Phytochemicals are often promoted generally as antioxidants and demonstrate variable levels of reactive oxygen species (ROS) sequestration in vitro, which attributes to their neuroprotective bioactivity. Sesquiterpenes from cannabis and essential oils may demonstrate bifunctional properties towards cellular oxidative stress, possessing pro-oxidant activities by generating ROS or scavenging ROS directly. Sesquiterpenes can also oxidize forming sesquiterpene oxides, however the relative contribution they make to the bioactivity or cytotoxicity of complex botanical extracts more generally is unclear, while selected cannabis-prevalent terpenes such as beta-caryophyllene may also activate cannabinoid receptors as part of their biological activity. In the present study, we investigated selected sesquiterpenes beta-caryophyllene and humulene and their oxidized forms (beta-caryophyllene oxide and zerumbone, respectively) against established antioxidants (ascorbic acid, alpha-tocopherol, and glutathione) and in the presence of cannabinoid receptor 1 and cannabinoid receptor 2 antagonists, to gain a better understanding of the molecular and cellular mechanisms of neuroprotection versus neurotoxicity in semi-differentiated rat neuronal phaeochromocytoma (PC12) cells. Our results demonstrate that the sesquiterpenes beta-caryophyllene, humulene and zerumbone possess concentration-dependent neurotoxic effects in PC12 cells. Both beta-caryophyllene-and humulene-evoked toxicity was unaffected by CB1 or CB2 receptor antagonism, demonstrating this occurred independently of cannabinoid receptors. Both glutathione and alpha-tocopherol were variably able to alleviate the concentration-dependent loss of PC12 cell viability from exposure to beta-caryophyllene, humulene and zerumbone. During 4-hour exposure to sesquiterpenes only modest increases in ROS levels were noted in PC12 cells, with glutathione co-incubation significantly inhibiting intracellular ROS production. However, significant increases in ROS levels in PC12 cells were demonstrated during 24-hour incubation with either antioxidants or sesquiterpenes individually, and with additive toxicity exhibited in combination. Overall, the results highlight a concentration-dependent profile of sesquiterpene neurotoxicity independent of cannabinoid receptors and dissociated from the formation of reactive oxygen species as a marker or correlate to the loss of cell viability.
