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Chronic abuse of psychoactive drugs including cocaine causes addiction, dependence, and brain disorders through the alteration of neuronal plasticity. Mitochondrial fission and fusion, collectively called as mitochondrial dynamics, participates in not only the maintenance of neuronal homeostasis but also reorganization of neuronal circuits. The purpose of this study is to examine effects of direct and repeated exposure of cocaine on mitochondrial dynamics in neuronal cells in vitro. Repeated exposure to 600 μM cocaine (2∼3 times per week) of Neuro2a mouse neuroblastoma cells for 3 weeks resulted in decrease of mitochondrial transmembrane potential, activation of autophagy, and upregulation of Parkin, a protein involved in mitochondrial autophagy. Increased expression of mitochondrial fission genes and significant increase in the ser-616 phosphorylated-DRP1, the key regulator of mitochondrial fission, were observed in the cells exposed repeatedly to 600 μM cocaine. Electron microscopy showed significant increase in the number of mitochondria in cocaine-treated cells compared with control cells. Thus, our results show that repeated cocaine exposure not only causes mitochondrial dysfunction but also alters mitochondrial dynamics in Neuro2a cells. Changes in the mitochondrial dynamics might be involved in neural adaptation during repeated cocaine exposure.

GSDMD and GSDME, members of the gasdermin protein family, are involved in the formation of plasma membrane channels contributing to cell rupture during a certain type of necrosis called pyroptosis. GSDMD is activated in response to immunological stimulation such as lipopolysaccharides (LPS) treatment while GSDME is mainly involved in drug-induced tumor cell death. Here we show that the expression of the GSDMD gene increases significantly during LPS-induced pyroptosis in RAW264.7 murine macrophage cells. In contrast, GSDME expression is decreased in the same cells. The increasing effect of LPS on GSDMD expression was observed only when the cells were cultured in high glucose (4.5 g/l) medium, suggesting that glucose availability is important for this effect. The effect of LPS on GSDMD expression is abolished by 2-deoxyglucose (2DG), confirming that glycolysis plays crucial roles in the increasing effect of LPS. Small interference RNA-mediated knock down of GSDMD or overexpression of GSDME causes LPS-induced pyroptosis to take place through GSDME rather than through GSDMD. Taken together, LPS regulates GSDMD and GSDME expression in opposite directions through, at least in part, its effect on glycolysis. This transcriptional regulation may contribute to the execution of pyroptosis in a GSDMD-dependent manner.

Age estimation in adults based on aspartic acid racemization (AAR) provides fewer errors and higher precision than that based on bone morphology for the identification of cadavers. The technique has been established in some labs as a routine method. However, as the essential requisites for the technique, a wide age range of teeth of the same type as the target tooth must be collected for calibration for each examination. We investigated whether dentin standard samples could be prepared by increasing the AAR rate via heat. Powdered dentin was prepared from a maxillary first premolar (13 years) and heated for 0-72 h at 110 °C. The extent of AAR increased significantly with heating time and the correlation was strong (r = 0.913; p 0.01). Similar results were found for a mandibular canine (24 years, r = 0.948; p 0.01) and a maxillary third molar (20 years, r = 0.944; p 0.01). We attempted to estimate the age of four maxillary first premolars of persons aged 25-58 years by using the heated samples (18 years, 12 h to 7 days). The differences between the actual and estimated ages were within ±5 years. The stability of the AAR rates in the powdered dentin during storage at 22-25 °C, 4 °C, and -30 °C was examined after 1 year and no significant changes had occurred. We were able to prepare dentin standard samples and created a calibration curve. This is a pilot study that needs to be validated before it can be used in forensic practice.

Pyroptosis, a type of inflammatory cell death, is dependent on the inflammatory caspase-mediated cleavage of gasdermin D (GSDMD), and the subsequent pore formation on plasma membranes through which interleukin (IL)-1β and IL-18 are released from cells. During proinflammatory activation, macrophages shift their metabolism from aerobic oxidative phosphorylation to anaerobic glycolysis. Hypoxia-inducible factor (HIF)1α is involved in the induction of IL-1β gene expression as well as the metabolic shift towards glycolysis. However, the relationships between pyroptosis and glycolysis, as well as between pyroptosis and HIF1α are poorly investigated. Here we show that lipopolysaccharide (LPS) stimulation of RAW264.7 murine macrophage cells results in pyroptosis when cells are cultured in high glucose medium. During pyroptosis, HIF1α activation occurs transiently followed by downregulation to sub-basal levels. HIF1α downregulation and pyroptosis are observed when cells are stimulated with LPS under high glucose conditions. We also found that intracellular levels of methylglyoxal (MGO), a side product of glycolysis, increase when cells are stimulated with LPS under high glucose conditions. The addition of glycolysis inhibitor and rapamycin suppresses HIF1α downregulation and pyroptosis. These results show that glycolysis plays a crucial role not only in pro-inflammatory activation, but also in pyroptosis in LPS-stimulated RAW264.7 macrophages.

To investigate septic lung injuries and the possible relief from injury by carbon monoxide (CO), rats were intraperitoneally (i.p.) administered water or the water-soluble CO-releasing molecule CORM (30 mg/kg body weight), followed by the successive administration of PBS or lipopolysaccharide (LPS, 15 mg/kg body weight, 6 h). The results in four experimental groups (control, LPS, LPS + CORM, CORM, n = 3 or 4 in each groups) were examined. Histological examination revealed the intravascular aggregation of erythrocytes in the lungs of the LPS group, and serological analysis showed a significant increase in D-dimer in the LPS group. Both the aggregation and D-dimer increase were ameliorated in the LPS + CORM group, suggesting that LPS-induced DIC in the lung is ameliorated by CORM. Proteomic as well as immunoblot analyses revealed that the levels of annexin A2 (AnxA2) were significantly decreased in the LPS group, but were at control levels in the LPS + CORM group. Concordant with the levels of AnxA2, the levels of both LC3 and collagen VI (COL VI) were decreased in the LPS group but not in the LPS + CORM group. Given the established roles of AnxA2 in fibrinolysis as well as intracellular vesicle trafficking, AnxA2 down-regulation should play an important role in the pathogenesis of septic lung injuries.

