiological responses of lung fibroblasts remain insufficiently characterised. This study aimed to determine whether bupivacaine disrupts intracellular Ca2+ homeostasis and induces cytotoxicity in IMR-90 human foetal lung fibroblasts via Ca2+-dependent mechanisms. Cell viability was assessed using the CCK-8 assay, and intracellular Ca2+ levels ([Ca2+]ᵢ) were monitored in fura-2-loaded cells. Pharmacological inhibitors were used to dissect the signalling pathways involved. Bupivacaine (20-60 μM) caused a concentration-dependent rise in [Ca2+]ᵢ and a corresponding decline in cell viability. These effects were significantly attenuated by pre-treatment with the Ca2+ chelator BAPTA-AM, indicating a Ca2+-dependent cytotoxic mechanism. Mechanistically, bupivacaine induced Ca2+ influx through store-operated Ca2+ entry (SOCE), involving protein kinase C (PKC), and triggered Ca2+ release from the endoplasmic reticulum (ER) via a phospholipase C (PLC)-dependent pathway. Mn2+ quenching assays confirmed SOCE as the primary route of Ca2+ entry. Bupivacaine disrupts Ca2+ signalling through both extracellular influx and intracellular release, contributing to pulmonary cytotoxicity. These findings underscore the relevance of Ca2+ homeostasis in anaesthetic-related lung injury and suggest that Ca2+ chelation may be a potential protective strategy.

Flavonoids are plant-derived polyphenols widely found in fruits, vegetables, and herbs, and have attracted growing interest for their neuroprotective and anti-cancer properties. Among them, calycosin (7-hydroxy isoflavone), a major bioactive component of Astragalus membranaceus, has demonstrated anti-inflammatory, antioxidant, and estrogenic activities. While its protective effects have been reported in cardiovascular and neurodegenerative models, its pharmacological role in glioblastoma and calcium ions (Ca²⁺) signaling remains largely unexplored. In this study, we investigated the effects of calycosin on intracellular Ca²⁺ levels ([Ca²⁺]i), cytotoxicity, and Ca²⁺-related signaling mechanisms in DBTRG-05MG human glioblastoma cells. Calycosin (25–75 µM) induced a concentration-dependent elevation in [Ca²⁺]i and significantly reduced cell viability. These cytotoxic effects were reversed by the intracellular Ca²⁺ chelator BAPTA-AM, indicating Ca²⁺-dependent toxicity. In Ca²⁺-free conditions, calycosin-induced Ca²⁺ entry was suppressed, and this was further attenuated by 2-aminoethoxydiphenyl borate (2-APB), a modulator of store-operated calcium entry (SOCE), and GF109203X, a protein kinase C (PKC) inhibitor. Thapsigargin experiments confirmed that calycosin mobilized Ca²⁺ from the endoplasmic reticulum (ER), while phospholipase C (PLC) inhibition by U73122 completely abolished the [Ca²⁺]i response. These results indicate that calycosin-induced cell death involves PLC-dependent ER Ca²⁺ release and SOCE-mediated Ca²⁺ influx, with PKC as a downstream effector. These findings highlight intracellular Ca²⁺ as a critical mediator of calycosin’s cytotoxic effects and suggest that calycosin, or related flavonoids, may serve as promising candidates for modulating Ca²⁺-dependent oncogenic signaling in glioblastoma therapy.

Alternariol (AOH) is a low-molecular-weight mycotoxin produced by Alternaria species, frequently found in cereal grains, tomatoes, and processed foods. While its genotoxic and endocrine-disrupting effects are well established, emerging studies suggest a neurotoxic potential that remains poorly understood. Astrocytes-key regulators of neuronal support and calcium (Ca2+) balance in the central nervous system (CNS)-are an underexplored target of AOH toxicity. Here, we investigated the effects of AOH on Ca2+ signaling and viability in human astrocytes (Gibco Human Astrocyte, GHA cells). AOH exposure (25-125 μM) caused a concentration-dependent rise in cytosolic Ca2+ concentrations ([Ca2+]ᵢ) and induced significant cytotoxicity. These effects were reversed by the intracellular Ca2+ chelator BAPTA-AM, suggesting a Ca2+-dependent mechanism. Mechanistically, AOH mobilized Ca2+ from the endoplasmic reticulum (ER), as shown by thapsigargin blockade, and facilitated extracellular influx via store-operated calcium entry (SOCE), inhibited by 2-APB (a SOCE inhibitor) and GF109203X (a protein kinase C, PKC inhibitor). Notably, inhibition of phospholipase C (PLC) by U73122 completely abolished the Ca2+ rise, implicating the PLC-ER axis as a key upstream trigger. Together, our findings reveal a novel Ca2+-dependent mechanism by which AOH disrupts astrocyte homeostasis, providing new insights into its neurotoxic potential and highlighting Ca2+ signaling as a potential therapeutic target in glial toxicology.

