Incomplete autophagy and increased cholesterol synthesis during neuronal cell death caused by a synthetic cannabinoid, CP-55,940.

There is a propensity for synthetic cannabinoid abuse to spread worldwide. CP-55,940, a synthetic cannabinoid having the ability to activate both CB1 and CB2 receptors, has been shown to induce cell death in neurons as well as other cells. Here we investigate molecular events underling the adverse effects of CP-55,940 on neuronal cells. Exposure of mouse neuroblastoma Neuro2a cells to 10-50 µM CP-55,940 results in concentration-dependent cell death that is not accompanied by an induction of apoptosis. CP-55,940 also stimulates autophagy, but the stimulation is not followed by an increase in autophagic degradation. Transcriptome analysis using DNA microarray revealed the increased expression of genes for the cholesterol biosynthesis pathway that is associated with the activation of SREBP-2, the master transcriptional regulator of cholesterol biosynthesis. However, free cholesterol is localized mainly to cytoplasmic structures, although it is localized to the plasma membrane in healthy cells. Thus, cellular trafficking of cholesterol seems to be somewhat disrupted in CP-55,940 stimulated cells. These results show for the first time that CP-55,940 stimulates autophagy as well as cholesterol biosynthesis, although not all the processes involved in the cellular response to CP-55,940 seem to be complete in these cells.

N-acetylcysteine alleviates arsenic trioxide-induced reductions in hepatic catalase gene expression both in vitro and in vivo.

Arsenic trioxide (ATO), one of the oldest and most frequently used poisons, is well-known in forensic science for inducing hepatotoxicity. The regulation of peroxisomal antioxidative enzyme catalase (CAT) involves intricate mechanisms at both transcriptional and post-transcriptional levels. However, the molecular mechanisms underlying the regulation of CAT gene expression in hepatic cells remain elusive. Furthermore, the regulation of CAT gene expression evident in animals administered with ATO in vivo is not well-explored, although several studies have revealed ATO-induced reductions in CAT enzymatic activity in rat livers. In this study, we revealed ATO-dependent reductions in CAT gene expression in both rat liver and Huh-7 human hepatoma cells. Our results indicate that the decline in CAT enzymatic activity can be attributed, at least in part, to the downregulation of its gene expression. The ATO-induced reduction in CAT expression was concurrent with the reduction in peroxisome proliferator-activated receptor-gamma (PPARγ) coactivator (PGC)-1α and inactivation of PPARγ, both considered as positive regulators of CAT gene expression. Moreover, antioxidant N-acetylcysteine (NAC) demonstrated the capability to alleviate the downregulation of CAT gene expression both in vivo and in vitro. Additionally, NAC played a role in alleviating ATO-induced hepatotoxicity, potentially by mitigating the transcriptional downregulation of the CAT gene. Altogether, these results indicate that ATO exerts toxicity by inhibiting the antioxidant defense mechanism, which may be useful for forensic diagnosis of arsenic poisoning and clinical treatment of mitigating ATO-induced hepatotoxicity.

Increased fatty acid synthesis and disturbed lipid metabolism in Neuro2a cells after repeated cocaine exposure: A preliminary study.

Chronic use of cocaine prompts neurodegeneration and neuroinflammation. Lipids play pivotal roles in neuronal function and pathology. Although evidence correlates cocaine use with the alteration of lipid metabolism in blood and brain, the precise mechanism remains to be elucidated. In this study, we explore the effect of cocaine on neuronal fatty acid profiles in vitro. Neuro2a cells following seven days of repeated exposure to cocaine (0, 600, 800, 1000 µM) showed apoptosis-irrelevant cell death, dysregulated autophagy, activation of atypical endoplasmic reticulum stress response, increased saturated and unsaturated fatty acid synthesis, and disrupted lipid metabolism. These preliminary findings indicated the association between lipid metabolism and cocaine-induced neurotoxicity, which should be beneficial for understanding the neurotoxicity of cocaine.

Ectopic myogenesis and fibrosis accompany adipogenesis during thymic involution induced by repeated cocaine administration.

