Chronic exposure to U18666A induces apoptosis in cultured murine cortical neurons

Niemann-Pick disease type C (NPC) is a juvenile neurodegenerative disorder characterized by premature neuronal loss and altered cholesterol metabolism. Previous reports applying an 8-h exposure of U18666A, a cholesterol transport-inhibiting agent, demonstrated a dose-dependent reduction in beta-amyloid (Abeta) deposition and secretion in cortical neurons, with no significant cell injury. In the current study, we examined the chronic effect of 24-72h of U18666A treatment on primary cortical neurons and several cell lines. Our results showed caspase-3 activation and cellular injury in U18666A-treated cortical neurons but not in the cell lines, suggesting cell death by apoptosis only occurred in cortical neurons after chronic exposure to U18666A. We also demonstrated through filipin staining the accumulation of intracellular cholesterol in cortical neurons treated with U18666A, indicating the phenotypic mimic of NPC by U18666A. However, additions of 10 and 25microM pravastatin with 0.5microg/ml U18666A significantly attenuated toxicity. Taken together, these data showed for the first time that U18666A induces cell death by apoptosis and suggested an important in vitro model system to study NPC.     

Homocysteine potentiates β-amyloid neurotoxicity: role of oxidative stress

The cause of neuronal degeneration in Alzheimer's disease (AD) has not been completely clarified, but has been variously attributed to increases in cytosolic calcium and increased generation of reactive oxygen species (ROS). The beta-amyloid fragment (Abeta) of the amyloid precursor protein induces calcium influx, ROS and apoptosis. Homocysteine (HC), a neurotoxic amino acid that accumulates in neurological disorders including AD, also induces calcium influx and oxidative stress, which has been shown to enhance neuronal excitotoxicity, leading to apoptosis. We examined the possibility that HC may augment Abeta neurotoxicity. HC potentiated the Abeta-induced increase in cytosolic calcium and apoptosis in differentiated SH-SY-5Y human neuroblastoma cells. The antioxidant vitamin E and the glutathione precursor N-acetyl-L-cysteine blocked apoptosis following cotreatment with HC and Abeta, indicating that apoptosis is associated with oxidative stress. These findings underscore that moderate accumulation of excitotoxins at concentrations that alone do not appear to initiate adverse events may enhance the effects of other factors known to cause neurodegeneration such as Abeta.     

Insensitivity of Ubiquinone Biosynthesis in Glioblastoma Cells to an Epileptogenic Drug, U18666A

Abstract: To investigate the perturbation of ubiquinone biosynthesis by a hypocholesterolemic drug, 3β-(2-di-ethylaminoethoxy)androst-5-en-17-one hydrochloride (U18666A), we measured the incorporation of radioactive mevalonate, methionine, tyrosine, and 4-hydroxybenzoic acid into ubiquinone in glioblastoma cells. These four precursors unanimously showed that ubiquinone biosynthesis was not significantly altered by U18666A, which blocked cholesterol biosynthesis at steps beyond mevalonate formation. The fluctuation of the endogenous mevalonate level had little effect on ubiquinone biosynthesis, implying the relative stability of cellular ubiquinone biosynthesis. Furthermore, exogenously added mevalonate did not have an appreciable effect on ubiquinone biosynthesis. The major ubiquinone produced in rat glioblastoma cells was identified as ubiquinone-9. The mevalonate-derived products accumulated in the U18666A-treated cells differed significantly from those reported in a broken cell study, suggesting the existence of delicate mechanisms regulating the formation of cholesterol intermediates.     

Beta-amyloid increases somatostatin expression in cultured cortical neurons

In beta-amyloid (Abeta)-induced neurotoxicity, activation of the NMDA receptor, increased Ca2+ and oxidative stress are intimately associated with neuronal cell death as normally seen in NMDA-induced neurotoxicity. We have recently shown selective sparing of somatostatin (SST)-positive neurons and increased SST expression in NMDA agonist-induced neurotoxicity. Accordingly, the present study was undertaken to determine the effect of Abeta25-35-induced neurotoxicity on the expression of SST in cultured cortical neurons. Cultured cortical cells were exposed to Abeta25-35 and processed to determine the cellular content and release of SST into medium by radioimmunoassay and SST mRNA by RT-PCR. Abeta25-35 induces neuronal cell death in a concentration- and time-dependent fashion, increases SST mRNA synthesis and induces an augmentation in the cellular content of SST. No significant changes were seen on SST release at any concentration of Abeta25-35 after 24 h of treatment. However, Abeta25-35 induces a significant increase of SST release into medium only after 12 h in comparison with other time points. Most significantly, SST-positive neurons are selectively spared in the presence of a lower concentration of Abeta25-35, whereas, in the presence of higher concentrations of Abeta25-35 for extended time periods, SST-positive neurons decrease gradually. Furthermore, Abeta25-35 induces apoptosis at lower concentrations (5 and 10 micromol/L) and necrosis at higher concentrations (20 and 40 micromol/L). Consistent with the increased accumulation of SST, these data suggest that Abeta25-35 impairs cell membrane permeability. Selective sparing of SST-positive neurons at lower concentrations of Abeta25-35 at early time points directly correlates with the pathophysiology of Alzheimer's disease.     