Aberrant cellular accumulation of cholesterol is associated with neuronal lysosomal storage disorders such as Niemann-Pick disease Type C (NPC). We have shown previously that l-norephedrine (l-Nor), a sympathomimetic amine, induces necrotic cell death associated with massive cytoplasmic vacuolation in SH-SY5Y human neuroblastoma cells. To reveal the molecular mechanism underling necrotic neuronal cell death caused by l-Nor, we examined alterations in the gene expression profile of cells during l-Nor exposure. DNA microarray analysis revealed that the gene levels for cholesterol transport (LDL receptor and NPC2) as well as cholesterol biosynthesis (mevalonate pathway enzymes) are increased after exposure to 3 mm l-Nor for ∼6 h. Concomitant with this observation, the master transcriptional regulator of cholesterol homeostasis, SREBP-2, is activated by l-Nor. The increase in cholesterol uptake as well as biosynthesis is not accompanied by an increase in cholesterol in the plasma membrane, but rather by aberrant accumulation in cytoplasmic compartments. We also found that cell death by l-Nor can be suppressed by nec-1s, an inhibitor of a regulated form of necrosis, necroptosis. Abrogation of SREBP-2 activation by the small molecule inhibitor betulin or by overexpression of dominant-negative SREBP-2 efficiently reduces cell death by l-Nor. The mobilization of cellular cholesterol in the presence of cyclodextrin also suppresses cell death. These results were also observed in primary culture of striatum neurons. Taken together, our results indicate that the excessive uptake as well as synthesis of cholesterol should underlie neuronal cell death by l-Nor exposure, and suggest a possible link between lysosomal cholesterol storage disorders and the regulated form of necrosis in neuronal cells.

The postmortem interval (PMI) of victims is a key parameter in criminal investigations. However, effective methods for estimating the PMI of skeletal remains have not been established because it is determined by various factors, including environmental conditions. To identify effective parameters for estimating the PMI of skeletal remains, we investigated the change in bone focusing on the amount of DNA, element concentrations, and bone density that occurred in the bone samples of bovine femurs, each maintained under one of five simulated environmental conditions (seawater, freshwater, underground, outdoors, and indoors) for 1 year. The amount of extracted mitochondrial DNA (mtDNA, 404 bp fragment) decreased over time, and significant DNA degradation (p &lt, 0.01), as estimated by a comparison with amplification results for a shorter fragment (128 bp), was detected between 1 month and 3 months. Eleven of 30 elements were detected in samples by inductively coupled plasma optical emission spectrometry, and Na and Ba showed significant quantitative differences in terms of environmental conditions and time (p &lt, 0.01). This preliminary study suggests that the level of DNA degradation determined by real-time polymerase chain reaction and element concentrations determined by inductively coupled plasma optical emission may be useful indices for estimating the PMI of victims under a wide range of environmental conditions. However, this study is a limited experimental research and not applicable to forensic cases as it is. Further studies of human bone with longer observation periods are required to verify these findings and to establish effective methods for PMI estimation.

The aim of this study was to examine the possible involvement of smooth muscle cell remodeling and the induction of MFG-E8 (milk fat globule protein epidermal growth factor-VIII) in vascular pathophysiology during cocaine administration in cultured cells and rats. Cocaine exerts bifurcate effects on vascular cells; it stimulates vasoconstriction through enhancement of catecholamine release at low doses, while it suppresses cardiovascular functions through inhibition of ion channels at high doses. Short-term exposure to a high concentration of cocaine (3 mM, 24 hr) resulted in cell death of A7r5 rat aorta-derived smooth muscle cells. On the other hand, long-term exposure of the same cells to a low concentration (0.3 mM, ~7 days) resulted in a transient increase in MFG-E8 expression followed by an increased tendency toward cyclin D1, PCNA (proliferating cell nuclear antigen), and CDK4 (cyclin-dependent protein kinase-4) expression. Interestingly, autophagy was not induced, but rather was impaired, in cocaine-treated cells. Increased expressions of MFG-E8, PCNA, and CDK4 were also observed in the aortic vascular cells of rats administered cocaine (50 mg/kg, 2 days, i.v.), confirming that cocaine induced MFG-E8 expression in vivo. Taken together, the results show that MFG-E8 is induced in vascular cells exposed to cocaine, and that this induction is likely to be involved in the vascular toxicity elicited by cocaine abuse.

Paraquat (PQ) is an herbicide that was once used worldwide, but is now prohibited in many nations due to its high toxicity to humans. However, there are still rare cases of the fetal intoxication of PQ, which was purchased prior to the prohibition in Japan. In this study, several cell death pathways, the mitochondrial stress response, and autophagy were examined in SH-SY5Y cells exposed to PQ. The results reveal the decrease of a mitochondrial stress sensitive-BNIP3 (Bcl-2/adenovirus E1B 19-kDa-interacting protein 3) protein, the suppression of autophagic flux, and the lack of apoptosis as well as other regulated forms of necrosis, such as necroptosis and ferroptosis. Taken together, our preliminary survey of cellular responses against PQ shows that, although responses of mitochondria and autophagy are observed, subsequent cell death is necrosis. Mechanism of PQ-induced SH-SY5Y cell death should be complicated and cannot be explained thoroughly by already-known mechanisms.

There are many modes of cell death. In this article, we have morphologically classified the various modes of cell death into three categories (types I–III) that correspond roughly to apoptosis, autophagic cell death (also known as “cell death associated with autophagy” or “autophagy-dependent cell death”), and necrosis, respectively. In the 1980s and 1990s, the executional mechanism of apoptosis was elucidated through genetic analyses of a multicellular nematode, C . elegance . This was followed by the discovery of the molecular mechanism of autophagy in experiments on a unicellular organism, Saccharomyces cerevisiae , in the 1990s and 2000s. Although a biochemical characterization of necrosis was lacking, a number of molecular mechanisms that lead to necrosis have been reported over the last two decades. As cell death is the ultimate form of cytotoxicity and chemicals induce cell death in a variety of ways, diverse modes of cell death have been reported for chemical cytotoxicity. This article will describe cell death modalities with an emphasis on the similarities and diversity of their regulatory mechanisms.