We have shown previously that daily cocaine administration for 7-14 days in rats resulted in the acceleration of thymic involution, which is, alike to age-related thymic involution, accompanied by ectopic adipogenesis. Here we show that the thymic involution caused by repeated cocaine administration is accompanied by not only adipogenesis but also ectopic myogenesis and fibrosis. In accordance with fibrosis, we observed an increase of N-cadherin, a marker of mesenchymal cells, as well as a decrease of E-cadherin, an epithelial cell marker, in the thymus in response to cocaine administration, suggesting the occurrence of epithelial-to-mesenchymal transition (EMT). Levels of fibronectin was also increased in the thymus of cocaine group compared to control group. In addition, increases in the levels of the transcription factors for myogenic differentiation, myogenin, MYF5, and MYF6, were observed in the thymus administered cocaine for 14 days. These results indicate that the thymic involution induced by cocaine administration involves not only adipogenesis and fibrosis but also ectopic myogenesis, which is scarcely observed and rather pathological process during thymic involution.

Excessive N-acetylcysteine exaggerates glutathione redox homeostasis and apoptosis during acetaminophen exposure in Huh-7 human hepatoma cells.

Acetaminophen (APAP) hepatotoxicity is one of the biggest drawbacks of this relatively safe and widely used drug. In addition to its hepatotoxicity, APAP also cause comparable levels of toxicity on human hepatoma cells. Here we show activation of the intrinsic caspase-9/3 pathway of apoptosis followed by gasdermin E (GSDME) cleavage and subsequent ballooning in APAP (10 mM, 72 h)-treated Huh-7 human hepatocarcinoma cells. N-acetylcysteine (NAC), an antioxidant currently used as an antidote for APAP overdose, does not alleviate APAP toxicity in Huh-7 cells; NAC overdose (10 mM) rather aggravates APAP toxicity. NAC overdose not only aggravates cell death, but also decreases the cellular GSH/GSSG ratio, an indicator of redox homeostasis of glutathione. These results show for the first time that APAP-induced apoptosis in hepatoma cells is followed by secondary necrosis via the caspase-3/GSDME pathway. NAC overdose (10 mM) not only worsens the glutathione redox status, but also accelerates this pathway.

Cocaine induces vascular smooth muscle cells proliferation via DRP1-mediated mitochondrial fission and PI3K/HIF-1α signaling.

Long-term cocaine abuse is associated with cardiovascular and pulmonary vascular complications. The vascular toxicity of cocaine can lead to vascular remodeling characterized by excessive proliferation of vascular smooth muscle cells. Though hypoxia-inducible factor (HIF) signaling and mitochondrial fission have been suggested to play essential roles in the pathogenesis of hypoxia-induced vascular remodeling, pathogenetic mechanism for cocaine-related vascular remodeling remains to be elucidated. In this study, we explore the effect of cocaine on the proliferation of vascular smooth muscle cells by in vitro experiments. The findings indicated that the cocaine-induced vascular smooth muscle cell hyperproliferation is achieved by enhancing DRP1-mediated mitochondrial fission and activating PI3K/HIF-1α signaling. Current findings suggested that mitochondrial fission would play a pivotal role in cocaine-related vascular remodeling and would be helpful in understanding the vascular toxicity of cocaine.

Aristolochic acid induces an inflammatory response with prostaglandin E2 production and apoptosis in NRK-52E proximal tubular cells.

Aristolochic acid nephropathy (AAN) is a type of drug-induced nephropathy in which ingestion of aristolochic acid (AA) causes acute kidney injury, with progressive renal fibrosis and upper urothelial carcinoma. Although the pathological features of AAN have been reported to involve significant cell degeneration and loss in the proximal tubules, the details of the toxic mechanism in the acute phase of the disease remain unclear. This study investigates the cell death pathway and intracellular metabolic kinetics of AA exposure in rat NRK-52E proximal tubular cells. AA exposure induces dose- and time-dependent apoptotic cell death in NRK-52E cells. We examined the inflammatory response to further investigate the mechanism of AA-induced toxicity. AA exposure increased the gene expression of inflammatory cytokines IL-6 and TNF-α, suggesting that AA exposure induces inflammation. Furthermore, analysis of lipid mediators by LC-MS revealed increases in intra- and extra-cellular arachidonic acid and prostaglandin E2 (PGE2). To investigate the relationship between the AA-induced increase in PGE2 production and cell death, celecoxib, an inhibitor of cyclooxygenase-2 (COX-2), which is involved in the production of PGE2, was administered, and a marked inhibition of AA-induced cell death was observed. These results suggest that exposure to AA induces concentration- and time-dependent apoptosis in NRK-52E cells, which is attributed to inflammatory responses mediated by COX-2 and PGE2.

