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Commercial cannabis oil products are widely available in Canada even though there is a significant gap in scientific information regarding them. Oils, such as vegetable oils, are known to undergo oxidative changes through free radical mechanisms when they are heated or aged, but the cannabis oils used in this study did not have expiry dates or best-before usage dates. This led to the question of how these products would be affected with time. We hypothesized that cannabis oils would produce increased concentrations of free radicals in aging-simulated conditions, which would be related to a decrease in cannabidiol (CBD) or Δ 9 -tetrahydrocannabinol (THC) content. Cannabis oils and their respective vehicles (oils) were heated using two protocols: One (moderate aging method) used a 2-day heating protocol at 50 °C, and the other (enhanced aging method) used a 14-day heating protocol at 70 °C. We used electron paramagnetic resonance (EPR) spectroscopy for free radical analysis using the spin trapping technique using 200 mM PBN and 0.02 mM CuCl 2 (for peroxide breakdown to free radicals). For active ingredient analysis (CBD, THC), we used LC/MS. Cannabis oils that contained unsaturated oils as their vehicles, such as olive or sunflower oil, all showed varying degrees of free radical formation. In both aged and unaged oils containing CBD or THC, less free radical formation was detected compared to the vehicle controls. Cannabis oils using medium-chain triglycerides (MCT) showed little or no free radical formation. The most significant decrease in CBD or THC was observed in the products using sunflower oil, to a lesser extent in MCT oil, and THC also decreased in olive oil. These findings are important for consumers and policymakers considering using such products in hot beverages or cooking and highlighting the importance of appropriate storage conditions., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Edaravone is considered to be a potent antioxidant drug known to scavenge free radical species and prevent free radical-induced lipid peroxidation. In this study, we investigated the effect of edaravone on the myeloperoxidase (MPO) activity, an enzyme responsible for the production of an array of neutrophil-derived oxidants that can cause cellular damage. The addition of edaravone to the reaction of MPO and hydrogen peroxide (H 2 O 2 ) significantly enhanced the reduction of MPO Compound II back to native MPO. Interestingly, the MPO-mediated production of toxic hypochlorous acid exhibited a concentration-dependent biphasic effect, with the apparent optimal edaravone concentration at 10 μM. Oxidation of edaravone by MPO was examined by various analytical methods. An MPO-catalyzed product(s) of edaravone was identified at 350 nm by kinetic analysis of UV-Vis spectroscopy. Several MPO-catalyzed metabolites of edaravone were proposed from the LC-MS analyses, including oxidized dimers from edaravone radicals. Electron spin resonance (ESR) spin trapping detected a carbon-centred radical metabolite of edaravone. NMR studies revealed that there are two exchangeable hydrogens, one of which is on the α-carbon, justifying the carbon-centred edaravone radical produced from MPO. Despite the formation of an edaravone carbon-radical metabolite, it did not appear to effectively oxidize GSH (in comparison with phenoxyl radicals). Viability (ATP) and cytotoxicity (LDH release) assays showed a concentration-dependent effect of edaravone on HL-60 cells treated with either a bolus concentration of 30 μM H 2 O 2 or a flux of H 2 O 2 generated by 5 mM glucose and 10 mU/mL glucose oxidase. The H 2 O 2 -induced toxicity was ameliorated at high edaravone concentrations (100-200 μM). In contrast, low concentrations of edaravone (1-10 μM) exacerbated the H 2 O 2 -induced toxicity. However, the effect of edaravone at low concentration (0-10 μM) appeared more prominent with the LDH assay only. The cellular findings correlated with the biochemical studies with respect to hypochlorous acid formation. These findings provide interesting perspectives regarding the duality of edaravone as an antioxidant drug., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Lactoperoxidase (LPO) is believed to serve as a mediator of host defense against invading pathogens. The protein is more abundant in body fluids such as milk, saliva, and tears. Lactoperoxidase is known to mediate the oxidation of halides and (pseudo)halides in the presence of hydrogen peroxide to reactive intermediates presumably involved in pathogen killing. More recently, LPO has been shown to oxidize a wide diversity of thiol compounds to thiyl free radicals, which ultimately lead to the formation of a protein radical characterized by DMPO-immunospin trapping. In the same study by our group the authors claimed that a consequence of this protein radical formation was the inactivation of LPO (Guo et al., J. Biol. Chem.279:13272-13283; 2004). Here we demonstrate that although thiyl radical formation does lead to LPO radical production, the formation of this radical is unrelated to the enzyme's activity. We suggest the source of this misleading interpretation to be the binding of GSH to ELISA plates, which interferes with ABTS and guaiacol oxidation. In addition, DMPO-GSH-nitrone adducts bind to ELISA plates, leading to ambiguities of interpretation since we have demonstrated that DMPO-GSH nitrone does not bind to LPO, and only LPO-protein-DMPO-nitrone adducts can be detected by Western blot.