Inhaled carbon monoxide (CO) gas is highly toxic, but the human body produces low levels of CO for vasoregulation and other purposes. Given the established protective roles of low concentrations of CO gas against a panel of pathological insults, CO-releasing molecules (CORMs) have been developed and examined in disease models both in vitro and in vivo. Among CORMs, CORM-3 [Ru(CO)3Cl(glycinate)], a ruthenium carbonyl compound, has been extensively studied since it is water-soluble and is suitable for in vivo application. As one of the most prominent features of CO gas is its anti-fibrotic effect, we examined the effects of CORM-3 on mouse embryonic fibroblasts (MEFs). The application of 1 mM CORM-3 to MEFs resulted in the decreased syntheses of collagens I and III within 24 h, confirming an anti-fibrotic effect. To our surprise, CORM-3 caused a rapid (within 1 h) dissociation of cell-associated plasma fibronectin (FN) from the cells, which is associated with formation of a reduction-resistant oligomer of plasma FN. This aberrant oligomerization of plasma FN was reproduced using purified FN in vitro. Furthermore, we showed that RuCl3, but not another water-soluble CORM, CORM-A1 [Na2H3BCO2], also oligomerized plasma FN in vitro. FN depletion from the serum substantially ameliorates cell death by prolonged (72 h) exposure to CORM-3, suggesting a detrimental role of FN oligomerization on cell death. Taken together, we reveal for the first time that FN is a CORM-3-interactive plasma protein, and that the CORM-3-FN interaction is involved in the death of fibroblasts.

We reported previously that when mouse atrium-derived HL-1 cardiomyocytes undergo apoptosis upon exposure to 2% ethanol, the cellular cytoskeleton is severely disrupted and the anti-apoptotic transcriptional co-activator Yes-associated protein (YAP) is inactivated. Consistent with our previous observations, the expression of connective tissue growth factor (CTGF), an anti-apoptotic growth factor and a target of YAP, decreases in a time-dependent manner during exposure to 2% ethanol. The restoration of YAP activation rescues the cells from apoptosis: both the retrovirus-mediated expression of constitutively active YAP and the stabilization of the actomyosin cytoskeleton by jasplakinolide prevent cell death. In contrast, YAP inhibitors have no effect on cell death, confirming the inactivation of YAP in ethanol-exposed cells. Thus, a decrease in actin tension and YAP inactivation should be crucially involved in the cytotoxicity of ethanol on HL-1 cardiomyocytes.

To examine the in vivo responses of promyelocytic leukemia protein (PML) to arsenic, rats (male, 6 weeks old, Sprague Dawley) were administered a single intraperitoneal dose of 5 mg/kg arsenic trioxide (ATO). The protein was examined in the heart, lung, liver, and brain 6 and 48 hours after administration: a significant response of PML was observed in the brain. Oxidative DNA modification was also observed in the brain as revealed by increased immunoreactivity to anti-8-hydroxy-2'-deoxyguanosine (8-OHdG) antibody. In contrast, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) stain reactivity was only slightly increased, suggesting oxidative cellular stress without apoptotic cell death in the ATO-administered rat brain. Among the DNA damage response pathways, the ATR-Chk1 axis was activated, while the ATM-Chk2 axis was not, implying that the PML response is associated with activation of the ATR-Chk1 DNA repair pathway in the brain.

CORM-3 is a water-soluble carbon monoxide (CO)-releasing molecule developed for possible therapeutic use of CO. CORM-3 belongs to a group of metal carbonyl compounds that contain transition metals and carbonyls as the central scaffold and coordinated ligands, respectively. CORM-3 has been reported to be reactive with many proteins in eukaryotes including mammals. Among them, several extracellular proteins, such as lysozyme, as well as plasma albumin and fibronectin, have been shown to interact directly with CORM-3. p62 is an intracellular adaptor protein required for targeting ubiquitinated (Ub) proteins to lysosomal degradation through autophagy. p62 has been shown to undergo self-oligomerization via covalent crosslinking in response to treatment with verteporfin, a benzoporphyrin derivative used for photodynamic therapy. Here we show that CORM-3 also interacts directly with p62. When applied to mouse embryonic fibroblasts (MEFs) at a high concentration (1 mM), CORM-3 causes the formation of reduction- and detergent-resistant high molecular weight (HMW)-p62. HMW-p62 accumulates more in atg5-/- MEFs than in wild type (WT) MEFs, showing the elimination of HMW-p62 through autophagy. HMW-p62 is also generated in H9c2 rat cardiomyoblastoma as well as A549 human alveolar epithelial cells, suggesting that HMW-p62 formation is not specific to MEFs, but, rather, is a general event in mammalian cells. HMW-p62 formation by CORM-3 can be reproduced using purified p62 in vitro, demonstrating the direct interaction between CORM-3 and p62. These results show that p62 is a CORM-3-interactive intracellular protein.