The effect of para (p)-cresol, an essential oil component, on calcium ion (Ca2+) signaling in human glioblastoma is unknown. The present study aimed to investigate how p-cresol influences intracellular Ca2+ levels ([Ca2+]i) and viability in DBTRG-05MG human glioblastoma cells. Cells were treated with p-cresol to assess its impact on cell viability and [Ca2+]i. Cell viability was evaluated using a WST-1 assay. [Ca2+]i was measured using a fluorescence-based Ca2+ indicator. Cells were loaded with the Ca2+-sensitive dye (fura-2), and fluorescence intensity was recorded before and after p-cresol treatment to determine changes in [Ca2+]i. p-Cresol induced concentration-dependent increases in [Ca2+]i between 50 and 150 µM. At 50-250 µM, p-cresol triggered cell death; this effect was reversed by pretreating the cells with the Ca2+ chelator BAPTA-AM. The removal of extracellular Ca2+ inhibited Ca2+ entry. p-Cresol-induced Ca2+ influx was confirmed by Mn2+-induced quenching of fura-2 fluorescence. Store-operated Ca2+ channel modulators SKF96365 and 2-aminoethoxydiphenyl borate and the protein kinase C inhibitor GF109203X inhibited p-cresol-induced Ca2+ entry, but voltage-gated Ca2+ channel blocker nifedipine did not. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin in Ca2+-free medium inhibited p-cresol-induced [Ca2+]i rises; conversely, treatment with p-cresol decreased thapsigargin-induced [Ca2+]i rises. Furthermore, phospholipase C (PLC) inhibition with U73122 abolished p-cresol-induced [Ca2+]i rises. In DBTRG-05MG cells, p-cresol triggered Ca2+-associated cell death. The process involved the entry of Ca2+ through PKC-regulated store-operated Ca2+ channels and release of Ca2+ from the endoplasmic reticulum, which depends on PLC. Additionally, BAPTA-AM, which has Ca2+-chelating properties, may be a promising compound in preventing p-cresol-induced cytotoxicity, a potential breakthrough in neurotoxic research in glioblastoma cell model.

Fipronil (FIP), a widely used agricultural insecticide, has raised significant concerns due to its harmful environmental residues and neurotoxic effects. Although FIP's toxicological impact has been studied in some neuronal cell models, its specific effects on human glial cells remain poorly understood. This study sought to investigate the mechanisms of FIP-induced toxicity and evaluate the protective potential of vitamin E (VE), a known antioxidant. Using human astrocyte (GHA) cells, we treated cells with FIP (5-25 μM), VE (20 μM), or both and compared their responses to untreated controls. Our findings revealed that FIP significantly reduced cell viability and caused morphological changes in astrocytes, including cellular shrinkage and detachment. FIP also increased the production of reactive oxygen species (ROS) and depleted intracellular glutathione (GSH) levels, indicating a disruption of cellular redox balance and the onset of oxidative stress. Furthermore, FIP triggered the activation of apoptotic pathways through upregulation of Bax, caspase-9, and caspase-3, coupled with downregulation of the antiapoptotic protein Bcl-2. Concurrently, FIP disrupted antioxidant defense mechanisms by modulating the Nrf2/HO-1/NQO1 signaling pathway. Interestingly, pretreatment with VE effectively reversed these effects. VE reduced ROS levels, replenished GSH, mitigated apoptosis, and restored antioxidant protein expression, protecting astrocytes against FIP-induced cytotoxicity and oxidative damage. These findings highlight oxidative stress as a critical factor in FIP-induced astrocyte toxicity and position VE as a promising protective agent. Further research is essential to explore VE's therapeutic potential in mitigating oxidative stress-related neurotoxicity caused by environmental toxins like FIP.

