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You have full access to this open access article. Synthetic cathinones are psychoactive substances, derivatives of a natural psychostimulant cathinone. Although many synthetic cathinones have lost their legal status in many countries, their abuse still continues worldwide. Recently, they have been reported to exert neurotoxic effects in vitro and in vivo. The molecular mechanisms of their action have not been fully elucidated. Recently, they have been linked to the induction of oxidative stress, autophagy, and apoptosis. The aim of this study was to investigate whether 3-fluoromethcathinone 3-FMC , a synthetic cathinone, is able to induce oxidative stress, autophagy, and apoptosis in HT22 immortalized mouse hippocampal cells. We found that treatment of HT22 cells with this compound results in a concentration-dependent increase in the intracellular production of reactive oxygen species. Our results also showed that 3-FMC at millimolar concentration is able to induce caspase-dependent apoptotic cell death in HT22 cells. Our findings suggest that abuse of 3-FMC may disturb neuronal homeostasis and impair functioning of the central nervous system. Synthetic cathinones are psychoactive substances, derivatives of a naturally occurring alkaloid cathinone Balint et al. Although many cathinones, e. The molecular mechanism of action of synthetic cathinones is based on their interaction with transporters of monoamine neurotransmitters such as dopamine, serotonin, and norepinephrine Cozzi et al. Many synthetic cathinones such as mephedrone, methylone, and MDPV were shown to exhibit high blood-brain barrier permeability in an in vitro model Simmler et al. However, it should be emphasized that adverse effects were also reported, regarding mostly the cardiovascular, nervous, and gastrointestinal systems. For instance, in the cardiovascular system, hypertension, tachycardia, chest pain, palpitations, cardiac arrest, and myocarditis were reported after the use of cathinones Assi et al. Symptoms regarding the gastrointestinal system were abdominal pain, nausea, vomiting, and anorexia Zawilska and Wojcieszak ; Winstock et al. Many drugs of abuse have been demonstrated to impair cognitive skills and exert neurotoxic effects den Hollander et al. Additionally, chronic methamphetamine abuse was shown to reduce hippocampal volume and hippocampal deficits correlated with impaired memory performance in its users Thompson et al. Accumulating data suggest that synthetic cathinones may also be neurotoxic and impair functions of the nervous system Hadlock et al. Noteworthy, mephedrone was reported to impair working memory in humans Freeman et al. Interestingly, there are different results regarding its neurotoxicity in animal models Hadlock et al. Some studies on long-term neurochemical effects of mephedrone in rodents suggest lack of neurotoxicity Baumann et al. Probably, the discrepancies are due to different experimental design, e. Recent in vitro studies have also shown that synthetic cathinones may exhibit neurotoxic properties. However, the precise molecular mechanisms of their action have not been fully elucidated. Our recent investigation revealed that 3-FMC inhibits growth and induces cell cycle arrest in HT22 immortalized mouse hippocampal cells Siedlecka-Kroplewska et al. Recently, some synthetic cathinones have been demonstrated to induce oxidative stress, autophagy, and apoptosis in neuronal cells Valente et al. Taking into account these findings, the aim of this study was to examine whether 3-FMC induces oxidative stress, autophagy, and apoptosis in HT22 hippocampal cells. Our results provide evidence that the mechanism of action of this synthetic cathinone in HT22 cells involves induction of oxidative stress as well as activation of autophagy. We also found that 3-FMC at millimolar concentrations is able to induce caspase-dependent apoptotic cell death. Stock solutions of this compound were prepared in sterile physiological saline solution and diluted to indicated concentrations shortly before use. Mouse anti-p62 antibody was obtained from Santa Cruz Biotechnology, Inc. Cy3-conjugated goat anti-rabbit secondary antibodies were obtained from Jackson ImmunoResearch Laboratories, Inc. Horseradish peroxidase-conjugated mouse anti-GAPDH primary