The aim of this study is to investigate the molecular mechanism underling the cardiotoxicity of methamphetamine, a psychostimulant drug that is currently abused in the world. A mouse atrial cardiac cell line, HL-1, which retains phenotypes of cardiac cells and serves as a useful model for examining cardiac pathophysiology, was used for this purpose. During treatment with 1 mM methamphetamine (MAP) for 3–48 h, massive but transient cytoplasmic vacuolization (3–12 h) followed by an intracellular accumulation of granules (24–48 h) was observed under light microscopy. The vacuoles were surrounded by the lysosome membrane marker LAMP1, while the granules colocalized with the autophagy markers LC3 and p62 as well as ubiquitinated proteins. Western blot analysis showed that LC3 was activated during MAP administration, although p62 was not degraded but rather accumulated. Concordant with p62 accumulation, the nuclear translocation of an anti-oxidative transcription factor, Nrf2, and the subsequent induction of its target gene, HO-1, was observed, suggesting an impairment of autophagic protein degradation and the subsequent activation of the p62/Nrf2/HO-1 pathway. In addition, proteomic analysis revealed a reduction in myosin heavy chain (MHC) protein levels during MAP administration. The ubiquitination of MHC and the induction of the muscle sarcomere protein-specific E3 ubiquitin ligases MuRF1 and atrogin-1 were proved by immunoprecipitation and quantitative real-time PCR, respectively. Taken together, the vacuolization of lysosomes and the subsequent accumulation of autophagosomes indicate an impairment of autophagic protein degradation during MAP administration; on the other hand, the ubiquitination and subsequent degradation of MHC indicate the proper progression of proteasomal degradation.

At first, the molecular mechanism of autophagy was unveiled in a unicellular organism Saccharomyces cerevisiae (budding yeast), followed by the discovery that the basic mechanism of autophagy is conserved in multicellular organisms including mammals. Although autophagy was considered to be a non-selective bulk protein degradation system to recycle amino acids during periods of nutrient starvation, it is also believed to be an essential mechanism for the selective elimination of proteins/organelles that are damaged under pathological conditions. Research advances made using autophagy-deficient animals have revealed that impairments of autophagy often underlie the pathogenesis of hereditary disorders such as Danon, Parkinson's, Alzheimer's, and Huntington's diseases, and amyotrophic lateral sclerosis. On the other hand, there are many reports that drugs and toxicants, including arsenic, cadmium, paraquat, methamphetamine, and ethanol, induce autophagy during the development of their toxicity on many organs including heart, brain, lung, kidney, and liver. Although the question as to whether autophagic machinery is involved in the execution of cell death or not remains controversial, the current view of the role of autophagy during cell/tissue injury is that it is an important, often essential, cytoprotective reaction; disturbances in cytoprotective autophagy aggravate cell/tissue injuries. The purpose of this review is to provide (1) a gross summarization of autophagy processes, which are becoming more important in the field of toxicology, and (2) examples of important studies reporting the involvement of perturbations in autophagy in cell/tissue injuries caused by acute as well as chronic intoxication.

Aim: Mitochondrial damage and subsequent oxidative stresses play important roles in the pathogenesis of sepsis-induced organ failure. Recently, autophagy, the major degradation pathway involved in mitochondrial quality control, was reported as a cellular adaptive response to oxidative stresses. The aim of the present study was to elucidate the molecular mechanism that underlies hepatic damage during lipopolysaccharide (LPS) treatment. We also try to determine if the damage can be attenuated by administration of cobalt protoporphyrin (CoPP), a potent heme oxygenase-1 (HO-1) inducer. Methods: Five-week-old male Sprague–Dawley rats were injected i.p. with 15 mg/kg LPS. To determine if hepatic damage following LPS administration can be attenuated by HO-1, CoPP was injected s.c. for 4 days consecutively at 24-h intervals. After treatment with LPS, the liver was obtained and analyzed. Results: A large reduction in liver mitochondrial protein and induction of autophagy were observed in LPS-treated rats. Electron microscopic and immunohistochemical analyses demonstrated autophagic vacuoles in LPS-treated rat liver. Oxidative stress markers (4-hydroxy-2-nonenal and 8-hydroxy-2′-deoxyguanosine) were increased in LPS-treated animals; CoPP treatment ablated these alterations. An inhibitor for the opening of mitochondrial permeability transition pore, cyclosporine A, suppressed oxidative stress as well as liver damage during LPS administration. CoPP promoted autophagy and prevented rats from liver damage during LPS administration. Conclusion: HO-1 promotes autophagy and elimination of damaged mitochondria thereby repressing oxidative stress in LPS-treated rat liver, revealing a novel mechanism for protection by HO-1 against septic liver damage.

Hydrogen sulfide (H2S) is a highly toxic gaseous molecule that causes death to humans exposed to high concentrations. H2S is absorbed into the body through the alveolar epithelium and other tissues. The aim of this study is to evaluate the molecular mechanism underling acute lung injury caused by the inhalation of high concentrations of H2S. Rat lung epithelium-derived L2 cells were exposed to a H2S donor, NaHS, at concentrations of 2-4 mM for 1-6 hr. NaHS caused shrinkage and death of the cells without caspase activation. An actin-binding protein, transgelin, was identified as one of the NaHS-inducible proteins in the cells. NaHS increased myosin light chain (MLC) phosphorylation, indicating that actomyosin-mediated cellular contractility and/or motility could be increased after NaHS exposure. The administration of ML-7, a myosin light chain kinase (MLCK) inhibitor, accelerated cell death after NaHS exposure. Based on these data, we conclude that the increase in MLC phosphorylation in response to NaHS exposure is a cellular protective reaction against NaHS toxicity. Enhancements in smooth muscle cell properties such as transgelin expression and actomyosin-mediated contractility/motility might be involved in cell survival after NaHS exposure.

Aim The purpose of the present study was to identify molecular markers of hepatic damage during lipopolysaccharide (LPS) treatment. Methods LPS (15 mg/kg of bodyweight) or vehicle was injected i.p. into 5-week-old male Sprague–Dawley rats. Proteins were extracted from the liver and were electrophoresed to examine the changes in the protein compositions during LPS treatment. Using a proteomic approach, major LPS-responsible protein in the liver was determined. Results A massive reduction in the levels of carbamoyl phosphate synthase-1 (CPS1), one of the most abundant proteins in liver mitochondria, was revealed during LPS administration. Electron microscopic and immunofluorescence analyses revealed large vacuoles, which were often localized in the vicinity of mitochondria, in the LPS-treated rat liver. Furthermore, we found that CPS1 is released into the circulation prior to liver damage marker alanine aminotransferase, indicating the active extrusion of CPS1 during LPS administration. Another liver mitochondrial protein, ornithine transcarbamylase, is also released into the circulation, implicating active extrusion of mitochondrial proteins. These phenomena are accelerated by a heme oxygenase inducer cobalt protoporphyrin whilst suppressed by a lysosome inhibitor chloroquine. Conclusion Plasma CPS1 should be a possible marker of septic liver damage and may be involved in systemic responses elicited by septic shock.