Flavonoids, found in fruits and vegetables, can potentially prevent brain diseases. Diosmin (diosmetin-7-O-rutinoside), a flavonoid, exhibits various pharmacological activities, but its impact on calcium ion (Ca2+) signaling and the associated mechanisms in human glioblastoma cells remain unclear. This study investigated the effect of diosmin on intracellular Ca2+ levels ([Ca2+](i)), cell viability, and the participation of Ca2+-related pathways in DBTRG-05MG human glioblastoma cells. It also investigated the connection between Ca2+ signaling and toxicity in cells treated with diosmin and the Ca2+ chelator BAPTA-AM. Research indicates that diosmin (20-60 mu M) caused an increase in [Ca2+](i) and induced cytotoxicity in a concentration-dependent manner. Furthermore, pre-treating the cells with the BAPTA-AM can intensify the cytotoxic effect. The removal of extracellular Ca2+ suppressed the entry of Ca2+. Agents that modulate store-operated Ca2+ channels, SKF96365 and 2-APB, can inhibit the entry of Ca2+ induced by diosmin. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin in a Ca2+-free environment inhibited the increase in [Ca2+](i) caused by diosmin; conversely, treatment with diosmin reduced the increase in [Ca2+](i) caused by thapsigargin. Moreover, inhibiting phospholipase C (PLC) with U73122 eliminated the increase in [Ca2+](i) triggered by diosmin. In DBTRG-05MG cells, diosmin-induced cell death is associated with Ca2+, a process involving the entry of Ca2+ through store-operated Ca2+ channels and the release of Ca2+ from the endoplasmic reticulum, which relies on the PLC. Additionally, BAPTA-AM, a compound with Ca2+-chelating properties, shows promise in enhancing diosmin-induced cytotoxicity, and this could represent a significant development in glioblastoma research.

The importance of fatty acids in human health and their potential in treating various brain diseases is increasingly acknowledged. Research indicates that ultra-long-chain fatty acids adversely affect dietary habits, while omega (ω)-3 polyunsaturated fatty acids confer health benefits. Eicosapentaenoic acid (EPA), an ω-3 polyunsaturated fatty acid, manifests diverse protective activities, including anti-oxidative effects and the attenuation of brain diseases. Previous studies have suggested that EPA can alleviate oxidative stress and forestall diseases stemming from oxidative damage. Nevertheless, EPA’s precise antioxidant mechanism and signaling pathway in human astrocytes remain elusive. To address this knowledge gap, we established an H2O2-induced oxidative damage model in Gibco® Human Astrocytes (GHA cells) and elucidated the underlying mechanisms and signaling pathways. Our assessments included cell viability through the CCK-8 assay, morphological examination via microscopy, ROS quantification using the DCFH-DA fluorescent probe, GSH content evaluation with the CMF-DA fluorescent probe, and protein expression analysis for antioxidant and apoptotic markers through Western blotting. The results showed that pretreatment with 3 µM of EPA countered the cytotoxicity, ROS production, and GSH depletion caused by H2O2 (250 µM) in GHA cells. Additionally, EPA pretreatment effectively reduced the cytotoxicity and oxidative stress resulting from H2O2 by modulating the Nrf2/HO-1/NQO1 and Bax/Bcl-2/caspase-9/caspase-3 signaling pathways in GHA cells. These findings enhance our understanding of EPA’s antioxidant mechanisms in the oxidative stress model of human astrocytes, illuminate the interplay between antioxidant and apoptotic signals, and offer promise for exploring potential preventive and therapeutic interventions for brain diseases.

