Safety evaluation of cannabidiol and industrial hemp extracts

Kanabidiol (ang. cannabidiolCBD) je prevladujoči fitokanabinoid industrijske konoplje (C. sativa). Zaradi številnih dokazanih pozitivnih lastnosti in odsotnosti psihoaktivnih učinkov, se je zanimanje za uporabo CBD v zadnjem času močno povečalo. Leta 2018 je bilo s strani regulatornih agencij odobreno prvo zdravilo na osnovi CBD, hkrati pa se je na trgu povečala tudi ponudba CBD izdelkov, registriranih kot prehranska dopolnila. Zaradi slabe regulacije ti izdelki vsebujejo različne vrste in količine kanabinoidov, kar lahko predstavlja potencialno tveganje za uporabnike. Obenem dosedanje raziskave toksičnosti CBD poročajo o nasprotujočih si ugotovitvah, zato varnost CBD še vedno ni povsem opredeljena. V magistrski nalogi smo preučevali vpliv dveh izdelkov s CBD na celice celične linije HepG2, ki smo jo uporabili kot in vitro model človeških hepatocit. Celice smo za 4 in 24 ur izpostavili CBD izolatu (CBD CP, z vsebnostjo CBD 99,6 %) in izvlečku konoplje (CBD EX, z vsebnostjo CBD 63,6 %). Izvedli smo test MTT za ugotavljanje citotoksičnosti ter test komet in test mikrojeder (CBMN) za ugotavljaje genotoksičnosti. Rezultati testa MTT so pokazali, da sta oba izdelka na celice delovala citotoksično, pri čemer je IC50 po 4-urni izpostavitvi, znašal 26.91 ± 2,27 μg/mL (CBD CP) in 26.20 ± 5,26 μg/mL (CBD EX), po 24-urni izpostavitvi pa 6.71 ± 0,59 μg/mL (CBD CP) in 7.90 ± 1,59 μg/mL (CBD EX). Koncentracije, ki so se pri testu MTT izkazale za ne-citotokisične, smo nato uporabili za preverjanje genotoksičnosti. CBD CP in CBD EX, pri ne-citotoksičnih koncentracijah, pri pogojih testiranja, nista povzročala DNK poškodb oziroma kromosomskih nepravilnosti, kot so mikrojedra (MNi), jedrni brsti (NBUDs) in nukleoplazmatski mostički (NPBs). V in vitro pogojih na HepG2 celično linijo tako nobeden od proučevanih CBD izdelkov ni deloval genotoksično.Cannabidiol (CBD) is the predominant phytocannabinoid found in industrial hemp (C. sativa). Recently, there has been a growing interest in CBD due to its numerous presumed benefits and the absence of psychoactive side effects. In 2018, regulatory agencies approved the first CBD-based medication for the treatment of severe seizure disorders. Furthermore, the market for CBD products, promoted as dietary supplements has witnessed significant growth in recent years. However, these products pose a potential risk to consumers as they are inadequately regulated and may contain a wide range of phytocannabinoids with varying concentrations. Moreover, current research on the toxicity of CBD has yielded conflicting findings, making it challenging to establish the safety of CBD products. The aim of this master study was to investigate the toxicity of two CBD products on the HepG2 cell line, which we used as an in vitro model for human hepatocytes. The cells were exposed to graded concentrations of CBD, (CBD CP, containing 99.6 % CBD) and hemp extract (CBD EX, containing 63.6 % CBD) for 4 and 24 hours. Subsequently, we employed the MTT test, comet assay, and micronucleus test (CBMN) to evaluate potential cytotoxic and genotoxic effects caused by these CBD samples. Our findings indicated that both samples exhibited cytotoxicity, with calculated IC50 values of 26.91 ± 2.27 μg/mL (CBD CP) and 26.20 ± 5.26 μg/mL (CBD EX) after 4 hours of exposure. After a 24-hour exposure, the IC50 values were 6.71 ± 0.59 μg/mL (CBD CP) and 7.90 ± 1.59 μg/mL (CBD EX). The MTT test enabled us to identify non-cytotoxic concentrations for further genotoxicity testing. The results of the comet assay and the CBMN test did not show an increase in DNA damage or chromosomal changes, such as micronuclei (MNi), nuclear buds (NBUDs), or nucleoplasmic bridges (NPBs). Therefore, we can conclude that none of the CBD products tested induced genotoxic effects in the HepG2 cell line after CBD exposure