Chelerythrine, formerly identified as a protein kinase C inhibitor, has also been shown to inhibit the anti-apoptotic Bcl-2 family proteins. However, recent studies have now demonstrated that chelerythrine can induce the loss of mitochondrial membrane potential (ΔΨm), a membrane permeability transition (MPT), and the subsequent activation of the mitochondrial apoptotic pathway, even in the cells deficient in Bax and Bak. This suggests the existence of an alternative Bax/Bak-independent pathway for apoptosis. The generation of reactive oxygen species (ROS) from the mitochondrial electron transport chain (ETC) is also implicated in the cytotoxity elicited by chelerythrine. In our current study, we show that chelerythrine induces the rapid apoptotic death of H9c2 cardiomyocyte-derived cells within 8 min of treatment. The proteolytic activation of caspase9 and caspase3, crucial mediators of the mitochondrial apoptotic pathway, are also observed within 6 min of exposure to this drug. The generation of ROS is detected but at only marginal levels in the treated cells. The inhibition of the mitochondrial ETC by rotenone and malonate had almost no effects on ROS generation but in both cases effectively inhibited both cell death and the caspase activation induced by chelerythrine. Hence, chelerythrine initiates the rapid mitochondrial apoptotic death of H9c2 cardiomyoblastoma cells in a manner that is likely independent of the generation of ROS from mitochondria.

Acute and chronic abuses of psychostimulant drugs such as methamphetamine (METH) have been known to cause cell death. In particular, neurotoxicity caused by such drugs is one of the most serious adverse events in humans. Although various effects on neuronal cells caused by METH have been studied, the cellular and molecular mechanisms of METH-induced neurotoxicity remain to be elucidated. To investigate the mechanism of METH-induced cytotoxicity, we studied cytological as well as biochemical changes in retinoic acid (RA)-differentiated SH-SY5Y human neuroblastoma cells. Marked cell death was observed with more than 7 mM METH, although caspase-dependent apoptotic cell death was not observed with any concentration of METH treatment. The most prominent cytomorphological effect by METH was the formation of large cytoplasmic vacuoles which were not colocalized with either GFP-LC3 or HSP47-GFP, autophagosome and ER markers respectively. In contrast, many of these vacuoles incorporated large molecular weight FITC-dextran and were confirmed as macropinosomes. Our results indicate that METH-induced cytomorphological effects on RA-differentiated SH-SY5Y human neuroblastoma cells involve macropinocytosis and the hyperstimulation of this process may be involved in METH-caused cytotoxicity.

We investigated the effect of glucose on hypoxic death of rat cardiomyocyte-derived H9c2 cells and found that there is an optimal glucose concentration for protection against hypoxic cell death. Hypoxic cell death in the absence of glucose is accompanied by rapid ATP depletion, release of apoptosis-inducing factor from mitochondria, and nuclear chromatin condensation, all of which are inhibited by glucose in a dose-dependent manner. In contrast, excessive glucose also induces hypoxic cell death that is not accompanied by these events, suggesting a change in the mode of cell death between hypoxic cells with and without glucose supplementation.

TPRA40/GPR175 is an orphan receptor whose physiological functions have not been found to date. In an attempt to generate transgenic mice that express an shRNA of TPRA40, we observed that the cell division of early mouse embryos that injected the short hairpin RNA expression vector was significantly accelerated compared with the control vector. The regulation of cell division by TPRA40 was also observed in HeLa cells. Since the C-terminal region of TPRA40 has been shown to be essential for the regulation of cell division, we performed yeast two-hybrid screening using the C-terminal region as bait. Nuclear antigen of 14 kDa (NA14), an autoantigen of Sjogren's syndrome, was identified as a binding protein to the C-terminal region of TPRA40. The binding of TPRA40 and NA14 was confirmed by GST pull-down assay and co-immunoprecipitation assay. FLAG-TPRA40 is transported from the cytosol to the plasma membrane in time-dependent manner and the translocation was inhibited by GFP-NA14ΔN, an N-terminal deletion mutant that cannot bind to microtubules but binds to TPRA40. TPRA40ΔC, which cannot bind to NA 14, shows impaired transport to the plasma membrane. Finally, we found that the effect of TPRA40 on mouse embryogenesis is strengthened by GFP-NA14, but not by GFP or GFP-NA14ΔN. These observations indicate that the functional plasma membrane transport of TPRA40 that regulates cell division of mouse embryos is mediated by NA14. J. Cell. Physiol. 217: 194–206, 2008. © 2008 Wiley-Liss, Inc.

Repeated hyperbaric oxygen (HBO) exposure prior to ischemia has been reported to provide neuroprotection against ischemic brain injury. The present study examined the time course of neuroprotection of HBO (3.5 atmosphere absolute, 100% oxygen, 1 h for 5 consecutive days) and the changes of gene/protein expression in rats. First, at 6 h, 12 h, 24 h, and 72 h after HBO sessions, rats were subjected to forebrain ischemia (8 min). Histopathological examination of hippocampal CA1 neurons was done 7 days after ischemia. Second, temporal genomic responses and protein expression were examined at the same time points after HBO sessions without subjecting animals to ischemia. HBO significantly reduced loss of hippocampal CA1 neurons that normally follows transient forebrain ischemia when the last HBO session was 6 h, 12 h, or 24 h before ischemia (survived neurons 55%, 75%, and 53%, respectively), whereas if there was a 72-h delay before the ischemic insult, HBO was not protective (survived neurons only 6%). Statistical analysis on microarray data showed significant upregulation in 60 probe sets including 7 annotated genes (p75NTR, C/EBPdelta, CD74, Edg2, Trip10, Nrp1, and Igf2), whose time course expressions corresponded to HBO-induced neuroprotection. The protein levels of p75NTR, C/EBPdelta, and CD74 were significantly increased (maximum fold changes 2.9, 2.0, and 7.9, respectively). The results suggest that HBO-induced neuroprotection against ischemic injury has time window, protective at 6 h, 12 h and 24 h but not protective at 72 h. Although the precise interaction is to be determined, the genes/proteins relevant to neurotrophin and inflammatory-immune system may be involved in HBO-induced neuroprotection.