Alantolactone, a bioactive sesquiterpene lactone derived from the roots of Inula helenium (elecampane), has garnered attention in biomedical and pharmacological research for its diverse therapeutic properties, including anticancer, anti-inflammatory, antimicrobial, and antioxidant activities. Despite its well-documented bioactivity, the effects of alantolactone on calcium ion (Ca2+) signaling and the underlying mechanisms in human breast cancer cells remain poorly understood. This study explored how alantolactone influences intracellular Ca2+ levels ([Ca2+]i), cell viability, and the role of Ca2+-dependent pathways in T-47D human breast cancer cells. Specifically, it examined the relationship between Ca2+ signaling and cytotoxicity in cells exposed to alantolactone, with or without the Ca2+ chelator BAPTA-AM. The findings reveal that alantolactone (25–75 μM) increases [Ca2+]i in a concentration-dependent manner, while concentrations of 25–100 μM induce cytotoxicity, an effect that can be reversed by BAPTA-AM pre-treatment. Removing extracellular Ca2+ significantly inhibits Ca2+ influx, and both SKF96365 and 2-APB, modulators of store-operated Ca2+ channels, block the alantolactone-induced Ca2+ entry. Additionally, in a Ca2+-free environment, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, suppresses the alantolactone-induced rise in [Ca2+]i, while alantolactone reduces the [Ca2+]i increase triggered by thapsigargin. Moreover, inhibiting phospholipase C (PLC) with U73122 abolishes the alantolactone-induced [Ca2+]i elevation. These results suggest that alantolactone-induced cell death in T-47D cells is Ca2+-dependent, involving Ca2+ entry via store-operated channels and Ca2+ release from the endoplasmic reticulum, with PLC playing a pivotal role. Importantly, the ability of BAPTA-AM to reverse alantolactone's cytotoxic effects highlights its potential therapeutic significance in breast cancer research.

BACKGROUND:Oxatomide, an antihistamine drug of the diphenylmethylpiperazine family, has anti-inflammatory effects in airway disease. Because oxatomide was shown to cause diverse physiological responses in several cell models, the impact of oxatomide on Ca2+ signaling and its related physiological effects has not been explored in IMR-90 human fetal lung fibroblasts. OBJECTIVES:This study assessed the effect of oxatomide on cell viability and intracellular free Ca2+ concentrations ([Ca2+]i) and examined whether oxatomide-induced cytotoxicity through Ca2+ signaling in IMR-90 cells. METHODS:Cell viability was measured by the cell proliferation reagent (WST-1). [Ca2+]i was measured by the Ca2+-sensitive fluorescent dye fura-2. RESULTS:Oxatomide (10-40 μM) concentration dependently reduced cell viability and induced [Ca2+]i rises in IMR-90 cells. This cytotoxic effect was reversed by chelation of cytosolic Ca2+ with BAPTA-AM. In terms of Ca2+ signaling, oxatomide-caused Ca2+ entry was inhibited by modulators of store-operated Ca2+ channels (2-APB and SKF96365) and protein kinase C (PKC) inhibitor (GF109203X). Furthermore, oxatomide-induced Ca2+ influx was confirmed by Mn2+-induced quench of fura-2 fluorescence. In a Ca2+-free medium, preincubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited oxatomide-evoked [Ca2+]i rises. Conversely, treatment with oxatomide abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 also inhibited oxatomide-caused [Ca2+]i rises. CONCLUSION:In IMR-90 cells, oxatomide-induced cytotoxicity by preceding [Ca2+]i rises involving PKC-sensitive store-operated Ca2+ entry and PLC-dependent Ca2+ release from the endoplasmic reticulum. BAPTA-AM, with its Ca2+ chelating effects, may be a potential compound for preventing oxatomide-induced cytotoxicity.

Ochratoxin A (OTA) is a type of mycotoxin commonly found in raw and processed foods. It is essential to be aware of this toxin, as it can harm your health if consumed in high quantities. OTA can induce toxic effects in various cell models. However, a more comprehensive understanding of the harmful effects of OTA on human astrocytes is required. This study evaluated OTA's neurotoxic effects on the Gibco® Human Astrocyte (GHA) cell line, its underlying mechanisms, and the antioxidant N-acetylcysteine (NAC) ability to prevent them. OTA exposure within 5-30 μM has induced concentration-dependent cytotoxicity. In the OTA-treated cells, the levels of reactive oxygen species (ROS) were found to be significantly increased, while the glutathione (GSH) contents were found to decrease considerably. The western blotting of OTA-treated cells has revealed increased Bax, cleaved caspase-9/caspase-3 protein levels, and increased Bax/Bcl-2 ratio. In addition, exposure to OTA has resulted in the induction of antioxidant responses associated with the protein expressions of Nrf2, HO-1, and NQO1. On the other hand, the pretreatment with NAC has partially alleviated the significant toxic effects of OTA. In conclusion, our findings suggest that oxidative stress and apoptosis are involved in the OTA-induced cytotoxicity in GHA cells. NAC could act as a protective agent against OTA-induced oxidative damage.

