Cytotoxicity and Metabolic Study of New Psychoactive Substances
- Publication Type:
- Thesis
- Issue Date:
- 2021
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The unprecedented growth of new psychoactive substances (NPS) render identification a challenging issue to both the forensic and clinical laboratories. NPS readily available in myriad of unknown formulation, posing serious threat and acute harm for the users. At present, there is paucity of information on the potential potency, toxicity mechanisms, and toxicokinetic parameters associated with the use of these drugs. The present study aimed to investigate the neurotoxicity potency and cellular mechanism of NPS. Hepatoxicity potency potential, metabolic stability and subsequent metabolism pathway of specific NPS is also explored for better understanding of the toxicokinetics of these NPS.
The neurotoxicity potential and mechanism of synthetic cathinones (SCs) butylone, pentylone and 3,4-methylenedioxypyrovalerone (MDPV) was investigated using differentiated SH-SY5Y cell line. Viability assays and end-point measurements that include markers of oxidative stress, mitochondrial bioenergetics, intracellular calcium (Ca²⁺) and cell death pathways were employed. All the three SCs displayed dose-dependent neurotoxicity with the following order of potency: butylone (least cytotoxic) < pentylone < MDPV (most cytotoxic). The activation of apoptotic cell death pathway implicated the orchestration of mitochondrial-mediated neurotoxicity mechanisms via oxidative stress, compromised bioenergetics balance and changes in Ca²⁺ homeostasis (𝘱 < 0.0001 vs. control).
The metabolism of synthetic cannabinoid (SCB), 4F-MDMB-BINACA was investigated using 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 models: HepG2 liver cells, fungus 𝘊𝘶𝘯𝘯𝘪𝘯𝘨𝘩𝘢𝘮𝘦𝘭𝘭𝘢 𝘦𝘭𝘦𝘨𝘢𝘯𝘴 (𝘊. 𝘦𝘭𝘦𝘨𝘢𝘯𝘴) and pooled human liver microsomes (HLM). Tentative structure elucidation of the 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 metabolites was performed using high-resolution mass spectrometry whilst twenty authentic human urine samples were retrospectively analysed using liquid chromatography-orbitrap mass spectrometry. A total of twenty-five 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 metabolites and eight 𝘪𝘯 𝘷𝘪𝘷𝘰 metabolites were tentatively identified. Ester hydrolysis and ester hydrolysis dehydrogenation 4F-MDMB-BINACA metabolites were recommended as urinary markers for 4F-MDMB-BINACA intake. 𝘊. 𝘦𝘭𝘦𝘨𝘢𝘯𝘴 has the potential to be used as a complementary model to predict and characterise human metabolites, as well as identifying possible drug toxicities for emerging SCBs.
The metabolic stability and hepatotoxicity potential of butylone, pentylone, MDPV and 4F-MDMB-BINACA were studied using HLM and HepG2 liver cells, respectively. Drug-treated HepG2 exhibited the following cytotoxicity potency: butylone (least cytotoxic) < pentylone < MDPV < 4F-MDMB-BINACA (most cytotoxic). For the metabolic stability study, NPS incubated in HLM were collected at various time points and subsequently analysed by liquid-chromatography tandem mass spectrometry. Calculated 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 half-lives together with estimated intrinsic clearance values categorised butylone, pentylone and MPDV as low clearance drugs and 4F-MDMB-BINACA as high clearance drug.
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