Sepsis/endotoxemia is elicited by the circulatory distribution of pathogens/endotoxins into whole bodies, and causes profound effects on human health by causing inflammation in multiple organs. Mitochondrial damage is one of the characteristics of the cellular degeneration observed during sepsis/endotoxemia. Elimination of damaged mitochondria through the autophagy-lysosome system has been reported in the liver, indicating that autophagy should play an important role in liver homeostasis during sepsis/endotoxemia. An increased appearance of mitochondrial DNA and proteins in the plasma is another feature of sepsis/endotoxemia, suggesting that damaged mitochondria are not only eliminated within the cells, but also extruded through currently unknown mechanisms. Here we provide evidence for the secretion of mitochondrial proteins and DNA from lipopolysaccharide (LPS)-stimulated rat hepatocytes as well as mouse embryonic fibroblasts (MEFs). The secretion of mitochondrial contents is accompanied by the secretion of proteins that reside in the lumenal space of autolysosomes (LC3-II and CTSD/cathepsin D), but not by a lysosomal membrane protein (LAMP1). The pharmacological inhibition of autophagy by 3MA blocks the secretion of mitochondrial constituents from LPS-stimulated hepatocytes. LPS also stimulates the secretion of mitochondrial as well as autolysosomal lumenal proteins from wild-type (Atg5(+/+)) MEFs, but not from atg5(-/-) MEFs. Furthermore, we show that direct exposure of purified mitochondria activates polymorphonuclear leukocytes (PMNs), as evident by the induction of IL1B/interlekin-1β, a pro-inflammatory cytokine. Taken together, the data suggest the active extrusion of mitochondrial contents, which provoke an inflammatory response of immune cells, through the exocytosis of autolysosomes by cells stimulated with LPS.

Recent research developments have revealed that caspase-dependent apoptosis is not the sole form of regulated cell death. Caspase-independent, but genetically regulated, forms of cell death include pyroptosis, necroptosis, parthanatos, and the recently discovered ferroptosis and autosis. Importantly, regulated necrosis can be modulated by small molecule inhibitors/activators, confirming the cell autonomous mechanism of these forms of cell death. The success of small molecule-mediated manipulation of regulated necrosis has produced great changes in the field of cell death research, and has also brought about significant changes in the fields of pharmacology as well as toxicology. In this review, we intend to summarize the modes of regulated cell death other than apoptosis, and discuss their implications in toxicology.

Direct exposure of cardiomyocytes to ethanol causes cardiac damage such as cardiac arrythmias and apoptotic cell death. Cardiomyocytes are connected to each other through intercalated disks (ID), which are composed of a gap junction (GJ), adherens junction, and desmosome. Changes in the content as well as the subcellular localization of connexin43 (Cx43), the main component of the cardiac GJ, are reportedly involved in cardiac arrythmias and subsequent damage. Recently, the hippo-YAP signaling pathway, which links cellular physical status to cell proliferation, differentiation, and apoptosis, has been implicated in cardiac homeostasis under physiological as well as pathological conditions. This study was conducted to explore the possible involvement of junctional intercellular communication, mechanotransduction through cytoskeletal organization, and the hippo-YAP pathway in cardiac damage caused by direct exposure to ethanol. HL-1 murine atrial cardiac cells were used since these cells retain cardiac phenotypes through ID formation and subsequent synchronous contraction. Cells were exposed to 0.5-2% ethanol; significant apoptotic cell death was observed after exposure to 2% ethanol for 48 hours. A decrease in Cx43 levels was already observed after 3 hours exposure to 2% ethanol, suggesting a rapid degradation of this protein. Upon exposure to ethanol, Cx43 translocated into lysosomes. Cellular cytoskeletal organization was also dysregulated by ethanol, as demonstrated by the disruption of myofibrils and intermediate filaments. Coinciding with the loss of cell-cell adherence, decreased phosphorylation of YAP, a hippo pathway effector, was also observed in ethanol-treated cells. Taken together, the results provide evidence that cells exposed directly to ethanol show 1) impaired cell-cell adherence/communication, 2) decreased cellular mechanotransduction by the cytoskeleton, and 3) a suppressed hippo-YAP pathway. Suppression of hippo-YAP pathway signaling should be effective in maintaining cellular homeostasis in cardiomyocytes exposed to ethanol.

In order to determine the amino-terminal sequence requirements for protein N-myristoylation, site-directed mutagenesis of the N-terminal region was performed using tumor necrosis factor (TNF) mutants as model substrate proteins. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system using rabbit reticulocyte lysate. A TNF mutant having the sequence MGAAAAAAAA at its N-terminus was used as the starting sequence to identify elements critical for protein N-myristoylation. Sequential vertical-scanning mutagenesis of amino acids at a distinct position in this model N-terminal sequence revealed the major sequence requirements for protein N-myristoylation: the combination of amino acids at position 3 and 6 constitutes a major determinant for the susceptibility to protein N-myristoylation. When Ser was located at position 6, 11 amino acids (Gly, Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, His) were permitted at position 3 to direct efficient protein N-myristoylation. In this case, the presence of Lys at position 7 was found to affect the amino acid requirement at position 3 and Lys became permitted at this position. When Ser was not located at position 6, only 3 amino acids (Ala, Asn, Gln) were permitted at position 3 to direct efficient protein N-myristoylation. The amino acid requirements found in this study were fully consistent with the N-terminal sequence of 78 N-myristoylated proteins in which N-myristoylation was experimentally verified. These observations strongly indicate that the combination of amino acids at position 3, 6 and 7 is a major determinant for protein N-myristoylation.

