RESUMEN
Background: Cannabis is one of the world's most commonly used substances; however, many questions remain unanswered as to how cannabis impacts the body. Recently, there has been a resurgence of research into the effects of plant-derived cannabinoids on mitochondrial health. In particular, a number of studies implicate mitochondrial-Δ9-tetrahydrocannabinol (Δ9-THC) interactions with altered memory, metabolism, and catalepsy in mice. Although the research in this field is expanding rapidly, there is little known about the effects of cannabis on mitochondria health in human subjects either in acute or chronic term use. Methods: Blood samples were obtained from a double-blind, placebo-controlled, parallel-group randomized clinical trial in which adults who regularly use cannabis (1-4 days/week) aged 19-25 years were randomized 2:1 to receive either an active (12.5% Δ9-THC) cigarette or placebo (<0.01% Δ9-THC) cigarette containing 750 mg of cannabis before driving simulator testing. DNA was extracted from whole blood using commercial spin columns, followed by measurement of mt-ND1, mt-ND4, and ß2M using quantitative polymerase chain reaction. One-way repeated measures analysis of variance (ANOVA) followed by Dunnett's multiple comparisons test was used to observe changes in mitochondrial DNA (mtDNA) copy number over time. A two-tailed Pearsons R test was used to assess correlations between mtDNA copy number and cannabinoid levels (Δ9-THC and metabolites) in blood. Results: We found that exposure to active cannabis containing Δ9-THC, as opposed to placebo, was associated with an acute reduction in mitochondrial DNA copy number in whole blood at 15 min and 1 h after smoking. The observed decrease in mtDNA copy number negatively correlated with blood concentrations of 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and 11-Nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH), the two primary metabolites of Δ9-THC, but not Δ9-THC itself. Further, the negative correlation between 11-OH THC and THC-COOH concentrations and mtDNA copy number was found in only a subgroup of participants who use cannabis infrequently, suggesting a tolerance effect. Conclusions: These results illuminate mitochondrial alterations attributed to Δ9-THC consumption, which may be mediated by metabolites. These results appear to suggest stronger effects in individuals who consume cannabis less frequently, suggesting some form of tolerance to the effects of Δ9-THC and its metabolites on mtDNA content in whole blood. Keywords: Mitochondria; mtDNA; cannabis; THC; THC metabolites; blood; THC-COOH; 11-OH-THC.
RESUMEN
BACKGROUND: Mitochondrial dysfunction is involved in several diseases ranging from genetic mitochondrial disorders to chronic metabolic diseases. An emerging approach to potentially treat mitochondrial dysfunction is the transplantation of autologous live mitochondria to promote cell regeneration. We tested the differential filtration-based mitochondrial isolation protocol established by the McCully laboratory for use in cellular models but found whole cell contaminants in the mitochondrial isolate. METHODS: Therefore, we explored alternative types of 5-µm filters (filters A and B) for isolation of mitochondria from multiple cell lines including HEK293 cells and induced pluripotent stem cells (iPSCs). MitoTracker™ staining combined with flow cytometry was used to quantify the concentration of viable mitochondria. A proof-of-principle mitochondrial transplant was performed using mitoDsRed2-tagged mitochondria into a H9-derived cerebral organoid. RESULTS: We found that filter B provided the highest quality mitochondria as compared to the 5-µm filter used in the original protocol. Using this method, mitochondria were also successfully isolated from induced pluripotent stem cells. To test for viability, mitoDsRed2-tagged mitochondria were isolated and transplanted into H9-derived cerebral organoids and observed that mitochondria were engulfed as indicated by immunofluorescent co-localization of TOMM20 and MAP2. CONCLUSIONS: Thus, use of filter B in a differential filtration approach is ideal for isolating pure and viable mitochondria from cells, allowing us to begin evaluating long-term integration and safety of mitochondrial transplant using cellular sources.
