Novel Psychoactive Phenethylamines: Impact on Genetic Material

Abstract

Psychedelic and stimulating phenethylamines belong to the family of new psychoactive substances (NPS). The acute toxicity framework has begun to be investigated, while studies showing genotoxic potential are very limited or not available. Therefore, in order to fill this gap, the aim of the present work was to evaluate the genotoxicity by treating TK6 cells with 2C-H, 2C-I, 2C-B, 25B-NBOMe, and the popular 3,4-Methylenedioxymethylamphetamine (MDMA). On the basis of cytotoxicity and cytostasis results, we selected the concentrations (6.25-35 µM) to be used in genotoxicity analysis. We used the micronucleus (MN) as indicator of genetic damage and analyzed the MNi frequency fold increase by an automated flow cytometric protocol. All substances, except MDMA, resulted genotoxic; therefore, we evaluated reactive oxygen species (ROS) induction as a possible mechanism at the basis of the demonstrated genotoxicity. The obtained results showed a statistically significant increase in ROS levels for all genotoxic phenethylamines confirming this hypothesis. Our results highlight the importance of genotoxicity evaluation for a complete assessment of the risk associated also with NPS exposure. Indeed, the subjects who do not have hazardous behaviors or require hospitalization by using active but still "safe" doses could run into genotoxicity and in the well-known long-term effects associated.

Keywords: 25B-NBOMe; 2C-B; 2C-H; 2C-I; MDMA; ROS; flow cytometry; genotoxicity; phenethylamine.

Figure 1

Chemical structures of 2C-H (A), 2C-I (B), 2C-B (C), 25B-NBOMe (D), and MDMA (E).

Figure 2

Cell viability on TK6 cells after 26 h treatment with 2C-H, 2C-I, 2C-B, 25B-NBOMe, and MDMA at the indicated concentrations with respect to the untreated control C (-). Each bar represents the mean ± SEM of five independent experiments. Data were analysed by ANOVA Repeated, followed by a Dunnet post-test. * p < 0.05 vs. C (-), ** p < 0.01 vs. C (-), *** p < 0.001 vs. C (-). The red line indicates the OECD threshold to be complied with for this genotoxicity test.

Figure 3

The apoptosis fold increase on TK6 cells after 26 h of treatment with 2C-H, 2C-I, 2C-B, 25B-NBOMe, and MDMA at the indicated concentrations with respect to the untreated control C (-). Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni or Dunnet post-tests. * p < 0.05 versus C (-).

Figure 4

The MNi frequency fold increase on TK6 cells after 26 h of treatment with 2C-H at the indicated concentrations with respect to the untreated negative control C (-) and to positive controls [MMC and VINB]. (A) A plot of the nuclei and MNi in the untreated control (B, left), and in 35 µM 2C-H-treated cultures (B, right). Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by the Bonferroni post-test. ** p < 0.01 vs. C (-); *** p < 0.001 vs. C (-).

Figure 5

The MNi frequency fold increase on TK6 cells after 26 h of treatment with 2C-I at the indicated concentrations with respect to the untreated negative control C (-) and to positive controls [MMC and VINB]. (A) A plot of the nuclei and MNi in the untreated control (B, left), and in 35 µM 2C-I-treated cultures (B, right). Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test. ** p < 0.01 vs. C (-); *** p < 0.001 vs. C (-).

Figure 6

The MNi frequency fold increase on the TK6 cells after 26 h treatment with 2C-B at the indicated concentrations with respect to the untreated negative control C (-) and to positive controls [MMC and VINB]. (A) A plot of the nuclei and MNi in the untreated control (B, left), and in 12.5 µM 2C-B-treated cultures (B, right). Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by the Bonferroni post-test. ** p < 0.01 vs. C (-); *** p < 0.001 vs. C (-).

Figure 7

The MNi frequency fold increase on TK6 cells after 26 h treatment with 25B-NBOMe at the indicated concentrations with respect to the untreated negative control [C (-)] and to positive controls [MMC and VINB]. (A) The plot of the nuclei and MNi in the untreated control (B), and in 12.5 µM 25B-NBOMe -treated cultures (C). Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test. * p < 0.05 vs. C (-), ** p < 0.01 vs. C (-); *** p < 0.001 vs. C (-).

Figure 8

MNi frequency fold increase on TK6 cells after 26 h treatment with MDMA at the indicated concentrations with respect to the untreated negative control C (-) and to positive controls [MMC and VINB]. (A) A plot of the nuclei and MNi in the untreated control (B, left) and in 35 µM MDMA-treated cultures (B, right). Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by Bonferroni post-test. ** p < 0.01 vs. C (-); *** p < 0.001 vs. C (-).

Figure 9

The ROS fold increase on TK6 cells after 1 h of treatment with 2C-H (A), 2C-I (B), 2C-B (C), 25B-NBOMe (D), and MDMA (E) at the indicated concentrations with respect to the untreated negative control C (-). Each bar represents the mean ± SEM of five independent experiments. Data were analysed using repeated ANOVA followed by t-test for paired data. * p < 0.05 vs. C (-).