Investigations of the genotoxic properties of two synthetic cathinones (3-MMC, 4-MEC) which are used as psychoactive drugs

Abstract

Synthetic cathinones (SCAs) are consumed worldwide as psychostimulants and are increasingly marketed as surrogates of classical illicit drugs via the internet. The genotoxic properties of most of these drugs have not been investigated. Results of earlier studies show that amphetamines which are structurally closely related to these compounds cause damage to the genetic material. Therefore, we tested the genotoxic properties of two widely consumed SCAs, namely, 3-MMC (2-(methylamino)-1-(3-methylphenyl) propan-1-one) and 4-MEC (2-(ethylamino)-1-(4-methylphenyl) propan-1-one) in a panel of genotoxicity tests. We found no evidence for induction of gene mutations in Salmonella/microsome assays, but both drugs caused positive results in the single cell gel electrophoresis (SCGE) assay which detects single and double strand breaks of DNA in a human derived buccal cell line (TR146). 3-MMC induced similar effects as 4-MEC and also caused significant induction of micronuclei which are formed as a consequence of structural and chromosomal aberrations. Negative results obtained in SCGE experiments with lesion specific enzymes (FPG and Endo III) show that these drugs do not cause oxidative damage of DNA. However, moderate induction of TBARS (which leads to the formation of DNA-reactive substances) was observed with 4-MEC, indicating that the drug causes lipid peroxidation while no clear effect was detected with 3-MMC. Results obtained with liver homogenate in SCGE-experiments show that phase I enzymes do not lead to the formation of DNA reactive metabolites. Taken together, our findings indicate that consumption of certain SCAs may cause adverse health effects in users as a consequence of damage to the genetic material.

Fig. 1. Chemical structures of the different compounds. A: Cathinone (2-amino-1-phenylpropan-1-one, CAS number: 16735-19-6), B: Amphetamine (1-phenylpropan-2-amine, CAS number: 2706-50-5), C: 3-MMC (2-(methylamino)-1-(3-methylphenyl)propan-1-one, CAS number: 1246816-62-5), D: 4-MEC (2-(ethylamino)-1-(4-methylphenyl)propan-1-one, CAS number: 1266688-86-1).

Fig. 2. Cytotoxic and genotoxic properties of 3-MMC and 4-MEC in TR146 cells. The indicator cells were exposed to different concentrations of the test compounds for 3 and 24 hours. Subsequently, the cytotoxic activities of the drugs were monitored with trypan blue (A–D) and comet formation was measured after electrophoresis (E–H, for details see Materials and methods). Bars indicate means ± SD of data obtained with three parallel cultures per experimental point (from each culture 3 slides were made and 50 cells were evaluated per slide). Stars indicate statistical significance (p ≤ 0.05, ANOVA).

Fig. 3. Impact of rat liver S9, bovine serum albumin (BSA) and heat inactivated S9 (HS9) on comet formation induced by 3-MMC and 4-MEC in TR146. The cells were treated with solutions of the drugs (50 μM 3-MMC or 4-MEC) with and without S9 mix, HS9 or BSA (final protein concentration 0.3 mg mL^–1). B[a]P (50 μM) was used as a positive control and induced a significant effect (with S9 the tail intensity was 21.6 ± 4.6 and without S9 9.6 ± 2.5). After treatment, DNA migration was determined in SCGE experiments under standard conditions. Bars represent means ± SD of results obtained with tree cultures in parallel. From each culture, three slides were made and 50 cells were analyzed for comet formation per slide.

Fig. 4. Impact of 3-MMC and 4-MEC on formation of oxidatively damaged pyrimidines (Endo III sensitive sites, Fig. 3A and B) and purines (FPG sensitive sites, Fig. 3C and D) in a human derived buccal cell line (TR146). The cells were exposed to the drugs for 24 h. Subsequently, the nuclei were isolated and treated with enzymes or with the buffers only as described in Materials and methods. Subsequently, DNA migration was monitored. Bars indicate means ± SD of results obtained with three cultures per experimental point (values obtained with the enzyme buffers were subtracted from values obtained with the enzymes). From each culture, three slides were made and 50 cells were evaluated per slide.

Fig. 5. Impact of the drugs on formation of TBARs. The cells were exposed to 3-MMC or 4-MEC for 3 h. Subsequently, they were homogenised and TBARS concentrations were determined spectrophotometrically. Bars indicate means ± SD of results obtained with three cultures per experimental point. Stars indicate statistical significance (p ≤ 0.05, ANOVA).