Neurotoxicological profile of the hallucinogenic compound 25I-NBOMe

4-Iodo-2,5-dimethoxy-N-(2-methoxybenzyl)phenethylamine (25I-NBOMe) is a new psychoactive substance with strong hallucinogenic properties. Our previous data reported increased release of dopamine, serotonin, and glutamate after acute injections and a tolerance development in the neurotransmitters release and rats’ behavior after chronic treatment with 25I-NBOMe. The recreational use of 25I-NBOMe is associated with severe intoxication and deaths in humans. There is no data about 25I-NBOMe in vivo toxicity towards the brain tissue. In this article 25I-NBOMe-crossing through the blood–brain barrier (BBB), the impact on DNA damage, apoptosis induction, and changes in the number of cortical and hippocampal cells were studied. The presence of 25I-NBOMe in several brain regions shortly after the drug administration and its accumulation after multiple injections was found. The DNA damage was detected 72 h after the chronic treatment. On the contrary, at the same time point apoptotic signal was not identified. A decrease in the number of glial but not in neural cells in the frontal (FC) and medial prefrontal cortex (mPFC) was observed. The obtained data indicate that 25I-NBOMe passes easily across the BBB and accumulates in the brain tissue. Observed oxidative DNA damage may lead to the glial cells’ death.

www.nature.com/scientificreports/ in comparison to control rats suggesting decreased neurogenesis in this brain region 13 . In the in vitro studies, 25I-NBOMe decreased the viability of H9c2 cells and primary mice cardiomyocytes. Moreover, in vitro activity of p21 (CDC42/RAC)-activated kinase 1 (PAK1) was also reduced, and the QT interval measured by electrocardiography in rats was prolonged. Thus, it may be stated that the drug produces cardiotoxicity 14 . Furthermore, in vitro studies with a close congener of this class, 25C-NBOMe (25-400 µM), showed a potent reduction of SN4741, SH-SY5Y, and PC12 cells viability indicating 50 times higher potency to reduce SH-SY5Y cells in respect to methamphetamine 15 . The incubation of the rat primary cortical cultures with 25B-NBOMe (30 μM) decreased neuronal activity that did not recover after 19 h of washout period 16 . Our in vivo studies indicated that the low dose of 25B-NBOMe (0.3 mg/kg) was potent in damaging DNA in the rat frontal cortex 17 . Although many studies report adverse effects like seizures 18 , intoxications, and deaths, aspects of the in vivo 25I-NBOMe-mechanism of neurotoxicity has to be explored in depth. Thus, the present study aimed to assess the induction of in vivo neurotoxicity in the rat brain after single and repeated 25I-NBOMe treatment. The distribution of the drug in brain regions was determined using mass spectrometry. DNA damage of 25I-NBOMe was evaluated using a comet assay test, and the apoptotic signal was detected using TUNEL assay. Moreover, an immunohistochemical assessment of cells number in the rat brain was studied.

Results
25I-NBOMe distribution in the rat blood plasma and brain regions. The performed analysis confirmed the presence of 25I-NBOMe with the precursor 428.14 m/z ion and two fragmentation ions 121.62 and 272.28 m/z (Supplementary, Fig. 1S). The drug was detected in the rat blood plasma and brain regions 15 min after 1 and 10 mg/kg single doses of 25I-NBOMe (Table 1). The most profound intensity of 25I-NBOMe precursor ion was found in the nucleus accumbens. A signal dose-dependence was observed in both the blood plasma sample and the frontal cortex, hippocampus, striatum, and nucleus accumbens. Representative spectral images of LC-MS/MS analyses from the blood plasma and frontal cortex are shown in Fig. 1. 25I-NBOMe presence was observed after single and chronic administration with 0.3 mg/kg 25I-NBOMe (Table 2). However, the signal intensity after 7-days treatment in all studied brain regions was higher in comparison to a single dose. The highest intensity after repeated injections was observed in the frontal cortex (Fig. 2).