U18666A Inhibits Intracellular Cholesterol Transport and Neurotransmitter Release in Human Neuroblastoma Cells

To determine if neurochemical function might be impaired in cell models with altered cholesterol balance, we studied the effects of U18666A (3--[(2-diethyl-amino)ethoxy]androst-5-en-17-one) on intracellular cholesterol metabolism in three human neuroblastoma cell lines (SK-N-SH, SK-N-MC, and SH-SY5Y). U18666A (0.2 g/ml) completely inhibited low density lipoprotein (LDL)-stimulated cholesterol esterification in SK-N-SH cells, while cholesterol esterification stimulated by 25-hydroxycholesterol or bacterial sphingomyelinase was unaffected or partially inhibited, respectively. U18666A also blocked LDL-stimulated downregulation of LDL receptor and caused lysosomal accumulation of cholesterol as measured by filipin staining. U18666A treatment for 18 h resulted in 70% inhibition of K⁺-evoked norepinephrine release in phorbol esterdifferentiated SH-SY5Y cells, while release stimulated by the calcium ionophore A23187 was only slightly affected. These results suggest that U18666A may preferentially block a voltage-regulated Ca²⁺ channel involved in norepinephrine release and that alterations in neurotransmitter secretion might be a feature of disorders such as Niemann-Pick Type C, in which intracellular cholesterol transport and distribution are impaired.     

Adaptation of neuronal cells to chronic oxidative stress is associated with altered cholesterol and sphingolipid homeostasis and lysosomal function

Chronic oxidative stress has been causally linked to several neurodegenerative disorders. As sensitivity for oxidative stress greatly differs between brain regions and neuronal cell types, specific cellular mechanisms of adaptation to chronic oxidative stress should exist. Our objective was to identify molecular mechanisms of adaptation of neuronal cells after applying chronic sublethal oxidative stress. We demonstrate that cells resistant to oxidative stress exhibit altered cholesterol and sphingomyelin metabolisms. Stress-resistant cells showed reduced levels of molecules involved in cholesterol trafficking and intracellular accumulation of cholesterol, cholesterol precursors, and metabolites. Moreover, stress-resistant cells exhibited reduced SMase activity. The altered lipid metabolism was associated with enhanced autophagy. Treatment of stress-resistant cells with neutral SMase reversed the stress-resistant phenotype, whereas it could be mimicked by treatment of neuronal cells with a specific inhibitor of neutral SMase. Analysis of hippocampal and cerebellar tissue of mouse brains revealed that the obtained cell culture data reflect the in vivo situation. Stress-resistant cells in vitro showed similar features as the less vulnerable cerebellum in mice, whereas stress-sensitive cells resembled the highly sensitive hippocampal area. These findings suggest an important role of the cell type-specific lipid profile for differential vulnerabilities of different brain areas toward chronic oxidative stress.     

Pink1 protects cortical neurons from thapsigargin-induced oxidative stress and neuronal apoptosis

Apoptosis mediates the precise and programmed natural death of neurons and is a physiologically important process in neurogenesis during maturation of the central nervous system. However, premature apoptosis and/or an aberration in apoptosis regulation are implicated in the pathogenesis of neurodegeneration. Thus, it is important to identify neuronal pathways/factors controlling apoptosis. Pink1 [phosphatase and tensin homologue (PTEN)-induced kinase 1] is a ubiquitously expressed gene and has been reported to have a physiological role in mitochondrial maintenance, suppressing mitochondrial oxidative stress, fission and autophagy. However, how Pink1 is involved in neuronal survival against oxidative stress remains not well understood. In the present paper, we demonstrate that thapsigargin, a specific irreversible inhibitor of endoplasmic reticulum (ER) calcium-ATPase, could lead to dramatic oxidative stress and neuronal apoptosis by ectopic calcium entry. Importantly, the neuronal toxicity of thapsigargin inhibits antioxidant gene Pink1 expression. Although Pink1 knockdown enhances the neuronal apoptosis by thapsigargin, its overexpression restores it. Our findings have established the neuronal protective role of Pink1 against oxidative stress and afford rationale for developing new strategy to the therapy of neurodegenerative diseases.     

