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.

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.