The effect of 25I-NBOMe on DNA damages in the rat frontal cortex and hippocampus. Single and repeated treatment with 0.3 mg/kg of 25I-NBOMe produced DNA damage by reactive oxygen species (ROS)    www.nature.com/scientificreports/ in the rat frontal cortex (F 2,29 = 237, p < 0.0001) and hippocampus (F 2,23 = 275, p < 0.0001), presented as a percent of tail moment measured at 72 h after the drug administration ( Fig. 5a,b, respectively). Only in the hippocampus was the DNA damage observed after multiple injections stronger than after a single dose of 25I-NBOMe (p < 0.01). LSD, as a comparative compound, caused DNA damage in the frontal cortex after a single and a 7-day treatment at the dose 0.05 mg/kg in comparison to control, and this damage was significantly greater after chronic administration (F 2,20 = 22, p < 0.0001). There was a significant increase in the tail moment value in the hippocampus after acute and chronic treatment with LSD (F 2,17 = 42, p < 0.0001). Similarly to the frontal cortex, the repeated injections caused a higher DNA damage than the single dose (p < 0.02). MDMA, as a positive control in this test, given acutely and chronically at the dose of 5 mg/kg exhibited DNA damaging properties in the Apoptotic signal in the rat frontal cortex and hippocampus after 25I-NBOMe administration. In this experiment, we evaluated the effect of 25I-NBOMe on apoptosis induction 72 h after the treatment. In contrast to a positive control (brain slice damaged with DNAse), the performed assay did not indicate apoptotic signal in brain slices from rats treated acutely or chronically with 25I-NBOMe given at the dose of 0.3 mg/kg. Since qualitative examination of sliced material did not provide any positive signal from the rat frontal cortex and anterior and posterior hippocampus (only a few isolated cells), the quantitative examination was not continued. The representative data are presented in Fig. 6.

Discussion
Our study indicated that 25I-NBOMe easily crosses the blood-brain barrier (BBB) since it was detected both in the blood serum and several brain regions 15 min after the injection. Repeated administration of 25I-NBOMe caused drug accumulation in the brain tissue. Seven-days treatment with 0.3 mg/kg of 25I-NBOMe decreased the number of astrocytes in the FC and mPFC and the number of microglia cells in the FC. On the other hand, the chronic treatment did not affect the number of cells in the hippocampus. Furthermore, the drug produced double-and single-strand DNA breaks by ROS after single and multiple injections in rats' frontal cortex and hippocampus. However, these changes did not lead to cell death by apoptosis. Our data, for the first time, presents the in vivo neurotoxicity of 25I-NBOMe. 25I-NBOMe was detected in the blood serum shortly after subcutaneous injections. The calculated spectral intensity was an order of magnitude higher after the 10 mg/kg dose than 1 mg/kg. Similarly, 15 min after injections, the drug was detected in the brain structures, mainly in the nucleus accumbens. It suggests fast and efficient penetration of 25I-NBOMe to the brain. The high intensity of 25I-NBOMe spectra from the nucleus accumbens and striatum could be explained by the first pass of the drug through more ventral subcortical brain regions and then crossing to more distant dorsal regions, such as cortex and hippocampus. Repeated administration with 0.3 mg/kg for 7 days of 25I-NBOMe resulted in the drug accumulation in all studied brain regions since the precursor ion intensity was increased in this experimental group compared to the single-injected one. In Ettrup et al. 19 study, carbon-11 labelled 25I-NBOMe known as 11 C-CIMBI-5 was located in the cortex and the lower range in the cerebellum shortly after the intravenous injection. In our study, among other brain regions, 25I-NBOMe accumulated mostly in the frontal cortex and hippocampus. This effect may be explained by the significant density of the 5-HT 2A receptors to which 25I-NBOMe exhibits a high binding affinity and which are   www.nature.com/scientificreports/ In vitro studies with the NBOMe series suggest toxic properties of these drugs. Twenty-four hour incubation with a wide range of 25C-NBOMe doses potently reduced the viability of cells 15 . Another congener 25B-NBOMe was also cytotoxic and attenuated neuronal activity of primary cortical cultures 16 . Our in vivo experiments indicated