Cholesterol accumulates in cell bodies,but is decreased in distal axons,of Niemann-Pick C1-deficient neurons

Niemann-Pick type-C (NPC) disease is characterized by a progressive loss of neurons and an accumulation of unesterified cholesterol within the endocytic pathway. Unlike other tissues, however, NPC1-deficient brains do not accumulate cholesterol but whether or not NPC1-deficient neurons accumulate cholesterol is not clear. Therefore, as most studies on cholesterol homeostasis in NPC1-deficient cells have been performed in fibroblasts we have investigated cholesterol homeostasis in cultured murine sympathetic neurons lacking functional NPC1. These neurons did not display obvious abnormalities in growth or morphology and appeared to respond normally to nerve growth factor. Filipin staining revealed numerous cholesterol-filled endosomes/lysosomes in NPC1-deficient neurons and the mass of cholesterol in cell bodies was greater than in wild-type neurons. Surprisingly, however, the cholesterol content of NPC1-deficient and wild-type neurons as a whole was the same. This apparent paradox was resolved when the cholesterol content of NPC1-deficient distal axons was found to be less than of wild-type axons. Cholesterol sequestration in cell bodies did not depend on exogenously supplied cholesterol since the cholesterol accumulated before birth and did not disperse when neurons were cultured without exogenous cholesterol. The altered cholesterol distribution between cell bodies and axons suggests that transport of cholesterol, particularly that synthesized endogenously, from cell bodies to distal axons is impaired in NPC1-deficient neurons.     

基于氧化应激与细胞凋亡探究双酚A慢性暴露致小鼠肾毒性作用机制

双酚A (bisphenol A, BPA)被广泛应用于生产环氧树脂和聚碳酸酯塑料等制品,在强酸、强碱或高温条件下,BPA被释放出来,然后渗入环境中。在大多数生物液体中都检测到了不同浓度的BPA,BPA的存在已被证明与许多慢性疾病密切相关,包括慢性肾病(chronic kidney disease,CKD)。然而,关于BPA的有害作用及其对CKD的不良影响知之甚少。为了探讨BPA对动物肾毒性的作用机制,本研究通过向饮水中加入0.01、0.1和1 mg/L的BPA,暴露于雌性小鼠4周后,交配和怀孕的雌性小鼠持续接触BPA,直到断奶;F1代3周龄雄性仔鼠继续口服相同剂量的BPA,持续10周。结果表明,0.1mg/L和1mg/LBPA处理组小鼠的肾脏损伤严重,血清中肾脏功能指标尿素氮(urea nitrogen,UN)、肌酐(creatinine,CR)和尿酸(uric acid,UA)的含量均发生显著升高(P<0.05);肾脏组织形态结构被损害;肾脏抗氧化相关基因在mRNA和蛋白水平上的表达显著降低(P<0.05),包括谷胱甘肽硫转移酶(glutathione-S-transf...     

Nonylphenol exposure is associated with oxidative and nitrative stress in pregnant women

ABSTRACT

Animal studies have shown that exposure to nonylphenol (NP) increases oxidative/nitrative stress, but whether it does so in humans is unknown. This study examines prenatal exposure to NP and its effects on oxidatively/nitratively damaged DNA, lipid peroxidation, and the activities of antioxidants. A total of 146 urine and blood specimens were collected during gestational weeks 27–38 and hospital admission for delivery, respectively. Urinary NP was analyzed by high-performance liquid chromatography (HPLC). Urinary biomarkers of oxidatively/nitratively damaged DNA and lipid peroxidation, including 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG), 8-nitroguanine (8-NO₂Gua), 8-iso-prostaglandin F_2α (8-isoPF_2α) and 4-hydroxy-2-nonenal-mercapturic acid (HNE-MA), were simultaneously analyzed using isotope-dilution liquid-chromatography/electron spray ionization tandem mass spectrometry. The activities of maternal plasma superoxide dismutase and glutathione peroxidase were analyzed by enzyme-linked immunosorbent assay. Urinary NP level was significantly associated with 8-oxodG and 8-NO₂Gua levels in late pregnancy, suggesting that NP may enhance oxidatively and nitratively damaged DNA. The adjusted odds ratios for high 8-oxodG level exhibited a significantly dose–response relationship with NP levels, stratified into four quartiles. 8-oxodG appears to be a more sensitive and effective biomarker of NP exposure than 8-NO₂Gua. These relationships suggest NP may play a role in the pregnancy complications.     

