Toxicity of cannabidiol and its metabolites in TM3 mouse Leydig cells: a comparison with primary human Leydig cells.

Cannabidiol (CBD), one of the major components extracted from the plant Cannabis sativa L., has been used as a prescription drug to treat seizures in many countries. CBD-induced male reproductive toxicity has been reported in animal models; however, the underlying mechanisms remain unclear. We previously reported that CBD induced apoptosis in primary human Leydig cells, which constitute the primary steroidogenic cell population in the testicular interstitium. In this study, we investigated the effects of CBD and its metabolites on TM3 mouse Leydig cells. CBD, at concentrations below 30 µM, reduced cell viability, induced G1 cell cycle arrest, and inhibited DNA synthesis. CBD induced apoptosis after exposure to high concentrations (≥ 50 µM) for 24 h or a low concentration (20 µM) for 6 days. 7-Hydroxy-CBD and 7-carboxy-CBD, the main CBD metabolites of CBD, exhibited the similar toxic effects as CBD. In addition, we conducted a time-course mRNA-sequencing analysis in both primary human Leydig cells and TM3 mouse Leydig cells to understand and compare the mechanisms underlying CBD-induced cytotoxicity. mRNA-sequencing analysis of CBD-treated human and mouse Leydig cells over a 5-day time-course indicated similar responses in both cell types. Mitochondria and lysosome dysfunction, oxidative stress, and autophagy were the major enriched pathways in both cell types. Taken together, these findings demonstrate comparable toxic effects and underlying mechanisms in CBD-treated mouse and primary human Leydig cells.

Induction of apoptosis by cannabidiol and its main metabolites in human Leydig cells.

Cannabidiol (CBD) is one of the most prevalent and abundant cannabinoids extracted from the plant Cannabis sativa. CBD has been reported to induce male reproductive toxicity in animal models. In this study, we examined the effects of CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD, on primary human Leydig cells, which play a crucial role in male reproductive health. Our results showed that CBD, at concentrations below the Bayesian benchmark dose (BMD)50, inhibited the growth of human Leydig cells by arresting the cell cycle at G1/S transition, disrupting cell cycle regulators, and decreasing DNA synthesis. Concentration-response transcriptomic profiling identified that apoptosis was one of the top biological processes significantly affected by treatment with CBD for 24 h. The occurrence of apoptosis was confirmed by increased activation of caspase-3/7 and an increased proportion of annexin V and propidium iodide (PI)-positive cells. Similar to CBD, both 7-carboxy-CBD and 7-hydroxy-CBD decreased cell viability and induced apoptosis after treatment for 24 h. 7-Hydroxy-CBD and 7-carboxy-CBD showed lower cytotoxicity than CBD, and 7-carboxy-CBD had the lowest cytotoxicity among the three compounds. Our findings revealed that CBD and its main metabolites can cause adverse effects on primary human Leydig cells.

Review of the oral toxicity of cannabidiol (CBD).

Information in the published literature indicates that consumption of CBD can result in developmental and reproductive toxicity and hepatotoxicity outcomes in animal models. The trend of CBD-induced male reproductive toxicity has been observed in phylogenetically disparate organisms, from invertebrates to non-human primates. CBD has also been shown to inhibit various cytochrome P450 enzymes and certain efflux transporters, resulting in the potential for drug-drug interactions and cellular accumulation of xenobiotics that are normally transported out of the cell. The mechanisms of CBD-mediated toxicity are not fully understood, but they may involve disruption of critical metabolic pathways and liver enzyme functions, receptor-specific binding activity, disruption of testosterone steroidogenesis, inhibition of reuptake and degradation of endocannabinoids, and the triggering of oxidative stress. The toxicological profile of CBD raises safety concerns, especially for long term consumption by the general population.

Cannabidiol-induced transcriptomic changes and cellular senescence in human Sertoli cells.