astrocytic cells loss as labeled with S100β after repeated treatment with 25I-NBOMe in the FC and mPFC and decreased the number of microglia cells as labeled with IBA-1 in the FC. Presented results do not indicate neurons loss in both cortical regions and the rats' hippocampus. S100β is a monomeric protein primarily synthesized by the end feet process of the astrocytes. It was found mainly in the nucleus and cytoplasm of astrocytes. S100β released from the cell may have trophic or toxic effects depending on its concentration. It has a neuroprotective effect at low concentrations, while at higher doses, it inhibits neuronal proliferation and differentiation and induces neurodegeneration and apoptosis 23 . In our immunohistochemical studies, we observed a lower number of S100β-positive cells in the FC and mPFC that may correspond to a decreased number of astrocytes in response to 25I-NBOMe treatment. Furthermore, astrocytes may release S100β protein extracellularly, affecting both astrocytes itself and neurons 24 . It is believed that a high S100β protein level is observed in a wide variety of pathological conditions and is negatively correlated with BBB integrity 25 . On the other hand, IBA-1 is a cytoplasmic protein specifically expressed in microglia. The role of microglia is mainly related to phagocytosis and removing damaged and apoptotic cells, neurofibrillary tangles, and plaques, but also DNA fragments 26 . Moreover, apart from the phagocytosis of apoptotic cells, microglia are also a key molecules in proinflammatory activity, maintaining homeostasis and controlling the fate of neurons and their progenitors 27 . It could be speculated that decreased number of microglia and astrocytes observed in our experiments may be related to the weakened repair mechanisms and defense functions of these cell types. Thus, 25I-NBOMe may pass easily and quickly through the damaged BBB and accumulate after chronic administration in the brain tissue, as demonstrated in our study.
Observed DNA damage at 72 h after single and repeated administration suggests neurotoxic properties of 25I-NBOMe. Cortical cells seem to be less resistant to the influence of 25I-NBOMe since our immunohistochemical studies indicated changes in cells number only in the FC and mPFC but not in the hippocampus. What is more, mass spectrometry analyses showed a higher accumulation of the drug in the frontal cortex with respect to the hippocampus. The in vitro work of Cocchi et al. 28 suggests the genotoxic capacity of phenethylamines in high concentration and hypothesizes a possible involvement of oxidative stress that does not always minimize cell viability. Our data is in vivo confirmation of this study showing the DNA damaging effect of phenethylamine congener 25I-NBOMe. Furthermore, our findings show the regional difference in neurotoxicity of 25I-NBOMe, indicating its damaging impact on cortical microglia and astrocytes being the important defense line in the brain. The lack of apoptotic signal detection in the TUNEL assay may be related to the time point of 72 h as insufficient to induce programmed cell death. For better understanding of a time of the apoptosis induction, the research in different time points is needed. Nevertheless, our data indicate the decrease in markers of glial cells, which corresponds to the appearance of double-and single-strand DNA breaks and points to the defective defense system of these cells but not neurons. Notably, the difference between brain regions in cells resistance against damage is noticed. Interestingly, LSD was weaker in its damaging effect on DNA than 25I-NBOMe due to its different receptor profiles 6 and its different impact on glutamatergic and dopaminergic neurotransmission 29 . Although, LSD is considered to be a safe substance, there are some older reports pointing out the possibility of causing chromosomal aberrations in vitro and in vivo, especially in the higher doses and in the long-term use 30,31 . Moreover, it was proposed that LSD may act directly on DNA by intercalation within DNA helix producing conformational changes that were not sufficient to decrease internal stability 32 . Thus, our data are the first that assess LSD effect on DNA damage studied in the comet assay. MDMA, known as oxidative stress inductor 33,34 and used in our study as a comparator, was nearly equal to 25I-NBOMe in oxidative damage of DNA in both brain regions.

