Abstract 25C-NBOMe is a designer substituted phenethylamine and a high-potency psychedelic that acts on the 5-HT 2A receptor. Although 25C-NBOMe overdoses have been related to several deaths in the USA and Europe, very limited data exists on the in vitro neurotoxicity of 25C-NBOMe. In this study, we found that 25C-NBOMe potently reduced cell viability of SH-SY5Y, PC12, and SN4741 cells, with IC 50 values of 89, 78, and 62 μM, respectively. Methamphetamine decreased the cell viability of these cells with IC 50 values at millimolar range in the same tests, indicating that 25C-NBOMe is > 50 times more potent than methamphetamine in its ability to reduce viability of SH-SY5Y cells. The neurotoxicity of 25C-NBOMe on SH-SY5Y cells was further confirmed by using fluorescein diacetate/propidium iodide double staining. 25C-NBOMe elevated the expression of phosphorylated extracellular signal-regulated kinase (pERK), but decreased the expression of phosphorylated Akt and phosphorylated Ser9- glycogen synthase kinase 3β (GSK3β) in time- and concentration-dependent manners. Interestingly, either specific GSK3β inhibitors or specific mitogen-activated protein kinase kinase (MEK) inhibitors significantly prevented 25C-NBOMe-induced neurotoxicity in SH-SY5Y cells. These results suggest that 25C-NBOMe unexpectedly produced more potent neurotoxicity than methamphetamine and that the inhibition of the Akt pathway and activation of the ERK cascade might be involved in 25C-NBOMe-induced neurotoxicity. Most importantly, these findings further inform the toxicity of 25C-NBOMe abuse to the central nervous system for public health.

Phenethylamine is a natural monoamine alkaloid that acts as a central nervous system (CNS) stimulant in humans. Many substituted phenethylamine analogs have been designed and synthesized to mimic the effects of phenethylamine itself as a CNS stimulant. 25C-NBOMe is a novel designer psychedelic drug, which is a phenethylamine substituted by adding two methoxy (–OCH 3 ) groups at the 2- and 5-position, a 2-methoxybenzyl group at the nitrogen, and substituting Cl at the 4-position of phenethylamine (Bersani et al. 2014). Because 25C-NBOMe selectively binds to the 5-HT 2A receptor (5-HT 2A R) in the CNS, the 11C-radiolabeled form of this drug has been used to map the distribution of 5-HT 2A R in the brain by utilization of positron emission tomography (PET) scanning.

Since 2012, 25C-NBOMe has been found to be used illegally as a recreation drug in many Western countries (Zuba et al. 2013). Due to its specific action on the serotonin system, users of 25C-NBOMe typically experience psychological changes such as visual hallucinations, intensive auditory augmentation, aggression, and uncontrollable violent behavior (Forrester 2014; Suzuki et al. 2015). In particular, 25C-NBOMe appears to be psychoactive at a dose of 200–1000 μg when taken sublingually or intranasally, while doses from 50 to 500 μg have been reported by users via smoking. Particularly, when administered sublingually, the threshold effect is displayed after a dose of 100–250 μg, while mild, strong, and very strong effects are achieved following 250–450, 450–800, and over 800 μg doses, respectively (Zuba et al. 2013; Kyriakou et al. 2015). Moreover, results from animal studies have shown that 25C-NBOMe can induce a head twitch response, which is used as a behavioral marker of hallucinogenic effects induced by 5-HT 2A R over-excitation (Bersani et al. 2014). In the designer drug market of the European Union and USA, 25C-NBOMe was sold as a “legal” replacement of LSD, or even sold and misrepresented as LSD, such that users may even be unaware of using 25C-NBOMe (Wood et al. 2015). Importantly, the use of 25C-NBOMe can lead to serious toxicity in humans. From 2012 to 2013, 25C-NBOMe alone, or in combination with other psychedelics, was associated with 32 non-fatal intoxications and four deaths in the European Union (Andreasen et al. 2015; Rajotte et al. 2017).