Melatonin increases survival and inhibits oxidative and amyloid pathology in a transgenic model of Alzheimer's disease

Increased levels of a 40-42 amino-acid peptide called the amyloid beta protein (A beta) and evidence of oxidative damage are early neuropathological markers of Alzheimer's disease (AD). Previous investigations have demonstrated that melatonin is decreased during the aging process and that patients with AD have more profound reductions of this hormone. It has also been recently shown that melatonin protects neuronal cells from A beta-mediated oxidative damage and inhibits the formation of amyloid fibrils in vitro. However, a direct relationship between melatonin and the biochemical pathology of AD had not been demonstrated. We used a transgenic mouse model of Alzheimer's amyloidosis and monitored over time the effects of administering melatonin on brain levels of A beta, abnormal protein nitration, and survival of the mice. We report here that administration of melatonin partially inhibited the expected time-dependent elevation of beta-amyloid, reduced abnormal nitration of proteins, and increased survival in the treated transgenic mice. These findings may bear relevance to the pathogenesis and therapy of AD.     

Early-phase redistribution of the cation-independent mannose 6-phosphate receptor by U18666A treatment in HeLa cells

It has been shown that the treatment with 3-[2-(diethylamino)ethoxy] androst-5-en-17-one (U18666A) causes the accumulation of cholesterol and the cation-independent mannose 6-phosphate receptor (CIMPR) in late endosomal/lysosomal compartments in BHK cells. The present study reports on a study of the effect of U18666A on CIMPR distribution in more detail in HeLa cells. When cells were treated with U18666A for 20 h, the intense perinuclear signal for CIMPR corresponding to the trans-Golgi network (TGN) disappeared and lamp1-negative punctate signals, scattered in the perinuclear region were detected. CIMPR then began to accumulate in lamp1-positive compartments 48 h after addition of the drug. Double immunofluorescence microscopy showed that U18666A-induced mannose 6-phosphate receptor-containing compartments (U-MPRCs), which were formed in the early phase of the redistribution, contained no marker for the TGN, late endosomes or lysosomes. Approximately half of the structures contained transferrin that had been internalized for 20 min, and cathepsin D, the majority of which appeared to be its precursor form. Immunoelectron-microscopic analysis revealed that U-MPRCs are composed of multivesicular bodies, irregularly shaped structures, and vesicular structures adjacent to the multivesicular bodies. These results suggest that U18666A treatment primarily suppresses the CIMPR transport pathways to late endosomes and from transferrin-containing endosomes, both of which may be dependent on cholesterol function.This work was supported by grants from Japan Ministry of Education, Culture, Sports, Science, and Technology.     

Tumour necrosis factor alpha mediated apoptosis in murine macrophages by Salmonella enterica serovar Typhi under oxidative stress

Invasive Salmonella has been reported to induce apoptosis of macrophages as part of its infection process, which may allow it to avoid detection by the innate immune system. However, the induction of apoptosis under the different host environments remains to be examined, including the oxidative stress experienced by pathogens in the macrophage milieu. To simulate in vivo oxidative conditions, Salmonella enterica serovar Typhi was grown in the presence of hydrogen peroxide and its ability to induce apoptosis of murine macrophages was assessed. Analysis of data revealed that oxidative stressed S. Typhi caused apoptotic cell death in 51% of macrophages, whereas S. Typhi grown under normal conditions accounted for apoptotic cell death in only 32% of macrophages. A significant increase in the levels of oxidants and decrease in the antioxidant was also observed which correlated with the increased generation of tumour necrosis factor alpha, interleukin-1alpha and interleukin-6. These results suggest that tumour necrosis factor alpha in conjunction with other cytokines may induce apoptotic cell death through the up-regulation of lipid peroxidation and down-regulation of superoxide dismutase. This finding may help us to understand better the host-pathogen interactions and may be of clinical importance in the development of preventive intervention against infection.     