Beauvericin (BEA) is a newly identified mycotoxin produced by various Fusarium species, and its contamination in food and animal feed is widespread globally. This mycotoxin demonstrates cytotoxic effects by inducing oxidative stress in multiple models. Furthermore, evidence indicates that BEA possesses diverse toxic activities, making it a promising candidate for toxicological research. Recent studies have highlighted the ability of BEA to traverse the blood-brain barrier, suggesting its potential neurotoxicity. However, limited information is available regarding the neurotoxic effects of BEA on human astrocytes. Therefore, this study aimed to assess the neurotoxic effects of BEA on the Gibco® Human Astrocyte (GHA) cell line and elucidate the underlying mechanisms. Additionally, the study aimed to investigate the protective effects of the antioxidant N-acetylcysteine (NAC) against BEA-induced toxicity. The data show that exposure to BEA within the 2.5-15 μM concentration range resulted in concentration-dependent cytotoxicity. BEA-treated cells exhibited significantly increased levels of reactive oxygen species (ROS), while intracellular glutathione (GSH) content was significantly reduced. Western blot analysis of cells treated with BEA revealed altered protein levels of Bax, cleaved caspase-9, and caspase-3, along with an increased Bax/Bcl-2 ratio, indicating the induction of apoptosis. Additionally, BEA exposure triggered antioxidant responses, as evidenced by increased protein expression of Nrf2, HO-1, and NQO1. Significantly, pretreatment with NAC partially attenuated the significant toxic effects of BEA. In conclusion, our findings suggest that BEA-induced cytotoxicity in GHA cells involves oxidative stress-associated apoptosis. Furthermore, NAC demonstrates potential as a protective agent against BEA-induced oxidative damage.

Mycotoxins are secondary metabolites produced by various kinds of fungi that can induce disease in humans. The fungal species Penicillium expansum produces patulin (C7H6O4), a polyketide lactone mycotoxin found in fruits. Patulin is classified as noncarcinogen; however, recently, it has been associated with harmful effects on the central nervous system. Patulin's toxic action has been established in various brain models; however, its effect on human glioblastoma remains elusive. This study explores whether patulin induces cytotoxicity through oxidative stress in DBTRG-05MG human glioblastoma cells. This study also evaluates whether the antioxidant N-acetylcysteine (NAC) protects against patulin-induced cytotoxicity. In DBTRG-05MG cells, patulin concentration (10-60 & mu;M) dependently induced cytotoxicity. Concerning oxidative stress, patulin (10 and 20 & mu;M) increased the production of intracellular reactive oxygen species (ROS) but depleted reduced glutathione (GSH) contents and regulated the expressions of antioxidant-related proteins (Nrf2 and HO-1). Furthermore, patulin induced cytotoxicity via modulation of apoptosis-related protein expressions (Bax, cleaved caspase-9, and cleaved caspase-3). These cytotoxic responses were partially reversed via pretreatment with NAC (10 & mu;M). In summary, these data help us understand the toxicology of patulin in human glioblastoma and evaluate whether NAC could clinically reduce patulin-affected brain damage.