antibodies, horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies, and horseradish peroxidase-conjugated rabbit anti-mouse secondary antibodies were purchased from Sigma-Aldrich USA. All other reagents, obtained from commercial suppliers, were of analytical grade. The immortalized mouse hippocampal HT22 cell line was kindly provided by Professor M. Next, cells were incubated with 3-FMC for 45 or 90 min. Simultaneously, control cells were incubated in the absence of 3-FMC. HT22 cells were incubated in the absence control or presence of 3-FMC for 24 h. The total concentration of proteins in cell lysates was determined using the Bradford protein assay. After washing with TBST 0. The membrane was also incubated with horseradish peroxidase-conjugated anti-GAPDH primary antibodies ,, 1 h at RT for loading control. Next, cells were incubated in the absence control or presence of 3-FMC for 24 h. After washing with PBS, cells were incubated with Cy3-conjugated anti-rabbit secondary antibodies for 1 h RT in the dark. Next, samples were mounted in the Permafluor mounting medium Thermo Scientific, USA and covered with glass coverslips. Slides were examined by the confocal microscope system FV10i Olympus, Japan. Adherent cells undergoing cell death tend to detach from the surface of tissue culture flasks. In order to prevent cell loss and better examine nuclear morphology of 3-FMC-treated cells, we tried to improve our standard procedure of cell staining for confocal microscopy. After incubation, all cells were collected, i. Statistical analysis was performed using Statistica 12 software StatSoft, Poland. Statistical differences between samples were evaluated using the non-parametric Mann-Whitney U test. To find out whether the mechanism of action of 3-FMC involves oxidative stress, we examined the effect of this compound on the intracellular production of reactive oxygen species ROS. HT22 cells were treated with 3-FMC for 45 min a or 90 min b. Cells were analyzed by flow cytometry as described in Materials and Methods. The microtubule-associated protein 1 light chain 3 LC3 plays an important role in autophagy Eskelinen Detection of LC3-II is a hallmark of the formation of autophagic vacuoles. This effect was concentration-dependent and was most pronounced at the 3-FMC concentration of 4 mM Fig. Detection of autophagy. Similar results were obtained in three independent experiments. C—control, untreated cells. It was particularly evident after exposure to 4 mM 3-FMC. In control cells, LC3 staining was mostly diffuse, indicative of cytosolic localization of LC3 protein Fig. Immunofluorescent analysis. Cells were incubated with primary anti-LC3 antibodies. Following incubation with Cy3-conjugated secondary antibodies and Hoechst , cells were examined by confocal microscopy as described in Materials and Methods. Data are representative of three independent experiments. The p62 protein, also known as sequestosome-1 SQSTM1 , interacts with ubiquitinated proteins targeting them for degradation by autophagy Klionsky et al. Our previous results revealed that treatment of HT22 cells with 3-FMC led to an increase in the number of cells in the sub-G 1 fraction, indicative of apoptosis Siedlecka-Kroplewska et al. In line with this finding, in the present study, we examined markers of apoptotic cell death such as phosphatidylserine externalization, caspase-3 activation, chromatin condensation, and fragmentation of cell nuclei. Loss of the plasma membrane asymmetry manifested by phosphatidylserine externalization belongs to early apoptotic events Galluzzi et al. As shown in Fig. Detection of cell death. Cells were treated with 1, 2, or 4 mM 3-FMC for 24 h. Cells were stained with Hoechst as described in Materials and Methods. We found that 3-FMC-induced cell death was associated with caspase-3 activation Fig. The active caspase-3 participates in the executive stage of apoptosis Galluzzi et al. Changes of the nuclear morphology typical for apoptotic cell death include chromatin condensation and nuclear fragmentation Prokhorova et al. The immunofluorescent analysis revealed that after exposure to 1 or 2 mM 3-FMC, the majority of HT22 cells exhibited intact cell nuclei with well visible nucleoli Fig. Noteworthy, at the same time, LC3-positive structures were present, indicative of autophagic vacuoles. Both accumulation