Neuroprotection of Insulin against Oxidative Stress-induced Apoptosis in Cultured Retinal Neurons： Involvement of Phosphoinositide 3-kinase/Akt Signal Pathway

In order to investigate the neuroprotection of insulin in retinal neurons,we used retinal neuronalculture as a model system to study the protective effects of insulin against H_2O_2-induced cytotoxicity andapoptotic death.Primary retinal neuronal cultures were grown from retinas of 0-2-day old Sprague-Dawleyrats.Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay.Apoptotic cell death was evaluated by the TdT-mediated digoxigenin-dUTP nick-end labeling assay,and byDNA laddering analysis.Phosphoinositide 3-kinase (PI3K) activity was measured using phosphoinositide4,5-bisphophate and [γ-~(32)P]ATP as substrate.Western blot analysis with anti-phospho-Akt (pS473) antibodywas performed to examine the level of phosphorylated Akt.We observed that treatment with 100μM H_2O_2for 24 h significantly decreased cell viability and induced apoptotic death of retinal neurons,and that pretreatmentwith 10 nM insulin significantly inhibited or attenuated H_2O_2-induced cytotoxicity and apoptosis.Pretreatmentwith LY294002,a specific PI3K inhibitor,abolished the cytoprotective effect of insulin.Insulin also stronglyactivated both PI3K and the downstream effector Akt.These results suggest that insulin protects retinalneurons from oxidative stress-induced apoptosis and that the PI3K/Akt signal pathway is involved in insulin-mediated retinal neuroprotection.     

Tolerance to oxidative stress is associated with both oxidative stress response and inherent growth in a fungal wheat pathogen

Reactive oxygen species are toxic byproducts of aerobic respiration that are also important in mediating a diversity of cellular functions. Reactive oxygen species form an important component of plant defenses to inhibit microbial pathogens during pathogen–plant interactions. Tolerance to oxidative stress is likely to make a significant contribution to the viability and pathogenicity of plant pathogens, but the complex network of oxidative stress responses hinders identification of the genes contributing to this trait. Here, we employed a forward genetic approach to investigate the genetic architecture of oxidative stress tolerance in the fungal wheat pathogen Zymoseptoria tritici. We used quantitative trait locus (QTL) mapping of growth and melanization under axenic conditions in two cross-populations to identify genomic regions associated with tolerance to oxidative stress. We found that QTLs associated with growth under oxidative stress as well as inherent growth can affect oxidative stress tolerance, and we identified two uncharacterized genes in a major QTL associated with this trait. Our data suggest that melanization does not affect tolerance to oxidative stress, which differs from what was found for animal pathogens. This study provides a whole-genome perspective on the genetic basis of oxidative stress tolerance in a plant pathogen.     

过量皮质酮致原代培养的大鼠海马神经元死亡方式的研究

目的和方法：以体外原代培养的大鼠海马神经元为研究对象,采用原位染色的方法,对不同剂量的皮质酮（CORT）致海马神经元死亡的方式进行研究。结果：在CORT作用下,海马神经元不仅会发生快速的坏死,而且还会发生慢性的凋亡;并且,随着CORT剂量增大和作用时间延长,海马神经元坏死和凋亡的发生率会随之增高。结论：海马神经细胞坏死和凋亡的发生,可能与CORT抑制神经元能量代谢的程度和增高神经元对谷氨酸神经毒性的敏感性有关。     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Induction by beta-bungarotoxin of apoptosis in cultured hippocampal neurons is mediated by Ca(2+)-dependent formation of reactive oxygen species

The component of the venom of the Taiwanese banded krait Bungarus multicinctus, beta-bungarotoxin (beta-BuTx), acts as an extremely potent inducer of neuronal apoptosis when applied to rat hippocampal cultures. While induction of cell death is dependent on toxin binding to voltage-activated K+ channels and subsequent internalization, the pro-apoptotic signals triggered by picomolar concentrations of beta-BuTx are not understood. Following toxin binding, a dramatic increase in intracellular Ca2+ became detectable after 30 min, and in reactive oxygen species (ROS) after 3-4 h. Conversely, Ca2+ chelators, radical quenchers and antioxidants efficiently antagonized beta-BuTx induced apoptosis. As shown for the antioxidant 2,3-dihydroxybenzoic acid, analysis by matrix assisted laser desorbtion-time of flight (MALDI-TOF) mass spectrometry excluded the protective effects to be due to reductive cleavage of the toxic beta-BuTx dimer. Inhibitors of the intracellular antioxidant defence system enhanced neuronal susceptibility to beta-BuTx, supporting the essential role of ROS in beta-BuTx-initiated apoptosis. Cell damage was accompanied by an accumulation of markers of oxidative cell stress, phospholipid hydroxyperoxides and the lipid peroxidation product, malonyl dialdehyde. These observations indicate that beta-BuTx-induced cell death resulted from an intracellular signalling cascade involving subsequent stages of a dramatic rise in free Ca2+, the accumulation of ROS, membrane lipid peroxidation and, finally, apoptosis.