Organophosphate pesticides (OPs), which are among the most widely used synthetic chemicals for the control of a wide variety of pests, are however associated with various adverse reactions in animals and humans. Chlorpyrifos, an OP, has been shown to cause various health complications due to ingestion, inhalation, or skin absorption. The mechanisms underlying the adverse effect of chlorpyrifos on neurotoxicity have not been elucidated. Therefore, we aimed to determine the mechanism of chlorpyrifos-induced cytotoxicity and to examine whether the antioxidant vitamin E (VE) ameliorated these cytotoxic effects using DBTRG-05MG, a human glioblastoma cell line. The DBTRG-05MG cells were treated with chlorpyrifos, VE, or chlorpyrifos plus VE and compared with the untreated control cells. Chlorpyrifos induced a significant decrease in cell viability and caused morphological changes in treated cultures. Furthermore, chlorpyrifos led to the increased production of reactive oxygen species (ROS) accompanied by a decrease in the level of reduced glutathione. Additionally, chlorpyrifos induced apoptosis by upregulating the protein levels of Bax and cleaved caspase-9/caspase-3 and by downregulating the protein levels of Bcl-2. Moreover, chlorpyrifos modulated the antioxidant response by increasing the protein levels of Nrf2, HO-1, and NQO1. However, VE reversed the cytotoxicity and oxidative stress induced by chlorpyrifos treatment in DBTRG-05MG cells. Overall, these findings suggest that chlorpyrifos causes cytotoxicity through oxidative stress, a process that may play an important role in the development of chlorpyrifos-associated glioblastoma.

Bioallethrin belongs to the family of pyrethroid insecticides. Previous studies have shown that bioallethrin affected the function of muscarinic receptor and subsequently induced neurotoxicity in different brain models. Reactive oxygen species (ROS) are generated in the metabolic course of the human body, which can cause human damage when overactivated. However, whether bioallethrin evokes cytotoxicity through ROS signaling and whether the antioxidant Vitamin E (VE) protects these cytotoxic responses in human glial cell model are still elusive. This study investigated the effect of bioallethrin on cytotoxicity through ROS signaling and evaluated the protective effect of the antioxidant VE in DBTRG-05MG human glioblastoma cells. The cell counting kit-8 (CCK-8) was used to measure cell viability. Intracellular ROS and glutathione (GSH) levels were measured by a cellular assay kit. The levels of apoptosis- and antioxidant-related protein were analyzed by Western blotting. In DBTRG-05MG cells, bioallethrin (25-75 μM) concentration-dependently induced cytotoxicity by increasing ROS productions, decreasing GSH contents, and regulating protein expressions related to apoptosis or antioxidation. Furthermore, these cytotoxic effects were partially reversed by VE (20 μM) pretreatment. Together, VE partially lessened bioallethrin-induced apoptosis through oxidative stress in DBTRG-05MG cells. The data assist us in identifying the toxicological mechanism of bioallethrin and offer future development of the antioxidant VE to reduce brain damage caused by bioallethrin.

Chlorpromazine, an antipsychotic medication, is conventionally applied to cope with the psychotic disorder such as schizophrenia. In cellular studies, chlorpromazine exerts many different actions through calcium ion (Ca2+) signaling, but the underlying pathways are elusive. This study explored the effect of chlorpromazine on viability, Ca2+ signaling pathway and their relationship in glial cell models (GBM 8401 human glioblastoma cell line and Gibco® Human Astrocyte (GHA)). First, chlorpromazine between 10 and 40 μM induced cytotoxicity in GBM 8401 cells but not in GHA cells. Second, in terms of Ca2+ homeostasis, chlorpromazine (10–30 μM) increased intracellular Ca2+ concentrations ([Ca2+]i) rises in GBM 8401 cells but not in GHA cells. Ca2+ removal reduced the signal by approximately 55%. Furthermore, chelation of cytosolic Ca2+ with BAPTA-AM reduced chlorpromazine (10–40 μM)-induced cytotoxicity in GBM 8401 cells. Third, in Ca2+-containing medium of GBM 8401 cells, chlorpromazine-induced Ca2+ entry was inhibited by the modulators of store-operated Ca2+ channel (2-APB and SKF96365). Lastly, in Ca2+-free medium of GBM 8401 cells, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin completely inhibited chlorpromazine-increased [Ca2+]i rises. Conversely, treatment with chlorpromazine abolished thapsigargin-increased [Ca2+]i rises. Inhibition of phospholipase C (PLC) with U73122 abolished chlorpromazine-increased [Ca2+]i rises. Together, in GBM 8401 cells but not in GHA cells, chlorpromazine increased [Ca2+]i rises by Ca2+ influx via store-operated Ca2+ entry and PLC-dependent Ca2+ release from the endoplasmic reticulum. Moreover, the Ca2+ chelator BAPTA-AM inhibited cytotoxicity in chlorpromazine-treated GBM 8401 cells. Therefore, Ca2+ signaling was involved in chlorpromazine-induced cytotoxicity in GBM 8401 cells.