The establishment of oxidants as mediators of signal transduction has renewed the interest of investigators in oxidant production and metabolism. In particular, H(2)O(2) has been demonstrated to play pivotal roles in mediating cell differentiation, proliferation, and death. Intracellular concentrations of H(2)O(2) are modulated by its rate of production and its rate of decomposition by catalase and peroxidases. In inflammation and infection, some of the H(2)O(2) is converted to hypochlorous acid, a key mediator of the host immune response against pathogens. In vivo HOCl production is mediated by myeloperoxidase, which uses excess H(2)O(2) to oxidize Cl(-). Mashino and Fridovich (Biochim. Biophys. Acta 956:63-69; 1988) observed that a high excess of HOCl over catalase inactivated the enzyme by mechanisms that remain unclear. The potential relevance of this as an alternative mechanism for catalase activity control and its potential impact on H(2)O(2)-mediated signaling and HOCl production compelled us to explore in depth the HOCl-mediated catalase inactivation pathways. Here, we demonstrate that HOCl induces formation of catalase protein radicals and carbonyls, which are temporally correlated with catalase aggregation. Hypochlorite-induced catalase aggregation and free radical formation that paralleled the enzyme loss of function in vitro were also detected in mouse hepatocytes treated with the oxidant. Interestingly, the novel immuno-spin-trapping technique was applied to image radical production in the cells. Indeed, in HOCl-treated hepatocytes, catalase and protein-DMPO nitrone adducts were colocalized in the cells' peroxisomes. In contrast, when hepatocytes from catalase-knockout mice were treated with hypochlorous acid, there was extensive production of free radicals in the plasma membrane. Because free radicals are short-lived species with fundamental roles in biology, the possibility of their detection and localization to cell compartments is expected to open new and stimulating research venues in the interface of chemistry, biology, and medicine.

The rat hepatocyte catalyzed oxidation of 2',7'-dichlorofluorescin to form the fluorescent 2,7'-dichlorofluorescein was used to measure endogenous and xenobiotic-induced reactive oxygen species (ROS) formation by intact isolated rat hepatocytes. Various oxidase substrates and inhibitors were then used to identify the intracellular oxidases responsible. Endogenous ROS formation was markedly increased in catalase-inhibited or GSH-depleted hepatocytes, and was inhibited by ROS scavengers or desferoxamine. Endogenous ROS formation was also inhibited by cytochrome P450 inhibitors, but was not affected by oxypurinol, a xanthine oxidase inhibitor, or phenelzine, a monoamine oxidase inhibitor. Mitochondrial respiratory chain inhibitors or hypoxia, on the other hand, markedly increased ROS formation before cytotoxicity ensued. Furthermore, uncouplers of oxidative phosphorylation inhibited endogenous ROS formation. This suggests endogenous ROS formation can largely be attributed to oxygen reduction by reduced mitochondrial electron transport components and reduced cytochrome P450 isozymes. Addition of monoamine oxidase substrates increased antimycin A-resistant respiration and ROS formation before cytotoxicity ensued. Addition of peroxisomal substrates also increased antimycin A-resistant respiration but they were less effective at inducing ROS formation and were not cytotoxic. However, peroxisomal substrates readily induced ROS formation and were cytotoxic towards catalase-inhibited hepatocytes, which suggests that peroxisomal catalase removes endogenous H(2)O(2) formed in the peroxisomes. Hepatocyte catalyzed dichlorofluorescin oxidation induced by oxidase substrates, e.g., benzylamine, was correlated with the cytotoxicity induced in catalase-inhibited hepatocytes.