UR and XLR are the new generation of synthetic cannabinoids and are chemically different to the first generation. New generations of synthetic cannabinoids are continuously emerging to replace the synthetic cannabinoids that have been made illegal. Home About Who Are We? Search for:. Recent Posts. Basal values of extracellular DA levels in the nucleus accumbens were 1. Locomotor activity was measured from 5 min before to 15 min after XLR degradant using an implanted Nano-Tag device.
Biosensor currents reflecting glutamate levels were obtained every 10 s from 2. Statistical analysis was performed using the Bonferroni test.
Locomotor activity was measured from 5 min before to 15 min after administration. Glutamate levels in the hippocampus were measured in vivo using an enzyme biosensor to investigate whether excessive glutamatergic transmission is involved in the excitatory effect of XLR degradant.
A decrease, in contrast to increase, was observed in the extracellular glutamate levels after XLR degradant treatment Fig. These results suggest that glutamatergic transmission does not play a major role in the XLR degradant-induced excitatory behavior. Next, a series of experiments were conducted to investigate the involvement of GABAergic transmission using gabapentin, which increases and maintains the GABAergic neuronal function along with inhibiting the voltage-dependent calcium channel.
Gabapentin significantly inhibited the XLR degradant-induced excitatory behavior between 2 and 3 min after treatment Fig. Some synthetic cannabinoids were reported to induce a generalized seizure in drug abusers immediately after smoking [ 21 ]. These compounds impart nonselective agonistic effects on centrally and peripherally located CB 1 and CB 2 receptors, although psychoactive effects are mainly mediated via the CB 1 receptor.
It has been reported that i. On the other hand, it has not been reported that XLR exhibits excitatory action.
In addition, the main psychoactive component in the XLR smoke was found to be XLR degradant produced by pyrolysis. The stimulated psychomotor function could be likened to the seizures in humans [ 26 , 27 ] and has been shown to be associated with electrographic seizures in mice [ 22 ].
Therefore, XLR degradant produced during smoking may cause seizures in mice and possibly in humans as well. It is suggested that the previous studies underestimated XLR potency which would increase by smoking. This study shows the importance of experimental design in line with the actual situation of human abuse.
The present study also demonstrated that the excitatory effect of XLR degradant was mediated mainly through the CB 1 receptor, a result consistent with that reported by Malyshevskaya et al. UR Fig. In the previous study, this UR degradant Fig. Therefore, smoking certain synthetic cannabinoids leads to greater psychoactive effects than inhaling their respective parent compounds.
Based on this finding, DA levels in the nucleus accumbens after XLR degradant administration were measured in the present study by microdialysis. The results showed that the onset of excitatory behavior did not coincide with the time of increase in DA levels Fig.
These results suggest that an increase in DA levels in the nucleus accumbens is not involved in the excitatory effect of XLR degradant. Reportedly, the synthetic cannabinoid AM causes seizures in mice in the early course of treatment when glutamate levels in the hippocampus increase significantly [ 11 ].
However, in the present study, glutamate levels in the hippocampus decreased significantly after XLR degradant administration Fig. This result suggests that the excitatory effect of XLR degradant is caused by a mechanism not mediated by glutamate. This study also demonstrated that gabapentin significantly suppressed the enhanced locomotor activity induced by XLR degradant Fig. In this study, because glutamate levels in the hippocampus significantly decreased, instead of increasing, after XLR degradant administration, suppressed GABAergic function may be involved in the excitatory effect of XLR degradant.
Although the overall mechanism of synthetic cannabinoid-mediated seizures or excitatory action is still unclear, CB 1 receptor-dependent neurotransmission and possibly the GABAergic function are likely involved in the transient hyperreflexic behavior. In conclusion, our results indicate that the inhalation of XLR smoke, the main active component of which is XLR degradant, causes hyperreflexia as well as hypothermia and catalepsy in mice.
The XLR degradant is more potent than XLR, demonstrating the importance of this study in line with the actual situation of human abuse. Pharmacol Biochem Behav — Neurotoxicol Teratol — Numazawa S The forefront of drug abuse—recent trends in illegal drug markets in Japan, Google Scholar. Accessed 28 Dec Agenda item 4. Accessed 9 Apr Accessed 5 Apr Drug Test Anal — Am J Med — J Toxicol Sci — Prog Neuropsychopharmacol Biol Psychiatry — Toxicol Appl Pharmacol —8. Exp Anim.
