Cellular effects of hemp and its cannabinoids \(in\) \(vitro\)

Natural non-psychoactive cannabinoids, including cannabigerol (CBG), cannabidiol (CBD), cannabichromene (CBC), cannabidivarin (CBDV), and cannabinol (CBN), are gaining popularity as dietary products due to their purported health benefits. These cannabinoids are believed to alleviate specific types of pain, reduce nausea, and ease anxiety while also displaying potential anti-inflammatory and even anti-carcinogenic properties. However, the lack of comprehensive data on their potential toxicity raises important questions about their safety. To address these concerns, we tested CBG, CBD, CBC, CBDV, and CBN for their genotoxic and cytotoxic potential in human lymphoblastoid TK6 cells. Additionally, we wanted to investigate their possible genotoxicity mechanism. Furthermore, as cannabinoid dietary products often contain several types of cannabinoids, we wanted to elucidate if their genotoxic activity changes when present as a mixture or within the hemp matrix. The findings revealed that all tested cannabinoids, except for CBDV, led to an increase in micronuclei formation, though this effect was diminished with the addition of a metabolic activation system. Notably, CBDV-induced micronuclei only occurred after metabolic activation. The combination of cannabinoids did not increase micronuclei formation additively, but the investigation was limited by increased toxicity. Cannabinoids incorporated in the hemp matrix caused increased micronuclei formation following metabolic activation. The mechanism of cannabinoid genotoxicity was examined using several assays, including anti-centromere staining, evaluation of mitotic disturbances, visualization of cytoskeletal structures and chromosomes, and analysis of the cell cycle. The results of these assays indicated that all cannabinoids caused predominantly aneugenic effects, mitotic disturbances, and disrupted microtubules and centrosomes, while an accumulation of cells in the G1 phase was observed only for CBG, CBD, and CBDV. The most probable conclusion based on these results is that their genotoxic effects are mediated by disruption of the mitotic mechanism, which leads to disturbed mitotic cells that go through abnormal segregation of chromosomes, resulting in micronuclei formation. The cytotoxic potential of cannabinoids was observed as a decrease in the proliferation index and cell growth of TK6 cells. Oxidative stress induction, which may occur in the context of cytotoxicity, was not detected in cannabinoid-treated cells, apart from CBDV-treated cells, which exhibited elevated levels of reactive oxygen species (ROS). Additionally, changes in mitochondrial membrane potential served as another indicator of cytotoxicity. Only CBD, CBDV, and CBN caused a decline in mitochondrial membrane potential. These results suggest a possible link between cannabinoid-induced mitochondrial damage and reduced cell proliferation, i.e. cytotoxicity. Finally, it was found that one cannabinoid led to the formation of unexpected cellular structures. Specifically, CBN caused the development of vacuoles within the cells. The presence of these vacuoles indicated that CBN may induce a particular type of cell death known as paraptosis. Several characteristics associated with paraptosis were observed in the CBN-treated cells, including cytoplasmic vacuolization, mitochondrial calcium overload, reduced mitochondrial membrane potential, and changes to the cytoskeleton. The observed genotoxic effects in this study were reached with micromolar concentrations of cannabinoids (1000 times higher than cannabinoid concentrations found in consumers’ blood), which was diminished with the addition of metabolic enzymes (with the exception of CBDV). This indicates that oral intake of cannabinoids may not pose a genotoxic risk for consumers. However, certain oil preparations of cannabinoids are meant to be held in the mouth, which could lead to absorption through the oral mucosa and systemic distribution without liver metabolism. Furthermore, as a suspected mechanism of cannabinoid genotoxicity is through the disruption of the mitotic machinery, it suggests that there may be a dose range below which there are no adverse effects, implying the possibility of safe ingestion of low amounts of cannabinoids. Overall, this study offers insights into the genotoxicity and cytotoxicity of selected cannabinoids. While the mechanism underlying cannabinoid genotoxicity is partially understood, there are still gaps in the data. It remains unclear whether cannabinoids interact with microtubules and centrosomes directly or indirectly and whether they disrupt tubulin synthesis or structure. Additionally, there are limitations in our understanding of cannabinoid cytotoxicity; although all cannabinoids influence cell proliferation, they do not uniformly affect ROS production or mitochondrial function. Lastly, it is suggested here that CBN may induce paraptosis based on observed characteristics, but further research is necessary to confirm this speculation.