Induction of filamin-C and its involvement in the regulation of cellular senescence and apoptosis in Huh-7 hepatoma cells during arsenic trioxide exposure.

Arsenic trioxide (ATO) is one of the most toxic inorganic arsenic compounds. In this study, we examined the effects of long-term (7 days) exposure to low dose (5 µM) ATO on a human hepatocellular carcinoma cell line, Huh-7. Along with apoptosis accompanied by secondary necrosis though GSDME cleavage, we observed enlarged and flattened cells adhering to the culture dish and surviving even after exposure to ATO. An increase in cyclin-dependent kinase inhibitor p21 levels as well as positive staining for senescence-associated ß-galactosidase activity were observed in ATO-treated cells, indicating cellular senescence. Screening for both ATO-inducible proteins by MALDI-TOF-MS analysis and ATO-inducible genes by DNA microarray analysis showed a marked increase in filamin-C (FLNC), an actin cross-linking protein. Interestingly, the increase in FLNC was observed in both dead and surviving cells, suggesting that the upregulation of FLNC by ATO occurs in both apoptotic and senescent cells. Small interference RNA-mediated knock down of FLNC resulted in not only a reduction of senescence-associated enlarged morphology of the cells, but also an exacerbation of cell death. Taken together, these results suggest a regulatory role of FLNC in the execution of senescence as well as apoptosis during ATO exposure.

Aggregation-prone A53T mutant of α-synuclein exaggerates methamphetamine neurotoxicity in SH-SY5Y cells: Protective role of cellular cholesterol.

The aim of this study is to examine the effects of wild type as well as a mutant (A53T) form of α-synuclein (α-syn) on neuronal cells exposed to methamphetamine (METH). SH-SY5Y human dopaminergic neuronal cells stably expressing exogenously added wild type (WT) or A53T α-syn were established for this purpose. Among the three types of cells, parental, WT α-syn-overexpressing, and A53T α-syn overexpressing SH-SY5Y cells (hereafter referred to as SH-SY5Y, WT SH-SY5Y, and A53T SH-SY5Y, respectively), only A53T SH-SY5Y cells showed significant loss of cell viability after exposure to 2 mM METH for 24 h. Transcriptome analysis using DNA microarray showed that METH induced genes for cholesterol biosynthesis in all of these three cell lines, suggesting that METH upregulates cellular cholesterol biosynthesis independently from cellular α-syn levels. Visualization of the cellular localization of free cholesterol showed that METH induces an aberrant intracellular accumulation of free cholesterol in all three cell lines. In addition, we observed the aggregation of α-syn into cytoplasmic granules, which was more apparent with A53T α-syn than WT α-syn, in cells exposed to METH. Furthermore, the cell death observed in METH-treated A53T SH-SY5Y cells was exaggerated by the addition of 2-hydroxypropyl-ß-cyclodextrin (CD), a substance used to extract cholesterol from cells. These results suggest that the aggregation of A53T α-syn in METH-treated cells should be involved in cell death. The upregulation of cellular biosynthesis and cholesterol accumulation by METH should play a protective role against A53T α-syn neurotoxicity in METH-treated SH-SY5Y cells.

Thymic involution caused by repeated cocaine administration includes apoptotic cell loss followed by ectopic adipogenesis.