A 54-year-old man, who lived alone, was hospitalized due to rapid deterioration of the general condition over a three-week period caused by alcoholic cirrhosis. One month after he left hospital, he was found dead in his house by his friend. Three days before he was found dead, he had met his friend and seemed to be in poor condition. Autopsy was conducted by a medical examiner to clarify the cause of death. Externally, signs of severe jaundice were apparent over the whole body, along with extensive abdominal swelling and edema of the extremities. Autopsy findings demonstrated that the abdominal cavity contained an amount of massive turbid and slight pale reddish brown ascites (23 l). There were no findings of severe peritoneal inflammation. The liver (650 g) was elastic hard and had a micro-nodular surface, which showed severe atrophy. Microscopic examination of the liver showed clear pseudolobule with severe fibrosis in the stroma. There were no significant changes in the heart or brain. The stomach was empty and only a slight amount of intestinal contents. There was no ethanol detected in the blood or urine. The direct cause of his death was circulatory dysfunction due to massive accumulation of the ascites. The reasons for the massive ascites accumulation over 20 l in this case were (1) that he had no serious complications other than ascites; and (2) he did not have any medical treatment just before his death.

Parkin is an E3 ubiquitin ligase involved in the elimination of damaged mitochondria. Ubiquitination of mitochondrial substrates by Parkin results in proteasomal as well as lysosomal degradation of mitochondria, the latter of which is executed by the autophagy machinery and is called as mitophagy (mitochondrial autophagy). The aim of this study is to examine the possible role of Parkin against cardiotoxicity elicited by arsenic trioxide (ATO) exposure in HL-1 mouse atrial cardiomyocytes. HL-1 cells were administered 1-10μM ATO for up to 24h, and the involvements of apoptosis, and the ubiquitin-proteasome and autophagy-lysosome systems (UPS and ALS) were examined. ATO dose-dependently reduced mitochondrial membrane potentials (ΔΨm) in HL-1 cells, indicating that ATO works as a mitochondrial toxin in these cells. Apoptosis was evident in cells exposed to more than 6μM ATO for 24h. Levels of Parkin in mitochondria-rich fractions were increased, suggesting the recruitment of Parkin to mitochondria. Ubiquitination of the voltage-dependent anion channel1 (VDAC1), a substrate of Parkin, was also proved by immunoprecipitation. Accumulation of ubiquitinated proteins including both K48- and K63-lineages was observed in HL-1 cells after ATO exposure, implying an increased demand for proteasomal as well as lysosomal degradation of cellular proteins. Although UPS was activated by ATO as proved by increased proteasomal activity, only slight activation of the ALS marker LC3 was observed, suggesting differential reactions of UPS and ALS to ATO toxicity. The abrogation of UPS by the proteasome inhibitor bortezomib significantly sensitized HL-1 cells to ATO toxicity, showing the contribution of UPS to the maintenance of cellular homeostasis during ATO exposure. Taken together, our results reveal the activation of Parkin as well as UPS during ATO exposure in HL-1 cardiomyocytes, which contributes to the maintenance of mitochondrial as well as cellular homeostasis.

The nerve axon is a good model system for studying the molecular mechanism of organelle transport in cells. Recently, the new kinesin superfamily proteins (KIFs) have been identified as candidate motor proteins involved in organelle transport. Among them KIF1A, a murine homologue of unc-104 gene of Caenorhabditis elegans, is a unique monomeric neuron– specific microtubule plus end–directed motor and has been proposed as a transporter of synaptic vesicle precursors (Okada, Y., H. Yamazaki, Y. Sekine-Aizawa, and N. Hirokawa. 1995. Cell. 81:769–780). To elucidate the function of KIF1A in vivo, we disrupted the KIF1A gene in mice. KIF1A mutants died mostly within a day after birth showing motor and sensory disturbances. In the nervous systems of these mutants, the transport of synaptic vesicle precursors showed a specific and significant decrease. Consequently, synaptic vesicle density decreased dramatically, and clusters of clear small vesicles accumulated in the cell bodies. Furthermore, marked neuronal degeneration and death occurred both in KIF1A mutant mice and in cultures of mutant neurons. The neuronal death in cultures was blocked by coculture with wild-type neurons or exposure to a low concentration of glutamate. These results in cultures suggested that the mutant neurons might not sufficiently receive afferent stimulation, such as neuronal contacts or neurotransmission, resulting in cell death. Thus, our results demonstrate that KIF1A transports a synaptic vesicle precursor and that KIF1A-mediated axonal transport plays a critical role in viability, maintenance, and function of neurons, particularly mature neurons.

The purpose of this study is to examine the molecular mechanism underlying the toxicity of arsenic trioxide (ATO) in cardiac cells. H9c2 rat cardiomyoblastoma cells undergo apoptosis during exposure to the concentrations of ATO > 10 μM for 24 h. The process is accompanied by the activation of caspases and is suppressed by the pan-caspase inhibitor z-VAD. Since ATO-induced H9c2 cell death is suppressed by Rho-kinase (ROCK) inhibitor Y-27632, but not by any antioxidants tested, apoptosis by ATO seems to be initiated through a ROCK-dependent and reactive oxygen species-independent mechanism. During the execution of apoptosis by ATO, the induction of autophagy is also observed. Importantly, autophagy is accelerated in cells treated with ATO plus Y-27632, although Y-27632 alone does not induce autophagy. The cytoprotective effect of Y-27632 against ATO toxicity is abrogated by the co-administration of an autophagy inhibitor, 3-methyladenine, suggesting that autophagy contributes to the cytoprotection by Y-27632. Taken together, the data indicate that the activation of ROCK is required for apoptotic H9c2 cardiomyoblastoma cell death by ATO, and that the ROCK inhibition not only inhibits caspase-dependent apoptotic machinery, but also causes a rise in the cytoprotective autophagy processes during ATO exposure.