of autophagic vacuoles and an intact nucleus are characteristics of cells undergoing autophagy Liu and Ouyang After h exposure of HT22 cells to 4 mM 3-FMC, the majority of cells exhibited intact nuclear architecture, accompanied by the presence of autophagic vacuoles Fig. However, confocal micrographs revealed that the shape of cells treated with 4 mM 3-FMC changed and was more rounded in comparison to untreated cells and cells treated with lower concentrations of this drug, suggesting decreased cell adhesion to the surface of the culture flask. Considering that adherent cells undergoing death tend to detach from the surface of the culture flask and may be lost during washing steps, we modified our standard procedure of slides preparation for confocal microscopy as described in Materials and Methods. Noteworthy, the detached cells may be suspected of pronounced apoptotic nuclear alterations. Using our improved method, we detected the presence of nuclear fragmentation and chromatin condensation after 24 h of treatment with 4 mM 3-FMC Fig. The precise molecular mechanisms of their action have not been fully elucidated. In this study, we used HT22 immortalized mouse hippocampal cells as an in vitro model of neuronal cells. Noteworthy, the hippocampus is the unique region of the brain, where the neural stem cells can be found Kempermann et al. It plays a crucial role in the formation of memory Yonelinas Accumulating evidence suggests that drug abuse may lead to hippocampal atrophy and memory deficits den Hollander et al. Interestingly, other psychostimulant drugs such as amphetamine or methamphetamine were also shown to induce oxidative stress in neuronal cells Huang et al. It is important to note that the overproduction of reactive oxygen or nitrogen species triggers oxidative damage of cellular structures and disturbs cellular homeostasis Halliwell Noteworthy, the higher the 3-FMC concentration, the more pronounced were the markers of autophagy. The western blotting analysis revealed a concentration-dependent increase in LC3-II expression. The cytosolic LC3-I protein serves as a substrate to form the LC3-II protein, present in membranes of autophagosomes, nascent amphisomes, and nascent autolysosomes Eskelinen Both accumulation of autophagic vacuoles and an elevated LC3-II level may suggest upregulation of autophagy; however, they may also indicate inhibition of autophagic flux associated with impaired degradation and reduced turnover of autophagosomes Klionsky et al. Autophagy is an evolutionarily conserved process during which damaged or misfolded proteins as well as damaged cell organelles can be eliminated Yang and Klionsky It is active at basal level in virtually all cells and serves mainly as a prosurvival mechanism underlying cellular homeostasis. In neuronal cells, it is essential to maintain their functions. However, autophagy may also function as a cell death mode known as autophagic cell death Galluzzi et al. There is a limited number of studies demonstrating that cell death is executed by autophagy Galluzzi et al. In most cases, autophagy appears to be a cytoprotective response activated by dying cells Galluzzi et al. Autophagy can be upregulated in response to nutrient depletion, hypoxia, or oxidative stress Yang and Klionsky Many chemical compounds induce cellular stress and activate autophagy as an adaptive stress-response and a prosurvival mechanism Yang et al. The cytoprotective role of autophagy may be related then to clearance of oxidized or aggregated proteins and damaged cell organelles. Accumulating evidence suggests that autophagy may be implicated in the mechanism of action of drugs of abuse. In the present study, oxidative stress induced by 3-FMC in HT22 cells may result in damage of proteins or organelles. Thus, upregulation of autophagy in 3-FMC-treated HT22 cells may appear as a consequence of disturbed cellular homeostasis. Of note, p62 protein serves as a selective autophagy receptor involved in autophagic clearance of misfolded proteins, protein aggregates, or depolarized mitochondria, whose efficient elimination is critical for cellular homeostasis and survival Rogov et al. Therefore, it can be speculated that autophagy activated in our experimental model appears as a prosurvival process. However, this hypothesis requires further investigation. Valente