Elsevier

Toxicology Letters

Volume 207, Issue 1, 10 November 2011, Pages 12-17
Toxicology Letters

Cytotoxicity of synthetic cannabinoids found in “Spice” products: The role of cannabinoid receptors and the caspase cascade in the NG 108-15 cell line

https://doi.org/10.1016/j.toxlet.2011.08.021Get rights and content

Abstract

The worldwide distribution of “Spice” that contains synthetic cannabinoids with a pharmacological activity similar to Δ9-tetrahydrocannabinol has been reported. In the current study, we evaluated the cytotoxicity of the synthetic cannabinoids, CP-55,940, CP-47,497 and CP-47,497-C8 towards NG 108-15 cells and investigated their mechanism of cytotoxicity. CP-55,940, CP-47,497 and CP-47,497-C8 were all cytotoxic for NG 108-15 cells in a concentration-dependent manner. The cytotoxicity of these synthetic cannabinoids was suppressed by preincubation with the selective CB1 receptor antagonist AM251, but not with the selective CB2 receptor antagonist AM630. Preincubation with a caspase-3 inhibitor significantly suppressed the cytotoxicity of these synthetic cannabinoids for NG 108-15 cells. Induction of apoptosis by these cannabinoids was also confirmed by staining of the cells with annexin V. Our results indicate that the cytotoxicity of synthetic cannabinoids towards NG 108-15 cells is mediated by the CB1 receptor, but not by the CB2 receptor, and further suggest that caspase-cascades may play an important role in the apoptosis induced by these synthetic cannabinoids.

Highlights

► We examined the cytotoxicity of cannabinoids that are detected in “Spice” products. ► These cannabinoids induced cytotoxicity and apoptosis in NG 108-15 cells. ► The cytotoxicity of these compounds is mediated by the cannabinoid CB1 receptor. ► Caspase-cascades may be involved in the synthetic cannabinoids-induced apoptosis.

Introduction

An increase in marijuana abuse among young people has become an object of public concern in many countries. Moreover, the distribution of “Spice” products, illegal drugs that have an effect similar to that of marijuana, has been reported in many European countries (Dresen et al., 2010). The main psychoactive constituent of marijuana that affects the central nervous system is Δ9-tetrahydrocannabinol (Δ9-THC). The pharmacological effects of Δ9-THC are known to include impairments in cognition, memory and learning, as well as euphoria, hallucinations (Abel, 1970, Adams and Martin, 1996, Block and Ghoneim, 1993, Reisine and Brownstein, 1994), hypomotility (Wilson et al., 2002), hyperphagia (Williams and Kirkham, 2002) and disruption of sleep regulation (Murillo-Rodriguez, 2008). These pharmacological effects of Δ9-THC are mediated through cannabinoid receptors that have been identified in rats (Matsuda et al., 1993) and humans (Gerard et al., 1991). At least two different subtypes of cannabinoid receptors, CB1 (Herkenham et al., 1990, Matsuda et al., 1990) and CB2 (Brown et al., 2002, Munro et al., 1993), have been identified, and it has been shown that the phychoactive effects of Δ9-THC are mediated through the CB1 receptor in the brain (Devane et al., 1988, Martin et al., 1991, Moldrich and Wenger, 2000).

The so-called “Spice” product contains synthetic cannabinoids, which have similar psychoactive effects to Δ9-THC. Forensic investigation of “Spice” products have identified the presence of the synthetic cannabinoids CP-47,497, a C8 homolog of CP-47,479 (CP-47,497-C8), and HU-210 (Lindigkeit et al., 2009), which are hazardous to human health when abused. Δ9-THC and HU-210 are members of classical cannabinoid groups, which consist of ABC-tricyclic dibenzopyran derivatives (Howlett et al., 1988), while the synthetic cannabinoids CP-47,497 and CP-47,497-C8 are AC-bicyclic compounds, which include only part of the tricyclic structure of Δ9-THC, and are classified as non-classical cannabinoids (Melvin et al., 1984, Melvin et al., 1993). Although there are many compounds that are analogues of synthetic cannabinoids (Howlett et al., 2002), the toxicological properties of these synthetic cannabinoids remain poorly understood. It is therefore both necessary and urgent to establish a rapid and stable method for evaluation of synthetic cannabinoid-induced cytotoxicity.

In the current study, we examined the cytotoxicity of CP-47,497 and CP-47,497-C8, which are detected in “Spice”, as well as the cytotoxicity of CP-55,940, which is a compound in which substitutions have been made at position C-4 of the cyclohexanol ring of CP-47,497, using a neuroblastoma-glioma hybrid cell line, NG 108-15. The NG 108-15 cell line was used for these studies since it is known to express functional cannabinoid receptors (Ho and Zhao, 1996, Matsuda et al., 1990). Endogenous expression of cannabinoid receptors in these cells allows investigation of whether activation of these receptors can directly regulate synthetic cannabinoid-induced cytotoxicity. We further investigated apoptotic morphological changes induced by these synthetic cannabinoids and the role of caspase-3 in their cytotoxicity.

Section snippets

Drugs and reagents

The synthetic cannabinoid ligands CP-55,940 and CP-47,497 were purchased from Cayman Chemical Company (Ann Arbor, MI, USA). The selective CB1 receptor antagonist AM251, the selective CB2 receptor antagonist AM630 and the irreversible caspase-3 inhibitor Z-DEVD-FMK were purchased from Tocris Bioscience (Ellisville, MO, USA). CP-47,497-C8 was kindly supplied by Dr. Ruri Kikura-Hanajiri and Dr. Yukihiro Goda (National Institute of Health Sciences, Tokyo, Japan).

Cell culture

The NG 108-15 cell line was obtained

Expression of cannabinoid receptors in NG 108-15 cells

We first investigated the expression of cannabinoid receptors in cells of the neuroblastoma-glioma hybrid cell line, NG 108-15, by immunofluorescence analysis of CB1 and CB2 receptors. Expression of both the CB1 and the CB2 receptor in NG 108-15 cells was clearly observed (Fig. 1).

Analysis of synthetic cannabinoid-induced cytotoxicity

Time- and concentration-dependent cytotoxic effects of CP-55,940 on NG 108-15 cells were analyzed using the CytoTox Glo™ Cytotoxicity Assay kit. Treatment with 1, 10 or 30 μM of CP-55,940 over a period of 5 h was

Discussion

The biological effects of many synthetic cannabinoids are not fully understood in spite of the fact that “Spice” is abused by humans (Muller et al., 2010). We therefore examined the cytotoxicity of cannabinoids that have an AC-bicyclic structure; CP-55,940, CP-47,497 and CP-47,497-C8, as part of an evaluation of the biological effects induced by synthetic cannabinoid ligands. We confirmed that the treatment of NG 108-15 cells with CP-55,940, CP-47,497 or CP-47,497-C8 for 2 h was cytotoxic for

Conflict of interest

The authors declare that there are no conflicts of interest.

Acknowledgements

This study was supported in part by Grants-in-aid for Health Science Research on Regulatory Science of Pharmaceuticals and Medical Devices, Health and Labour Sciences Research Project (to M.F.) supported by the Ministry of Health Labour and Welfare of Japan. The funding sources had no involvement in study design, in the collection, analysis or interpretation of data, in the writing of the report or in the decision to submit the paper for publication.

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