Conclusion
Our data clearly indicate that phenethylamine hallucinogen 25I-NBOMe sold as a replacement for LSD and acting with similar potency as LSD may cause severe neurotoxicity by inducing oxidative stress and suppressing the defense role of astrocytic and microglial cells in rat frontal cortex and hippocampus. Drug administration. Animals received single or multiple (once per day for seven days) subcutaneous injections of 25I-NBOMe dissolved in 0.9% NaCl at a dose of 0.3 mg/kg/2 ml. The control group was treated with 0.9% NaCl solution in the same way. All procedures were performed 72 h after the last injection. The time point of 72 h was chosen due to the possible generation of neurotoxicity via oxidative stress caused by reactive oxygen species (ROS). Exceptionally, for initial 25I-NBOMe brain distribution studies, rats received the drug in the dose of 1 and 10 mg/kg and were sacrificed 15 min after the drug administration. The rat blood plasma and brain tissues were collected immediately after rats' decapitation. LSD (0.05 mg/kg, ip) was given daily for 7 days in a volume of 1 ml/kg, while MDMA 5 mg/kg, ip was injected in a volume of 2 ml/kg every second day (overall 4 injections).

LC-MS/MS with electrospray ion trap mass spectrometry (ESI-MS).
The presence of 25I-NBOMe in the rat brain regions and blood serum was analyzed with electrospray ion trap mass spectrometry (Amazon SL from Bruker Daltonics, Bremen, Germany). At 72 h after the last injection with 25I-NBOMe or 15 min after injection of single low and high (1 and 10 mg/kg) dose, animals were sacrificed by decapitation. Brains were removed and the frontal cortex, striatum, nucleus accumbens, and hippocampus were dissected. The blood samples were also collected after 25I-NBOMe injections. Tissue samples were homogenized in an ice-cold 0.1 M HClO 4 , and were centrifuged at 10,000×g for 10 min at 4 °C. Blood was incubated for 30 min and then centrifuged for 15 min, 3000 rpm at 4 °C. The collected blood serum and tissue supernatants were frozen at − 80 °C until the time of mass spectrometry analysis. Immunohistochemistry. Animals were deeply anesthetized and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde (PFA) in 0.1 M phosphate-buffered saline (PBS). After 24 h of fixation in 4% PFA (4 °C), 300 µm sections were cut through the frontal cortex (FC) and medial prefrontal cortex (mPFC) as well as hippocampus using a VT-1000S vibratome (Leica Microsystems, Heidelberg, Germany). Free-floating sections were processed for single staining of neurons with neuronal-specific nuclear protein (NeuN), glia with specific calcium-binding protein B (S100β), and microglia cells with ionized calcium-binding adaptor molecule 1 (IBA-1) antibodies. Subsequently, brain sections were rinsed and incubated for 1 h in a blocking buffer: 0.01 M PBS containing 0.3% Triton X-100 and 5% average horse, goat, or rabbit serum. After that, the sections were incubated for 48 h at 4 °C with one of the following primary antibodies: monoclonal anti-NeuN (1:1000), monoclonal anti-S100β-subunit (1:1000) or polyclonal IBA-1 (1:500) diluted in 0.01 M PBS containing 0.3% Triton X-100 (PBST) and 3% average horse or goat serum. Primary antibody binding was visualized with biotinylated secondary antibodies, the Avidin/Biotin Complex (Vectastain Elite ABC Kit) according to recommended by manufacturer concentration and 3,3′-diaminobenzidine tetrahydrochloride (DAB, 10 mg/50 ml and 0.025% H 2 O 2 ) solution to give a brown color to NeuN, S100β and IBA-1-immunoreactive cells. For data presentation, digital images were captured using a digital camera CX 9000 (Bioscience Microbrightfield, Inc., Germany) attached to a Leica microscope (CTR 6000) with 2.5 and 5.0 dry or 63 × and 100 × oil objectives (Leica) that was controlled by Stereo Investigator