Agonists of 5-HT 2A R, such as 3,4-methylenedioxymethamphetamine and 1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane, can induce neuronal death in vitro (Capela et al. 2006; Ishiguro et al. 2016). Activation of 5-HT 2A R can stimulate the mitogen-activated protein kinase (MAPK) signaling pathway. In particular, 5-HT 2A R agonists activate the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway, while 5-HT 2A R antagonists inhibit glycogen synthase kinase 3β (GSK3β) through activation of Akt. Considering their involvements in neuronal apoptosis, the alteration of MEK/ERK and Akt/GSK3β pathways might underlie the neurotoxicity induced by psychedelics binding the 5-HT 2A R. However, as a novel designer psychedelic drug, limited data exists on the neurotoxicity of 25C-NBOMe.

The neurotoxicity of 25C-NBOMe was tested in vitro against SH-SY5Y, PC12, and SN4741 cells, since these are widely used to evaluate the neurotoxic and neuroprotective effects of chemicals. Cells were treated with a concentration curve of 25C-NBOMe (25–400 μM) for 24 h, and cell viability was measured by the MTT assay. It was found that 25C-NBOMe at concentrations above 100 μM significantly decreased cell viability in all three cell lines (ANOVA, p < 0.01; Fig. 1). Moreover, 50 μM 25C-NBOMe significantly induced cell death in SN4741 cells, but not SH-SY5Y or PC12 cells, indicating the preferentially potent neurotoxicity of 25C-NBOMe in SN4741 dopaminergic cells (ANOVA, p < 0.01; Fig. 1). The IC 50 values of 25C-NBOMe on cell viability were calculated for all three cell lines, and 25C-NBOMe decreased cell viability of SH-SY5Y, PC12, and SN4741 cells with IC 50 values of 89, 78, and 62 μM, respectively (Fig. 1d). In the same test conditions, methamphetamine reduced cell viability of SH-SY5Y, PC12, and SN4741 cells with IC 50 values of 5.0, 2.0, and 4.0 mM, respectively (Fig. 1d). Therefore, 25C-NBOMe is 56, 25, and 64 times more potent than methamphetamine at reducing cell viability in SH-SY5Y, PC12 and SN4741 cells, respectively. Fig. 1 25C-NBOMe induces neuronal death in SH-SY5Y cells, PC12 cells, and SN4741 cells. a SH-SY5Y cells, b PC12 cells, and c SN4741 cells were treated with 25C-NBOMe at indicated concentrations. Cell viability was measured by the MTT assay 24 h after 25C-NBOMe challenge. d IC 50 values of methamphetamine and 25C-NBOMe on cell survival of various cell lines as indicated. e SH-SY5Y cells were treated with 50 or 100 μM 25C-NBOMe or vehicle control for 24 h. Then, SH-SY5Y cells were assayed with FDA/PI double staining. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; **p < 0.01 versus the control group (Dunnett’s post hoc test) Full size image

FDA/PI double staining was also used to confirm the neurotoxicity of 25C-NBOMe in SH-SY5Y cells. Cells were treated with 50 or 100 μM 25C-NBOMe for 24 h. It was found that 25C-NBOMe largely increased the number of PI-positive dead cells and decreased the number of FDA-positive live cells in SH-SY5Y cells, demonstrating that 25C-NBOMe can potently produce neurotoxicity in vitro (Fig. 1e).