Article PubMed Google Scholar. Eur J Pharmacol — Neuropharmacology — J Pharmacol Exp Ther — J Anal Toxicol — Kumar A, Lalitha S, Mishra J Hesperidin potentiates the neuroprotective effects of diazepam and gabapentin against pentylenetetrazole-induced convulsions in mice: possible behavioral, biochemical and mitochondrial alterations. Indian J Pharmacol — Clin Toxicol — Sci Rep ACS Chem Neurosci — Behav Pharmacol — Addiction — PMID Ann Emerg Med — J Med Toxicol — Eur J Pharmacol R1—R3.
J Neurosci — Neuroscience — Download references. The authors are grateful to Mr. You can also search for this author in PubMed Google Scholar. Results of gene mutation assays obtained with the bacterial indicator strain, Salmonella typhimurium TA98 and TA in the presence and absence of metabolic activation mix. The Salmonella typhimurium strains TA and TA98 were exposed to different concentrations of the SCs in the presence and absence of metabolic activation mix rat liver S9 as described in materials and methods.
Figure 2 depicts the results which were obtained with the drugs in the SCGE experiments with lymphocytes Fig. Lymphocytes were treated for 3 h, TR cells for 24 h. The cytotoxic effects of the drugs were monitored with the trypan blue exclusion test Lindl and Bauer Several investigations indicate that SCs cause inflammations which may lead to release of reactive oxygen species [for further details, see Bileck et al.
Therefore, we used a modified protocol of the SCGE technique to monitor formation of oxidatively damaged purines and pyrimidines. The results of these experiments are summarized in Fig.
It can be seen that no evidence for increased formation of oxidized DNA bases was detected in the present experiments. Impact of treatment of human-derived buccal cells line TR with synthetic cannabinoids on formation of oxidatively damaged purines FPG-sensitive sites, a , b and pyrimidines Endo III sensitive sites, c , d.
The cells were exposed to the drugs for 24 h. Subsequently, DNA migration was monitored. From each culture, two slides were made and 50 cells were evaluated per slide. To find out whether addition of phase I enzymes which are represented in liver-derived homogenate S9 leads to activation of the drugs, further experiments were carried out. The results are depicted in Fig. A similar effect was seen when the enzyme mix was replaced by BSA solution which contained an identical amount of protein as the activation mix.
From each culture, two slides were made and 50 cells were analyzed for comet formation per slide. The results of experiments in which the indicator cells were exposed in an air—liquid interface system are summarized in Table 2.
RCS-4 was in both indicator cell lines a more potent genotoxin, i. From each culture, 3 slides were made and 50 cells were evaluated per slide. In each experiment, positive controls were included; i. To find out whether treatment of human cells leads to formation of MN, which reflect structural and numerical chromosomal aberrations, cytome experiments were conducted.
The results with lymphocytes and with TR cells are summarized in Table 3. Impact of different synthetic cannabinoids on cytokinesis-block proliferation indices and on the rates of various nuclear aberrations in human mitogen-stimulated lymphocytes and in TR cells. Human mitogen-stimulated lymphocytes from three individuals were treated with different concentrations of the test compounds for 3 h. TR cells were exposed to different concentrations of the drugs for 24 h. Per experimental point, two slides were prepared and cells were evaluated from each slide.
Both compounds caused in both cell types induction of MNi. Also RCS-4 caused a positive result in both types of indicator cells. The rates of these anomalies were also elevated in both cell types as a consequence of treatment with RCS-4, but these effects were only moderate and did not reach significance.
Three different genotoxicity systems were used in the present study to investigate the genotoxic properties of XLR, one of the most widely consumed SCs and, of the benzoyl indole RCS Furthermore, attempts were made to characterize the molecular mechanisms which lead to damage of the genetic material and to find out whether adverse effects can be expected in drug users under realistic exposure conditions.
SCs with benzoyl structure like RCS-4 have not been studied before in microbial mutagenicity assays according to our knowledge, and the present findings show that the compound is devoid of activity under all experimental conditions. On the contrary, clear evidence for induction of DNA damage was found in SCGE experiments which reflect single- and double-strand breaks as well as apurinic sites Tice et al. In this context, it is notable that we observed induction of comet formation by other SCs in earlier experiments; for example, the cyclohexylphenol CP,C8 caused significant induction of DNA migration in lymphocytes and in TR cells Bileck et al.