Cocaine abuse has a negative impact on the immune system. To investigate the adverse effects of binge cocaine administration on lymphoid organs such as thymus and spleen, we examined the effects of repeated intravenous (i.v.) administration of cocaine on rats. Sprague Dawley rats (male, 8 weeks old) received 20 mg/kg body weight of cocaine hydrochloride per day for 7 or 14 days. In addition to a significant loss in the weight of the spleen, consistent with our previous intraperitoneal (i.p.) injection model of binge cocaine abuse (50 mg/kg cocaine for 7 days), we also found a significant loss of weight as well as apparent shrinkage of the thymus in the cocaine group. Transcriptome analysis of the thymus revealed increased expressions of genes involved in apoptosis, such as Ifi27 and Traf2, as well as decreased expressions of several genes related to lipid metabolism, such as Cd36, Adipoq, Scd1, and Fabp4, in the thymus of the cocaine group (7 days), suggesting an apoptotic loss of thymic cells as well as alterations in lipid metabolism. Paradoxically, cocaine activates PPARγ, a key transcriptional factor activating lipid metabolism, although ectopic adipogenesis was scarcely observed in the thymus. Further analysis of rats administered 20 mg/kg cocaine for 14 days revealed ectopic adipogenesis, which was accompanied with the activation of PPARγ as well as increased expression of Adipoq and Fabp4, in the thymus. Taken together, these results indicate that repeated cocaine administration induces thymic involution, which is initiated by the loss of thymic cells through apoptosis and subsequent ectopic adipocyte development.

Contraction Band Necrosis with Dephosphorylated Connexin 43 in Rat Myocardium after Daily Cocaine Administration.

Contraction band necrosis (CBN) is a common abnormality found in the myocardium of cocaine abusers, but is rarely reported in experimental models of cocaine abuse. Connexin 43 (Cx43) is essential for cardiac intercellular communication and the propagation of CBN. Under stress or injury, cardiac Cx43 is dephosphorylated, which is related to cardiomyocyte dysfunction and pathogenesis, whereas adiponectin exerts beneficial effects in the myocardium. In this study, we explore the effects of cocaine on cardiac Cx43 in vivo. Rats were administered cocaine via the tail vein at 20 mg/kg/day for 14 days, and showed widespread CBN, microfocal myocarditis and myocardial fibrosis, corresponding to a dysfunction of cardiac mitochondria under increased oxidative stress. The increase in dephosphorylated cardiac Cx43 and its negative correlation with the myocardial distribution of CBN after cocaine administration were determined. In addition, apoptosis and necroptosis, as well as increased adiponectin levels, were observed in the myocardium after cocaine exposure. Accordingly, we found altered profiles of cardiac Cx43, CBN and its negative correlation with dephosphorylated cardiac Cx43, and the possible involvement of adiponectin in the myocardium after 14 days of cocaine administration. The latter might play a protective role in the cardiotoxicity of cocaine. The current findings would be beneficial for establishing novel therapeutic strategies in cocaine-induced cardiac consequences.

Role of Mitochondrial Dynamics in Cocaine's Neurotoxicity.

The dynamic balance of mitochondrial fission and fusion maintains mitochondrial homeostasis and optimal function. It is indispensable for cells such as neurons, which rely on the finely tuned mitochondria to carry out their normal physiological activities. The potent psychostimulant cocaine impairs mitochondria as one way it exerts its neurotoxicity, wherein the disturbances in mitochondrial dynamics have been suggested to play an essential role. In this review, we summarize the neurotoxicity of cocaine and the role of mitochondrial dynamics in cellular physiology. Subsequently, we introduce current findings that link disturbed neuronal mitochondrial dynamics with cocaine exposure. Finally, the possible role and potential therapeutic value of mitochondrial dynamics in cocaine neurotoxicity are discussed.

The Role of Peroxiredoxins in the Regulation of Sepsis.

Oxidative stress, a result of a disturbance in redox homeostasis, is considered to be one of the main aggravating events in the pathogenesis of immune disorders. Peroxiredoxins (Prdxs) are an enzyme family that catalyzes the reduction of peroxides, including hydrogen peroxide, lipid peroxides, and nitrogen peroxides. Although the maintenance of cellular redox homeostasis through Prdxs is essential for surviving in adverse environments, Prdxs also participate in the regulation of cellular signal transduction by modulating the activities of a panel of molecules involved in the signal transduction process. Although Prdxs were discovered as intracellular anti-oxidative enzymes, recent research has revealed that Prdxs also play important roles in the extracellular milieu. Indeed, Prdxs have been shown to have the capacity to activate immune cells through ligation with innate immune receptors such as toll-like receptors (TLRs). In this review, we will summarize the intracellular as well as extracellular roles of Prdxs for and against the pathogenesis of inflammatory disorders including sepsis, hemorrhagic shock, and drug-induced liver injury.