To determine if the alteration of PI3-K/Akt and MEK/ERK signaling pathways was involved in 25C-NBOMe-induced neuronal death in SH-SY5Y cells, the effects of specific inhibitors on 25C-NBOMe-induced neurotoxicity were evaluated. SB415286 is a specific inhibitor of GSK3β, and PD98059 and U0126 are two specific inhibitors of MEK. SH-SY5Y cells were treated with inhibitors for 2 h and then exposed to 100 μM 25C-NBOMe for another 24 h. The results showed that all these inhibitors significantly prevented 25C-NBOMe-induced neuronal death in SH-SY5Y cells, suggesting that 25C-NBOMe produces neuronal death via the activation of GSK3β and MEK (ANOVA, p < 0.05; Fig. 2a). Fig. 2 25C-NBOMe inhibits the Akt and activates the ERK pathway in SH-SY5Y cells. a SH-SY5Y cells were treated with SB415286, U0126, or PD98059 at the indicated concentrations for 2 h and then exposed to 100 μM 25C-NBOMe. Cell viability was measured by MTT assay 24 h after 25C-NBOMe challenge. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *p < 0.05 and **p < 0.01 versus the control group (Dunnett’s post hoc test). b SH-SY5Y cells were incubated with 100 μM 25C-NBOMe for various durations as indicated. Western blotting analysis was performed to detect protein expression of pAkt, Akt, pERK, and ERK. c SH-SY5Y cells were incubated with 25C-NBOMe at the indicated concentration for 1 h. Western blotting analysis was performed to detect protein expression of pAkt, Akt, pSer9-GSK3β, GSK3β, ERK, and pERK. Data, expressed as percentage of control, were the mean ± SEM of three separate experiments; *p < 0.05 and **p < 0.01 versus the control group (Dunnett’s post hoc test) Full size image

Western blotting analysis was also used, to confirm the alteration of signaling pathways by 25C-NBOMe. The phosphorylation of Akt inhibits GSK3β by the phosphorylation of its Ser9 residue. ERK is the downstream molecule of MEK. SH-SY5Y cells were treated with 100 μM 25C-NBOMe and harvested at 15, 30, and 60 min after challenge with 25C-NBOMe. As shown in Fig. 2b, 25C-NBOMe time dependently induced the decrease of pAkt and the increase of pERK, with significant alteration at 1 h after 25C-NBOMe challenge. (ANOVA, p < 0.05; Fig. 2b). Therefore, we chose 1 h as the treatment duration in the further study.

The concentration-dependent effects of 25C-NBOMe on the alteration of signaling molecules were also investigated. SH-SY5Y cells were treated with 25–200 μM 25C-NBOMe and harvested 1 h after 25C-NBOMe challenge. As demonstrated in Fig. 2c, 25C-NBOMe concentration dependently decreased the expression of pAkt and pSer9-GSK3β, suggesting that 25C-NBOMe activates GSK3β by inhibiting the PI3-K/Akt pathway. Moreover, 25C-NBOMe concentration dependently increased the expression of pERK in SH-SY5Y cells, demonstrating that 25C-NBOMe activates the MEK/ERK cascade (ANOVA, p < 0.05; Fig. 2c).

Designer drugs are structural analogs of controlled substances which were designed to mimic the pharmacological effects of the original drugs. From the late 1990s, many designer drugs have been sold over the internet labeled as “research chemicals” in order to avoid violating laws and assuming liabilities for illicit use. However, because of the structural diversities, the safety profiles of these designer drugs are largely unknown. Many designer drugs produce unexpected side effects and have even been linked to deaths, due to the lack of toxicological studies prior to marketing and use. 25C-NBOMe is one such designer drug. Although the documented recent abuse of 25C-NBOMe and analogs of it have been widespread, and resulted in several deaths, no data have been reported regarding the in vitro toxicity of 25C-NBOMe.

The major target of 25C-NBOMe is 5-HT 2A R, while methamphetamine inhibits monoamine transporters. Many 5-HT psychedelics induce neuronal apoptosis via sustained activation of 5-HT 2A R. In order to evaluate the neurotoxicity of these chemicals in vitro, PC12 cells were used because these constitutively express 5-HT 2A R and the dopamine active transporter (DAT), and SH-SY5Y and SN4741 cells were also selected because they express DAT but not 5-HT 2A R. Methamphetamine produced neurotoxicity to all of these cell lines with IC 50 values of around 3 mM, which was expected and consistent with previous reports (Zhao et al. 2016; Li et al. 2018). At the same condition, however, 25C-NBOMe produced remarkably more potent neurotoxicity than methamphetamine, with IC 50 values of 50, 60, and 70 μM for PC12, SH-SY5Y, and SN4741 cells respectively. Most importantly, the toxic potencies of 25C-NBOMe to these three cell lines are roughly equivalent, regardless of expression of 5-HT 2A R. These results suggested that 25C-NBOMe might be more toxic to dopaminergic neurons than methamphetamine via certain pathways bypassing 5-HT 2A R, at least in vitro. This is the first study to discover that a psychedelic 5-HT 2A R activator might also produce neurotoxicity via 5-HT 2A R-independent pathways.