It is not known in general whether positive results in SCGE experiments lead to persisting alterations of the genetic material. In order to find out whether the two drugs cause damage at the chromosomal level which is associated with adverse health effects, we conducted cytome assays in which we studied the formation of MNi which are a consequence of structural and numerical aberrations Norppa and Falck as well as other nuclear anomalies.
As described in the results sections Table 2 , positive results were obtained with both drugs in the buccal cell line TR and also in the lymphocytes. It is known that these anomalies reflect genetic instability; i. The biological consequences of the formation of these anomalies are not known at present; in the case of MNi, it is notable and it was shown by Bonassi and co-workers that increased rates of MN in peripheral lymphocytes of humans are associated with elevated cancer risks Bonassi et al.
The results of the present experiments allow to draw some conclusions in regard to the molecular mechanisms which lead to damage of the genetic material. The negative results in the bacterial assays indicate that the drugs do not form bulky DNA adducts as other groups of genotoxic carcinogens e. Furthermore, it can be also excluded that the induction of the comets which was observed in the SCGE experiments is causally related to formation of oxidatively damaged purines and pyrimidines.
As shown in Fig. Many genotoxic carcinogens require metabolic activation via phase I enzymes. To mimic these metabolic processes which lead to formation of DNA-reactive metabolites in vivo, liver enzyme homogenate S9 mix is added in routine mutagenicity assays OECD , A similar effect was seen when BSA was added to the incubation mixtures.
These findings show that enzymes which are contained in liver homogenates do not convert these drugs to DNA-reactive intermediates. The observation of a reduction in their genotoxic activity which was seen with the enzyme mix and with pure proteins may be indicative of detoxification via non-enzymatic protein binding.
It is possible that these detoxification reactions play also a role in the human body in addition to metabolic processes which are catalyzed by enzymes e. The last part of this study concerned the question whether the drugs cause DNA damage under realistic exposure conditions. Only for few SCs including RCS-4 , data on their concentrations in body fluids of humans after consumption of the drugs are available Kneisel et al.
The concentrations which were detected are 2—3 orders of magnitude lower than those which caused positive results in the present experiments. In order to assess whether induction of genetic damage occurs in users as a consequence of inhalation of the drugs, we conducted with both SCs experiments with an air—liquid interface system.
As described above, the cells were grown on membranes and exposed to drugs under conditions which mimic the exposure of tissues in the respiratory tract. The amounts which were vaporized 5 and 20 mg are contained in 1—2 typical SC containing cigarettes Adamowicz et al.
As described in the results section, we found with both SCs clear evidence for induction of DNA migration in both types of indicator cells. Taken together, the results of the present study show that both drugs cause DNA instability in human-derived cells at the chromosomal level.
It is known that instability of the genetic material plays a key role in the etiology of human cancer Ferguson et al. Furthermore, it is also involved in neurodegenerative disorders Madabhushi et al.
Therefore, the findings of our investigation indicate that adverse health effects may be caused in drug users as a consequence of the DNA-damaging properties of the drugs, and further investigations are warranted to clarify this important issue.
Open access funding was provided by Medical University of Vienna. National Center for Biotechnology Information , U. Archives of Toxicology. Arch Toxicol. Published online Feb 8. Verena J. Author information Article notes Copyright and License information Disclaimer. Corresponding author. Received Oct 16; Accepted Jan 4. This article has been cited by other articles in PMC.
Abstract Aim of this study was the investigation of the genotoxic properties of XLR [1- 5-fluoropentyl -1 H -indolyl] 2,2,3,3-tetramethylcyclopropyl methanone, a widely consumed synthetic cannabinoid SC , and of the benzoyl indole RCS-4 4-methoxyphenyl 1-pentyl-1 H -indolyl methanone.
Open in a separate window. Collection of lymphocytes Peripheral blood samples were collected from three healthy, non-smoking male volunteers without any history of recent disease or exposure to toxic chemical agents. Table 1 Results of gene mutation assays obtained with the bacterial indicator strain, Salmonella typhimurium TA98 and TA in the presence and absence of metabolic activation mix.
Single cell gel electrophoresis experiments standard conditions Figure 2 depicts the results which were obtained with the drugs in the SCGE experiments with lymphocytes Fig. SCGE experiments with lesion-specific enzymes Several investigations indicate that SCs cause inflammations which may lead to release of reactive oxygen species [for further details, see Bileck et al. SCGE experiments with liver S9 mix and bovine serum albumin To find out whether addition of phase I enzymes which are represented in liver-derived homogenate S9 leads to activation of the drugs, further experiments were carried out.
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