Lipopolysaccharide (LPS)-induced myocardial dysfunction is caused, at least in part, by mitochondrial dysfunction. Mitochondrial dysfunction and the oxidative damage associated with it are scavenged through various cellular defense systems such as autophagy to prevent harmful effects. Our recent study has demonstrated that cobalt protoporphyrin IX (CoPPIX), a potent inducer of heme oxygenase-1 (HO-1), ameliorates septic liver injuries by enhancing mitochondrial autophagy in rats. In our current study, we show that CoPPIX (5 mg/kg s.c.) not only accelerates the autophagic response but also promotes lysosome reformation in the rat heart treated with LPS (15 mg/kg i.p.). Lysosomal membrane-associated protein-2 (LAMP2), which is essential to the maintenance of lysosomal functions in the heart, is depleted transiently but restored rapidly during LPS administration in the rat. Activation of transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy, was also observed, indicating a hyper consumption and subsequent reformation of the lysosome to meet the increased demand for autophagosome cleaning. CoPPIX was found to promote these processes and tended to restore the LPS-induced suppression of cardiac performances whilst chloroquine (CQ; 20 mg/kg i.p.), an inhibitor of lysosomes and autophagic protein degradation, abrogates these beneficial effects. The cardioprotective effect of CoPPIX against LPS toxicity was also observed via decreased levels of cardiac releasing enzymes in the plasma. Taken together, our current data indicate that lysosome reformation mediated by TFEB may be involved in cardioprotection against LPS-induced septic insults, and serve as a novel mechanism by which CoPPIX protects the heart against oxidative stress.

To determine whether tubulin molecules transported in axons are polymers or oligomers, we carried out electron microscopic analysis of the movement of the tubulin molecules after photoactivation. Although previous optical microscopic analyses after photobleaching or photoactivation had suggested that most of the axonal microtubules were stationary, they were not sufficiently sensitive to allow detection of actively transported tubulin molecules which were expected to be only a small fraction of total tubulin molecules in axons. In addition, some recent studies using indirect approaches suggested active polymer transport as a mechanism for tubulin transport (Baas, P.W., F.J. Ahmad. 1993. J. Cell Biol. 120:1427-1437; Yu, W., V.E. Centonze, F.J. Ahmad, and P.W. Bass, 1993, J. Cell Biol. 122:349-359; Ahmad, F.J., and P.W. Bass. 1995. J. Cell Sci. 108:2761-2769). So, whether transported tubulin molecules are polymers or not remain to be determined. To clear up this issue, we made fluorescent marks on the tubulin molecules in the axons using a photoactivation technique and performed electron microscopic immunocytochemistry using anti-fluorescein antibody. Using this new method we achieved high resolution and high sensitivity for detecting the transported tubulin molecules. In cells fixed after permeabilization, we found no translocated microtubules. In those fixed without permeabilization, in which oligomers and heterodimers in addition to polymers were preserved, we found much more label in the regions distal to the photoactivated regions than in the proximal regions. These data indicated that tubulin molecules are transported not as polymers but as heterodimers or oligomers by an active mechanism rather than by diffusion.

Mitochondrial damage and subsequent oxidative stresses play important roles in the pathogenesis of sepsis-induced organ failure. Recently, autophagy, the major degradation pathway involved in mitochondrial quality control, was reported as a cellular adaptive response to oxidative stresses. The aim of the present study was to elucidate the molecular mechanism that underlies hepatic damage during lipopolysaccharide (LPS) treatment. We also try to determine if the damage can be attenuated by administration of cobalt protoporphyrin (CoPP), a potent heme oxygenase-1 (HO-1) inducer. Five-week-old male Sprague-Dawley rats were injected i.p. with 15 mg/kg LPS. To determine if hepatic damage following LPS administration can be attenuated by HO-1, CoPP was injected s.c. for 4 days consecutively at 24-h intervals. After treatment with LPS, the liver was obtained and analyzed. A large reduction in liver mitochondrial protein and induction of autophagy were observed in LPS-treated rats. Electron microscopic and immunohistochemical analyses demonstrated autophagic vacuoles in LPS-treated rat liver. Oxidative stress markers (4-hydroxy-2-nonenal and 8-hydroxy-2'-deoxyguanosine) were increased in LPS-treated animals; CoPP treatment ablated these alterations. An inhibitor for the opening of mitochondrial permeability transition pore, cyclosporine A, suppressed oxidative stress as well as liver damage during LPS administration. CoPP promoted autophagy and prevented rats from liver damage during LPS administration. HO-1 promotes autophagy and elimination of damaged mitochondria thereby repressing oxidative stress in LPS-treated rat liver, revealing a novel mechanism for protection by HO-1 against septic liver damage.

The purpose of the present study was to identify molecular markers of hepatic damage during lipopolysaccharide (LPS) treatment.LPS (15 mg/kg of bodyweight) or vehicle was injected i.p. into 5-week-old male Sprague-Dawley rats. Proteins were extracted from the liver and were electrophoresed to examine the changes in the protein compositions during LPS treatment. Using a proteomic approach, major LPS-responsible protein in the liver was determined.A massive reduction in the levels of carbamoyl phosphate synthase-1 (CPS1), one of the most abundant proteins in liver mitochondria, was revealed during LPS administration. Electron microscopic and immunofluorescence analyses revealed large vacuoles, which were often localized in the vicinity of mitochondria, in the LPS-treated rat liver. Furthermore, we found that CPS1 is released into the circulation prior to liver damage marker alanine aminotransferase, indicating the active extrusion of CPS1 during LPS administration. Another liver mitochondrial protein, ornithine transcarbamylase, is also released into the circulation, implicating active extrusion of mitochondrial proteins. These phenomena are accelerated by a heme oxygenase inducer cobalt protoporphyrin whilst suppressed by a lysosome inhibitor chloroquine.Plasma CPS1 should be a possible marker of septic liver damage and may be involved in systemic responses elicited by septic shock.