Inverse regulation of GSDMD and GSDME gene expression during LPS-induced pyroptosis in RAW264.7 macrophage cells.

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.

Altered cardiac mitochondrial dynamics and biogenesis in rat after short-term cocaine administration.

Abuse of the potent psychostimulant cocaine is widely established to have cardiovascular consequences. The cardiotoxicity of cocaine is mainly associated with oxidative stress and mitochondrial dysfunction. Mitochondrial dynamics and biogenesis, as well as the mitochondrial unfolded protein response (UPRmt), guarantee cardiac mitochondrial homeostasis. Collectively, these mechanisms act to protect against stress, injury, and the detrimental effects of chemicals on mitochondria. In this study, we examined the effects of cocaine on cardiac mitochondrial dynamics, biogenesis, and UPRmt in vivo. Rats administered cocaine via the tail vein at a dose of 20 mg/kg/day for 7 days showed no structural changes in the myocardium, but electron microscopy revealed a significant increase in the number of cardiac mitochondria. Correspondingly, the expressions of the mitochondrial fission gene and mitochondrial biogenesis were increased after cocaine administration. Significant increase in the expression and nuclear translocation of activating transcription factor 5, the major active regulator of UPRmt, were observed after cocaine administration. Accordingly, our findings show that before any structural changes are observable in the myocardium, cocaine alters mitochondrial dynamics, elevates mitochondrial biogenesis, and induces the activation of UPRmt. These alterations might reflect cardiac mitochondrial compensation to protect against the cardiotoxicity of cocaine.

Cell Death and Survival Pathways Involving ATM Protein Kinase.

Cell death is the ultimate form of cellular dysfunction, and is induced by a wide range of stresses including genotoxic stresses. During genotoxic stress, two opposite cellular reactions, cellular protection through DNA repair and elimination of damaged cells by the induction of cell death, can occur in both separate and simultaneous manners. ATM (ataxia telangiectasia mutated) kinase (hereafter referred to as ATM) is a protein kinase that plays central roles in the induction of cell death during genotoxic stresses. It has long been considered that ATM mediates DNA damage-induced cell death through inducing apoptosis. However, recent research progress in cell death modality is now revealing ATM-dependent cell death pathways that consist of not only apoptosis but also necroptosis, ferroptosis, and dysfunction of autophagy, a cellular survival mechanism. In this short review, we intend to provide a brief outline of cell death mechanisms in which ATM is involved, with emphasis on pathways other than apoptosis.

Oxcarbazepine induces mitotic catastrophe and apoptosis in NRK-52E proximal tubular cells.

Certain medicines including anticancer drugs, NSAIDs and antiepileptic drugs are known to cause drug-induced nephropathy. For example, antiepileptic drugs such as carbamazepine (CBZ) and valproic acid have been reported to cause damage to the proximal tubular cells. Although there has been a great deal of research concerning the nephrotoxicity of CBZ, little is known about that of oxcarbazepine (OXC), a derivative of CBZ. To investigate the molecular mechanism underlying renal proximal tubular cell death caused by OXC, we examined alterations in the gene expression profile of NRK-52E proximal tubular cells during OXC exposure. DNA microarray analysis revealed that the levels of genes related to mitotic processes including chromosomal and cytoplasmic segregation, progression to G2/M phase, and formation of the mitotic spindle are increased after exposure to 50 µM OXC for 6 h. Cell cycle analysis by flow cytometry showed that OXC at concentrations between 25 and 100 µM induces G2/M arrest. We also found that OXC significantly increases histone H3 phosphorylation, indicative of mitotic cells. These results imply that OXC induces cell cycle arrest at the mitotic phase. Immunofluorescence analysis showed monopolar spindles, which are formed in response to centrosome separation defects, in OXC-treated cells. We also show that OXC suppresses the phosphorylation of PLK1, which is involved not only in the activation of the kinesin family of motor proteins for centrosome separation and bipolar spindle assembly, but also in the cleavage of centrosomal proteins. Thus, our results indicate that OXC inhibits centrosome separation by reducing the activation of PLK1, which leads to the formation of an abnormal spindle and induces mitotic catastrophe and apoptosis in NRK-52E cells.