et al. This effect was prominent after treatment of cells with 4 mM 3-FMC; whereas at a lower concentration of this drug, it was negligible. The cell death mechanism was associated with caspase-3 activation and phosphatidylserine externalization. Morphological changes characteristic for apoptotic cell death such as chromatin condensation and fragmentation of cell nuclei were also observed. These findings corroborate results of our previous study Siedlecka-Kroplewska et al. Taking into account the above findings, after treatment with 1 or 2 mM 3-FMC, only autophagy markers were observed in HT22 cells; whereas after exposure to 4 mM 3-FMC, both autophagic and apoptotic characteristics were detected. Thus, 3-FMC induced autophagy or both autophagy and apoptosis, depending on its concentration. In agreement with our results, recent in vitro studies also showed induction of both autophagy and apoptosis after treatment with other synthetic cathinones Valente et al. After treatment of HT22 cells with 1 or 2 mM 3-FMC, the number of dead cells was negligible, which supports the hypothesis that autophagy activation may be a cytoprotective cell response. However, toxicity of 4 mM 3-FMC was probably too high to be prevented by autophagy. Therefore, the number of dead cells dramatically increased. Induction of apoptosis by 4 mM 3-FMC in HT22 cells indicates that apoptotic pathways are involved in the mechanism of cell death. Numerous studies suggest that there is an interplay between autophagy and apoptosis Thorburn Autophagic and apoptotic signaling pathways share some mediators, e. Autophagy as a prosurvival mechanism may block or delay apoptotic cell death. Intriguingly, caspase activation may serve as a molecular switch between autophagy and apoptosis Wu et al. Activated caspases degrade autophagic proteins and inhibit autophagic response determining cell fate. In the present study, the number of HT22 cells with active caspase-3 significantly increased after treatment with 4 mM 3-FMC, suggesting a possible switch from autophagy to apoptosis. However, further studies are needed to confirm this hypothesis. Our finding that relatively high, millimolar concentrations of 3-FMC exerted significant biological effects in neuronal HT22 cells is consistent with reports of other authors concerning in vitro studies on drugs of abuse. Other drugs including MDMA or methamphetamine also exerted cytotoxic effects in vitro at millimolar concentrations Li et al. It is difficult to predict what would be the 3-FMC concentration in the human brain in vivo after its administration. It may depend on the administration route as well as the ability of this drug to penetrate the blood-brain barrier. The most common administration routes of synthetic cathinones reported by users are insufflation and oral ingestion Freeman et al. The less common routes are intravenous, subcutaneous, intramuscular injections as well as rectal insertion, smoking, and insertion in the eye eyeballing Assi et al. Assuming the average peripheral blood volume of 5 L and intravenous administration route, the calculated 3-FMC dose leading to its 1, 2, or 4 mM blood concentration is equal to 1. According to case reports concerning synthetic cathinone-related intoxications, users reported doses up to 7 g EMCDDA Some users reported to use drugs over several consecutive days Winstock et al. The risk of overdosing seems to be high, since users experience a desire to redose Winstock et al. It should be emphasized that there are numerous reports on acute and lethal intoxication with synthetic cathinones Boulanger-Gobeil et al. In conclusion, our results provide evidence that 3-FMC induces oxidative stress and activates autophagy in HT22 neuronal cells. We propose that autophagy triggered by oxidative damage in 3-FMC-treated HT22 cells may appear as a cellular defense mechanism, however, when it cannot prevent toxicity of a high dose of 3-FMC apoptotic pathways become activated. Further studies may help understand molecular mechanisms of 3-FMC neurotoxicity. Noteworthy, the implication of oxidative stress in the mechanism of action of 3-FMC strongly suggests that abuse of this synthetic cathinone may disturb neuronal homeostasis and impair functioning of the central nervous system. Consideration of the naphthylpyrovalerone analogues and related compounds. Accessed 