Cannabidiol (CBD), one of the major cannabinoids in the plant Cannabis sativa L., is the active ingredient in a drug approved for the treatment of seizures associated with certain childhood-onset epileptic disorders. CBD has been shown to induce male reproductive toxicity in multiple animal models. We previously reported that CBD inhibits cellular proliferation in the mouse Sertoli cell line TM4 and in primary human Sertoli cells. In this study, using a transcriptomic approach with mRNA-sequencing analysis, we identified molecular mechanisms underlying CBD-induced cytotoxicity in primary human Sertoli cells. Analysis of differentially expressed genes demonstrated that DNA replication, cell cycle, and DNA repair were the most significantly affected pathways. We confirmed the concentration-dependent changes in the expression of key genes in these pathways using real-time PCR. mRNA sequencing showed upregulation of a group of genes tightly associated with the senescence-associated secretory phenotype (SASP) and with the activation of the p53 signaling pathway, a key upstream event in cellular senescence. Prolonged treatment of 10 µM CBD-induced cellular senescence, as evidenced by the stable cessation of proliferation and the activation of senescence-associated ß-galactosidase (SA-ß-gal), 2 hallmarks of senescence. Additionally, using real-time PCR and Western blotting assays, we observed that CBD treatment increased the expression of p16, an important marker of cellular senescence. Taken together, our results show that CBD exposure disturbs various interrelated signaling pathways and induces cellular senescence in primary human Sertoli cells.

In vitro effects of cannabidiol and its main metabolites in mouse and human Sertoli cells.

Cannabidiol (CBD) is a major cannabinoid present in extracts of the plant Cannabis sativa (marijuana). While the therapeutic effects of CBD on epilepsy have been demonstrated, less is understood regarding its potential adverse effects. Recent studies revealed that CBD induced toxicity in the male reproductive system of animal models. In this study, we used TM4, an immortalized mouse Sertoli cell line, and primary human Sertoli cells to evaluate the toxicities of CBD and its main metabolites, 7-carboxy-CBD and 7-hydroxy-CBD. CBD induced concentration- and time-dependent cytotoxicity in mouse and human Sertoli cells, which mainly resulted from the inhibition of the G1/S-phase cell cycle transition. CBD also inhibited DNA synthesis and downregulated key cell cycle proteins. Moreover, CBD reduced the mRNA and protein levels of a functional marker, Wilms' tumor 1. Similar to CBD, 7-carboxy-CBD and 7-hydroxy-CBD inhibited cellular proliferation and decreased DNA synthesis. 7-Carboxy-CBD was less cytotoxic than CBD, while 7-hydroxy-CBD showed comparable cytotoxicity to CBD in both mouse and human Sertoli cells. Compared to mouse Sertoli cells, CBD, 7-hydroxy-CBD, and 7-carboxy-CBD were more cytotoxic in human Sertoli cells. Our results indicate that CBD and its main metabolites can inhibit cell proliferation in mouse and human Sertoli cells.

Studying NB-LRR immune receptor localization by agroinfiltration transient expression.

NB-LRR immune receptors in plants play dual roles as sentries and as activators of defense. The site in the cell where these activities take place can be different for different NB-LRRs. Furthermore, recognition and defense activation can occur in distinct subcellular compartments. Therefore, determining the subcellular localization of NB-LRRs is a key step toward understanding how they function. Recent advances in confocal microscopy enable high-resolution imaging of proteins in live cells. Agroinfiltration in the Nicotiana benthamiana model plant system is a convenient way of expressing proteins for localization studies. This chapter explains how to use N. benthamiana to transiently express NB-LRRs for confocal fluorescence microscopy.

The leucine-rich repeat domain in plant innate immunity: a wealth of possibilities.

The innate immune system of both plants and animals uses immune receptors to detect pathogens and trigger defence responses. Despite having distinct evolutionary origin, most plant and animal immune receptors have a leucine-rich repeat (LRR) domain. The LRR domain adopts a slender conformation that maximizes surface area and has been shown to be ideal for mediating protein-protein interactions. Although the LRR domain was expected to be a platform for pathogen recognition, the NB-LRR class of plant innate immune receptors uses its LRR domain to carry out many other roles. This review discusses the domain architecture of plant LRRs and the various roles ascribed to this motif.