Applied Molecular Biosciences Unit

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Adverse outcome pathways induced by 3,4‑dimethylmethcathinone and 4‑methylmethcathinone in differentiated human SH‑SY5Y neuronal cells

Publication . Soares, Jorge; Costa, Vera Marisa; Gaspar, Helena; Santos, Susana; Bastos, Maria de Lourdes

Cathinones (β-keto amphetamines), widely abused in recreational settings, have been shown similar or even worse toxicological profile than classical amphetamines. In the present study, the cytotoxicity of two β-keto amphetamines [3,4-dimethylmethcathinone (3,4-DMMC) and 4-methylmethcathinone (4-MMC)], was evaluated in differentiated dopaminergic SH-SY5Y cells in comparison to methamphetamine (METH). MTT reduction and NR uptake assays revealed that both cathinones and METH induced cytotoxicity in a concentration- and time-dependent manner. Pre-treatment with trolox (antioxidant) partially prevented the cytotoxicity induced by all tested drugs, while N-acetyl-l-cysteine (NAC; antioxidant and glutathione precursor) and GBR 12909 dopamine transporter inhibitor) partially prevented the cytotoxicity induced by cathinones, as evaluated by the MTT reduction assay. Unlike METH, cathinones induced oxidative stress evidenced by the increase on intracellular levels of reactive oxygen species (ROS), and also by the decrease of intracellular glutathione levels. Trolox prevented, partially but significantly, the ROS generation elicited by cathinones, while NAC inhibited it completely. All tested drugs induced mitochondrial dysfunction, since they led to mitochondrial membrane depolarization and to intracellular ATP depletion. Activation of caspase-3, indicative of apoptosis, was seen both for cathinones and METH, and confirmed by annexin V and propidium iodide positive staining. Autophagy was also activated by all drugs tested. Pre-incubation with bafilomycin A1, an inhibitor of the vacuolar H+- ATPase, only protected against the cytotoxicity induced by METH, which indicates dissimilar toxicological pathways for the tested drugs. In conclusion, the mitochondrial impairment and oxidative stress observed for the tested cathinones may be key factors for their neurotoxicity, but different outcome pathways seem to be involved in the adverse effects, when compared to METH.

2020Journal article

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Synthetic cannabinoids JWH-018, JWH-122, UR-144 and the phytocannabinoid THC activate apoptosis in placental cells

Publication . Almada, Almada; Alves, Patrícia; Fonseca, Bruno M.; Carvalho, Félix; Queirós, Cláudio R.; Gaspar, Helena; Amaral, Cristina; Teixeira, Natércia A.; Correia-da-Silva, Georgina

The increasing use of synthetic cannabinoids (SCBs) in recreational settings is becoming a new paradigm of drug abuse. Although SCBs effects mimic those of the Cannabis sativa plant, these drugs are frequently more potent and hazardous. It is known that endocannabinoid signalling plays a crucial role in diverse reproductive events such as placental development. Moreover, the negative impact of the phytocannabinoid Δ9-tetrahydrocannabinol (THC) in pregnancy outcome, leading to prematurity, intrauterine growth restriction and low birth weight is well recognized, which makes women of childbearing age a sensitive group to developmental adverse effects of cannabinoids. Placental trophoblast turnover relies on regulated processes of proliferation and apoptosis for normal placental development. Here, we explored the impact of the SCBs JWH-018, JWH-122 and UR-144 and of the phytocannabinoid THC in BeWo cell line, a human placental cytotrophoblast cell model. All the cannabinoids caused a significant decrease in cell viability without LDH release, though this effect was only detected for the highest concentrations of THC. Moreover, a cell cycle arrest at the G2/M phase was also observed. JWH-018 and JWH-122 increased reactive oxygen species (ROS) production and THC, UR-144 and JWH-122 caused loss of mitochondrial membrane potential. All the compounds were able to induce caspase-9 activation. The involvement of apoptotic pathways was further confirmed through the significant increase in caspase -3/-7 activities. For UR-144, this effect was reversed by the CB1 antagonist AM281, for JWH-018 and THC this effect was mediated by both cannabinoid receptors CB1 and CB2 while for JWH-122 it was cannabinoid receptor-independent. This work demonstrates that THC and SCBs are able to induce apoptotic cell death. Although they may act through different mechanisms and potencies, the studied cannabinoids have the potential to disrupt gestational fundamental events.

2020Journal article

Restricted access