5 Feb J Anal Toxicol — J Neurochem — Life Sci — Gen Hosp Psychiatry Article Google Scholar. Hum Psychopharmacol 32 3. Wien Klin Wochenschr — Article PubMed Google Scholar. Neuropsychopharmacology — J Med Toxicol — Drug Test Anal — Brown JM, Yamamoto BK Effects of amphetamines on mitochondrial function: role of free radicals and oxidative stress. Pharmacol Ther — Eur Rev Med Pharmacol Sci — PubMed Google Scholar. Neurochem Int — Autophagy — Mol Neurobiol — Drug Alcohol Depend — Chandramani Shivalingappa P, Jin H, Anantharam V, Kanthasamy A, Kanthasamy A N-acetyl cysteine protects against methamphetamine-induced dopaminergic neurodegeneration via modulation of redox status and autophagy in dopaminergic cells. Parkinsons Dis Google Scholar. Eur J Pharmacol — J Neurol Neurosurg Psychiatry — Pharmacol Biochem Behav — Eskelinen EL Maturation of autophagic vacuoles in mammalian cells. Eskelinen EL The dual role of autophagy in cancer. Curr Opin Pharmacol — Addiction — Cell Death Differ — Neurology — Cell Mol Neurobiol — J Pharmacol Exp Ther — Biochem Soc Trans — J Emerg Med — Oncotarget — Brain Res — Kanthasamy A, Anantharam V, Ali SF, Kanthasamy AG Methamphetamine induces autophagy and apoptosis in a mesencephalic dopaminergic neuronal culture model: role of cathepsin-D in methamphetamine-induced apoptotic cell death. Ann N Y Acad Sci — Cold Spring Harb Perspect Biol 7:a Kroemer G, Levine B Autophagic cell death: the story of a misnomer. Nat Rev Mol Cell Biol — PLoS One 7:e J Neurosci — PLoS One 9:e Neurotoxicology — Liu B, Ouyang L Involvement of autophagy and apoptosis in studies of anticancer drugs. In: Hayat MA ed Autophagy: cancer, other pathologies, inflammation, immunity, infection, and aging, vol 3. Academic, Elsevier, pp — Psychopharmacology — Toxicol Appl Pharmacol — Forensic Sci Int e93—e Marinetti LJ, Antonides HM Analysis of synthetic cathinones commonly found in bath salts in human performance and postmortem toxicology: method development, drug distribution and interpretation of results. Toxicology — Neuropsychobiology — Neurotox Res — Cell Mol Life Sci — Lancet — Mol Cell — J Clin Psychopharmacol — J Physiol Pharmacol — Br J Pharmacol — Neuropharmacology — Thorburn A Apoptosis and autophagy: regulatory connections between two supposedly different processes. Apoptosis —9. Tian C, Murrin LC, Zheng JC Mitochondrial fragmentation is involved in methamphetamine-induced cell death in rat hippocampal neural progenitor cells. PLoS One 4:e Arch Toxicol — BMC Psychiatry J Subst Abus Treat — Methods Cell Biol — Emerg Med J — Int J Biol Sci — Curr Opin Cell Biol — Cancer Biol Ther — PLoS One e Yonelinas AP The hippocampus supports high-resolution binding in the service of perception, working memory and long-term memory. Behav Brain Res — Forensic Sci Int — Download references. This work was supported by grant no. You can also search for this author in PubMed Google Scholar. Correspondence to Kamila Siedlecka-Kroplewska. Reprints and permissions. Siedlecka-Kroplewska, K. Neurotox Res 34 , — Download citation. Received : 07 February Revised : 27 March Accepted : 29 March Published : 14 April Issue Date : October Anyone you share the following link with will be able to read this content:. Sorry, a shareable link is not currently available for this article. Provided by the Springer Nature SharedIt content-sharing initiative. Neurotoxicity Research Aims and scope Submit manuscript. Download PDF. Abstract Synthetic cathinones are psychoactive substances, derivatives of a natural psychostimulant cathinone. Adverse outcome pathways induced by 3,4-dimethylmethcathinone and 4-methylmethcathinone in differentiated human SH-SY5Y neuronal cells Article 07 May Use our pre-submission checklist Avoid common mistakes on your manuscript. Introduction Synthetic cathinones are psychoactive substances, derivatives of a naturally occurring alkaloid cathinone Balint et al. Hoechst Staining Adherent cells undergoing cell death tend to detach from the surface of tissue culture flasks. Full size image. Funding This work was supported by grant no. View author publications. Ethics declarations Conflict of Interest The authors declare that they have no conflict of interest. About this article. Cite this article Siedlecka-Kroplewska, K. Copy to clipboard. 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