Sustained splenic contraction after daily cocaine administration in rats.

The purpose of this study is to examine the effect of repeated cocaine administration on the whole body of rats. Rats (male, 6 weeks old, Sprague Dawley) were injected intraperitoneally with cocaine (50 mg/kg) once a day for 1, 3 or 7 days, and major organs (heart, liver, lung, brain, kidney, spleen) were excised from the sacrificed animals. During autopsy, we found a reduction in spleen size, but not other organs, in cocaine-administered rats as compared to control rats. This reduction became to be noticed at 3 day and easily perceived at 7 day. No marked changes were observed in other organs examined. H&E and EMG staining showed a tendency for a decrease in the number of red blood cells (RBCs) as well as an increase in collagen fibers in the spleens of rats treated repeatedly with cocaine. Transcriptome analysis indicated that repeated cocaine administration depletes RBCs from the spleen. Immunoblot analysis showed that cocaine increases the phosphorylation of myosin light chain (MYL) as well as the levels of transgelin, both of which are involved in the contraction of myofibrils. Collectively, these results show that repeated cocaine administration results in sustained contraction of the spleen, which leads to the release of RBCs from the spleen into circulation.

Possible roles of AMPK and macropinocytosis in the defense responses against Δ9-THC toxicity on HL-1 cardiomyocytes.

Cannabinoids are some of the most popular recreationally used illicit drugs, and are frequently consumed along with alcoholic beverages. Although the whole body effects of cannabinoids depend largely on their effects on the central nerve system, cannabinoids could harm the heart directly, due to the presence of the endocannabinoid system including cannabinoid receptor1 and 2 (CB-R1 and CB-R2) in the heart. The aim of this study is to examine the mechanism of direct cardiotoxicity of Δ9-tetrahydrocannabinol (Δ9-THC), the main psychoactive ingredient of cannabis. For this purpose, HL-1 murine atrial cardiac muscle cells were treated with 10 or 30 µM Δ9-THC, along with 100 mM ethanol to examine the possible synergistic effects of Δ9-THC and ethanol. Transcriptome analysis showed upregulation of the genes involved in the unfolded protein response (UPR), including Bip, CHOP, ATF4 and ATF6, in cells treated with Δ9-THC. Immunoblot analysis showed caspase3 activation, indicating apoptosis caused by ER stress in Δ9-THC-treated cells. Microscopic analysis showed that Δ9-THC enhances macropinocytosis, a process involved in the uptake of extracellular fluids including nutrients. Moreover Δ9-THC seemed to activate AMPK, a sensor of intracellular energy status and an activator of macropinocytosis. Finally, we found that compound C (AMPK inhibitor) aggravated cell death by Δ9-THC while AICAR (AMPK activator) ameliorated it. Collectively, these results indicate that the activation of AMPK is necessary for the survival of HL-1 cells against Δ9-THC toxicity. Macropinocytosis might serve as one of the survival pathways downstream of AMPK.

Pyroptotic cell death by exposure to 1-butanol in H9c2 cardiomyoblastoma cells.

The aim of this study is to examine the molecular mechanism of cytotoxicity caused by direct exposure to short chain alcohol. We showed previously that exposing H9c2 cardiomyoblastoma cells to 150 mM 1-butanol results in cell death within 1 h through an intrinsic apoptotic pathway. The cell death is accompanied by plasma membrane blebbing and caspase-3 activation. Here we show that a higher concentration (200 mM) of 1-butanol, as well as prolonged exposure (3-6 h) to 150 mM 1-butanol, induces plasma membrane ballooning, a characteristic feature of pyroptosis. Although gasderminD (GSDMD) cleavage by caspase-1 was not observed, GSDME cleavage by caspase-3 was observed during exposure to 150 mM 1-butanol for 6 h. We conclude that pyroptotic cell death by 1-butanol in H9c2 cardiomyoblastoma cells should occur via the caspase-3-GSDME pathway, revealing that 1-butanol could induce not only apoptosis but also pyroptosis in the cells.