Rotenone, a plant-derived pesticide belonging to genera Derris and Lonchorcarpus, is an inhibitor of NADH dehydrogenase complex. Studies have shown that rotenone was applied as a neurotoxic agent in various neuronal models. Hydroxytyrosol [2-(3,4-dihydroxyphenyl)-ethanol] is a natural phenolic compound found in the olive (Olea europaea L.). Studies of hydroxytyrosol have dramatically increased because this compound may contribute to the prevention of neurodegenerative diseases. Although hydroxytyrosol has received increasing attention due to its multiple pharmacological activities, it is not explored whether hydroxytyrosol inhibited rotenone-induced cytotoxicity in the neuronal cell model. The aim of this study was to explore whether hydroxytyrosol prevented rotenone-induced Ca2+ signaling, cytotoxicity and oxidative stress in HCN-2 neuronal cell line. In HCN-2 cells, rotenone (5-30 mu M) concentration-dependently induced cytosolic Ca2+ concentrations ([Ca2+](i)) rises and cytotoxicity. Treatment with hydroxytyrosol (30 mu M) reversed rotenone (20 mu M)-induced cytotoxic responses. In Ca2+-containing medium, rotenone-induced Ca2+ entry was inhibited by 2-APB (a store-operated Ca2+ channel modulator) or hydroxytyrosol. In Ca2+-free medium, treatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) or hydroxytyrosol significantly inhibited rotenone-induced [Ca2+](i) rises. Furthermore, treatment with hydroxytyrosol reversed ROS levels, cytotoxic responses, and antioxidant enzyme activities (SOD, GPX and CAT) in rotenone-treated cells. Together, in HCN-2 cells, rotenone induced Ca2+ influx via store operated Ca2+ entry and Ca2+ release from the endoplasmic reticulum and caused oxidative stress. Moreover, hydroxytyrosol ameliorated Ca2+ or ROS-associated cytotoxicity. It suggests that hydroxytyrosol might have a protective effect on rotenone-induced neurotoxicity in human neuronal cells.

Hypaconitine, a neuromuscular blocker, is a diterpene alkaloid found in the root of Aconitum carmichaelii. Although hypaconitine was shown to affect various physiological responses in neurological models, the effect of hypaconitine on cell viability and the mechanism of its action of Ca2+ handling is elusive in cortical neurons. This study examined whether hypaconitine altered viability and Ca2+ signalling in HCN‐2 neuronal cell lines. Cell viability was measured by the cell proliferation reagent (WST‐1). Cytosolic Ca2+ concentrations [Ca2+]i was measured by the Ca2+‐sensitive fluorescent dye fura‐2. In HCN‐2 cells, hypaconitine (10–50 μmol/L) induced cytotoxicity and [Ca2+]i rises in a concentration‐dependent manner. Removal of extracellular Ca2+ partially reduced the hypaconitine's effect on [Ca2+]i rises. Furthermore, chelation of cytosolic Ca2+ with BAPTA‐AM reduced hypaconitine's cytotoxicity. In Ca2+‐containing medium, hypaconitine‐induced Ca2+ entry was inhibited by modulators (2‐APB and SKF96365) of store‐operated Ca2+ channels and a protein kinase C (PKC) inhibitor (GF109203X). Hypaconitine induced Mn2+ influx indirectly suggesting that hypaconitine evoked Ca2+ entry. In Ca2+‐free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin abolished hypaconitine‐induced [Ca2+]i rises. Conversely, treatment with hypaconitine inhibited thapsigargin‐induced [Ca2+]i rises. However, inhibition of phospholipase C (PLC) with U73122 did not inhibit hypaconitine‐induced [Ca2+]i rises. Together, hypaconitine caused cytotoxicity that was linked to preceding [Ca2+]i rises by Ca2+ influx via store‐operated Ca2+ entry involved PKC regulation and evoking PLC‐independent Ca2+ release from the endoplasmic reticulum. Because BAPTA‐AM loading only partially reversed hypaconitine‐induced cell death, it suggests that hypaconitine induced a second Ca2+‐independent cytotoxicity in HCN‐2 cells.