software (v. 8.10.2, 1995-2007 Bioscience Microbrightfield, Inc., Germany). The final photomicrographs were composed using the Adobe Photoshop program. The numbers of NeuN-, S100β-and IBA-1-immunopositive cells in the analyzed brain regions were estimated using unbiased stereological methods 35,36 . Briefly, every sixth section from the systematic random sampling along the rostrocaudal axis was analyzed with a 63 ×/1.4-0.7 lens using the Stereo Investigator stereology system software. The cells appearing in the upper focal plane were omitted to prevent the counting of cell caps (− 5 μm of the topmost surface of the section). Immunopositive cells were marked when they were within the optical dissectors, which comprised a focal plane of 1600 μm 2 × 15 μm. The total numbers of NeuN, S100β-and IBA-1-immunopositive cells in the FC, mPFC, and hippocampus were automatically calculated by the Stereo Investigator software. In addition, Alkaline comet assay. The alkaline comet assay was performed with the use of CometAssay ® Reagent Kit for Single Cell Gel Electrophoresis Assay. At 72 h after the acute and chronic treatment with 25I-NBOMe (0.3 mg/kg, sc × 7 days), LSD (0.05 mg/kg, ip × 7 days) or MDMA (5 mg/kg, ip × 4 doses every second day) animals were sacrificed by decapitation and the frontal cortex (that consist of the sum of FC and mPFC) and hippocampus were dissected. After homogenization and several purification and centrifugation stages (as described previously in 17 ), the nuclear suspension was obtained using a sucrose gradient (2.8 M/2.6 M, bottom to top). The nuclear fraction was mixed with low melting point agarose and transferred immediately onto CometSlides™.
The following steps, including membrane lysis, DNA unwinding, alkaline electrophoresis, and staining (SYBR ® Gold), were carried out according to Trevigen CometAssay ® protocol. Stained sections were acquired and analyzed under a fluorescence microscope (Nikon Eclipse50i, Japan) equipped with a camera and NIS Elements software. The data was analyzed using OpenComet software v.1.3 (cometbio.org), a plugin of ImageJ program v.1.47 (NIH, Bethesda, MD, USA). DNA damage was presented as a tail moment. Tail moment incorporates a measure of both the smallest detectable size of migrating DNA (reflected by the comet tail length) and the number of damaged pieces (represented by the intensity of DNA in the tail).
Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. As described above, animals were perfused transcardially and brains were fixed overnight in 4% PFA, then gradually dehydrated and stored in 70% ethanol (EtOH). Paraffin-embedded brain tissue was sectioned on a microtome (Leica, RM45) for 7-µm-thick sections. For further experiments following sections from the region of the frontal cortex, anterior and posterior hippocampus were collected. Next, chosen microscope slides with tissue sections were deparaffinized in xylene and declining concentrations of EtOH and remained in 0.01 M PBS. The apoptotic signal was assessed using Click-iT™ Plus TUNEL Assay for in situ apoptosis detection with Alexa Fluor™ 488 picolyl azide dye. For positive control, brain slices were incubated with 1 unit of DNAse I (A&A Biotechnology, Gdansk, Poland) as recommended by manufacturer. Vectashield Mounting Medium (Vector Laboratories) with DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) was used for slipping and counterstaining the slides. Stained slides were examined under a fluorescence microscope (Nikon Eclipse50i, Japan) equipped with a camera and NIS Elements software.

Data analysis.
Presented results were analyzed with one-way ANOVA followed by Tukey's post hoc test. The differences were considered significant if the p-value was smaller than 0.05. All statistical analyses were carried out using STATISTICA v.