There are many pro-survival and pro-apoptotic signaling pathways involved in neuronal death that may potentially explain how 25C-NBOMe could produce in vitro neurotoxicity. For example, the activation of the PI3-K/Akt pathway promotes neuronal survival and protects neurons against neurotoxic insults. Activated Akt protein further inhibits the activity of pro-apoptotic GSK3β. On the order hand, the activation of the MEK/ERK signaling mediates neuronal apoptosis. In this study, it was found that MEK and GSK3β inhibitors (i.e., activating Akt) could significantly prevent 25C-NBOMe-induced neurotoxicity, suggesting that the inhibition of the PI3-K/Akt pathway and the activation of the MEK/ERK cascade might be involved in the neurotoxicity produced by 25C-NBOMe.

The alteration of the signaling pathways mediating 25C-NBOMe-induced neurotoxicity was further examined based on protein levels. It was observed that 25C-NBOMe concentration and time dependently reduced the expression of pAkt and pSer9-GSK3β and elevated the expression of pERK. These results provided supporting evidence that 25C-NBOMe might produce in vitro neurotoxicity via the inhibition of PI3-K/Akt pathway and the activation of the MEK/ERK cascade. Interestingly, previous studies have shown that the stimulation of 5-HT 2A R could cause the activation of the MEK/ERK cascade. The current findings that 25C-NBOMe is a potent agonist of 5-HT 2A R and that it activates the MEK/ERK cascade are consistent with current understanding of this pathway (Watts 1998; Aringhieri et al. 2017).

25C-NBOMe has been detected at concentrations around 0.3–3 μg/kg in the peripheral blood and urine samples collected during the autopsies of abusers, which is much lower than the levels that produced neurotoxicity in vitro (Andreasen et al. 2015; Morini et al. 2017). It is accordingly unclear if 25C-NBOMe produces in vivo neurotoxicity in the brain of abusers. However, it should be noted that the blood and urine concentrations of 25C-NBOMe do not match its concentration in the brain. 25C-NBOMe can easily pass the blood–brain barrier and reach the CNS due to its high lipophilic property (Bersani et al. 2014). Moreover, 25C-NBOMe can be ingested nasally (Bersani et al. 2014). Therefore, it is reasonable to speculate that the use of 25C-NBOMe may result in its concentration in the CNS being considerably higher than in body fluids. Furthermore, the inherent delay between the time of drug intake and autopsy sample collection should be emphasized, since this should lead to lower body fluid concentrations due to metabolic of 25C-NBOMe and possibly a postmortem redistribution.

In conclusion, this is the first study to report the in vitro neurotoxicity of the designer psychedelic drug 25C-NBOMe in SH-SY5Y, PC12, and SN4741 cells. It was also found that the neurotoxicity of 25C-NBOMe might be mediated by the activation of the ERK pathway and the inhibition of the Akt cascade in SH-SY5Y cells. In combination with the fact that abuse of 25C-NBOMe has already resulted in several deaths, the findings of this in vitro study also warn for public health of the dangerousness to the central nervous system of 25C-NBOMe abuse.

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Funding This work was supported by the National Key Research and Development Program of China (2017YFC0803605), the National Natural Science Foundation of China (81673407, 81870853, 81850410553), Ningbo Sci & Tech Project for Common Wealth (2017C50042), Zhejiang Provincial Key Laboratory of Pathophysiology (201804), Ningbo municipal innovation team of life science and health (2015C110026), LiDakSum Marine Biopharmaceutical Development Fund, and the K. C. Wong Magna Fund in Ningbo University.

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