Zearalenone Induces Apoptosis And Cytoprotective Autophagy In Primary Leydig Cells

Zearalenone (ZEA) is a nonsteroidal estrogenic mycotoxin found in several food commodities worldwide. ZEA causes reproductive disorders, genotoxicity, and autoimmune toxicity in animals. However, little is known about the functions of apoptosis and autophagy after exposure to ZEA in Leydig cells. The apoptotic and autophagy induction effects of ZEA on primary Leydig cell models were evaluated. From the experiments, the following results were obtained:1. ZEA-induced apoptosis in primary rat Leydig cells. Leydig cells were treated with0μg/mL to20μg/mL of ZEA for12h. Cell viability was measured by the MTT assay. The morphological features of Leydig cells after treatment with20μg/mL ZEA for12h were visualized by Hoechst33258stainin and TEM analysis. Leydig cells were stained with Annexin V and propidium iodide, and the apoptotic rates were determined by flow cytometry. This results shown that, The cell viability significantly decreased when rat Leydig. cells were treated with different concentrations of ZEA (0μg/mL to20μg/mL) for12h (p<0.01). Leydig cells were treated with20μg/mL of ZEA for12h, followed by Hochest33258staining. The control cells did not show significant changes in their cell nuclei, whereas the ZEA-treated cells demonstrated chromatin condensation and nuclear fragmentation. The TEM images showed that the cells treated with ZEA demonstrated morphological changes during apoptosis, including the condensation of chromatin and the formation of membrane-embedded apoptotic bodies. ZEA-induced apoptosis was confirmed by flow cytometry using Annexin V/PI dual staining, the apoptotic rate significantly increased versus the control group (p<0.05). ZEA induced apoptosis in rat Leydig cells in a dose-dependent manner. This results suggest that, ZEA inhibit Leydig cells growth through apoptotic.2. ZEA-induced apoptosis signaling pathways in primary rat Leydig cells. Leydig cells were treated with0μg/mL to20μg/mL of ZEA for12h. Cell were stained with JC-1, and the mitochondrial membrane potential (MMP) were determined by flow cytometry. ZEA activates caspase-9and caspase-3, cleaves PARP, regulates Bcl-2family members and cytochrome c to induce apoptosis by western blot in rat Leydig cells. This results shown that, The MMP significantly decreased when rat Leydig cells were treated with different concentrations of ZEA (0μg/mL to20μg/mL) for12h (p<0.05) and MMP levels gradually decreased in a dose-dependent manner. Western blot analysis showed that the protein levels of activated caspase-9and caspase-3as well as cleaved PARP significantly increased in a dose-dependent manner. The results of western blot showed that Bax protein levels increased in the Leydig cells treated with ZEA for12h and that Bcl-2protein levels gradually decreased in a dose-dependent manner. Thus, the ratio of Bax/Bcl-2increased. Western blot analysis showed that the level of mitochondrial cytochrome c decreased in a dose-dependent manner and that the level of cytosolic cytochrome c concomitantly increased. This results suggest that ZEA treatment activated the mitochondria pathway of apoptosis by Bcl-2family proteins and eventually caused apoptosis.3. ZEA triggers autophagy in primary rat Leydig cells. The cells were treated with different concentrations of ZEA (2.5,5, and10μg/mL) for12h and then stained with AO. The fluorescent signal of the cells was measured with flow cytometer and Immunofluorescence. The levels of LC3and Beclin-1proteins were observed by western blot. This results shown that, The data showed that the volume percentage of cellular acids significantly increased after the cells were treated with ZEA for12h (p<0.05). Western blot analysis showed that the levels of LC3-II and Beclin-1proteins significantly increased after ZEA treatment (p<0.05). The specific punctate LC3-II proteins accumulated in the ZEA-treated groups (5μg/mL). In addition, more dots appeared in the ZEA-treated cells than in the control cells (p<0.05). The ZEA-treated Leydig cells exhibited several autophagosomes that were absent in the control cells by TEM. In general, these results indicate that ZEA induces autophagy in Leydig cells.4. Autophagy delays apoptosis in ZEA-treated rat Leydig cells. The specific autophagic inhibitor chloroquine (CQ) and inducer rapamycin (RAP) were introduced to the ZEA-treated rat Leydig cells to elucidate the relationship between apoptosis and autophagy in ZEA-induced toxicity. Western blot analysis was performed using LC-3antibody. Leydig cells were stained with Annexin V and propidium iodide, and the percentages of apoptotic cells were determined by flow cytometry. This results shown that, the levels of LC3-II proteins significantly increased after ZEA cotreated with RAP (p<0.05). Similar results were observed in ZEA cotreated with CQ. The apoptotic rate significantly increased after the cotreatment of5μg/mL ZEA and CQ compared with that after the ZEA treatment alone (5μg/mL)(p<0.01). By contrast, the apoptotic rate decreased after the cotreatment of20μg/mL ZEA and RAP (p<0.01). This result suggests that autophagy hinders apoptosis in ZEA-treated Leydig cells.Taken together, this study illustrated that ZEA induced rat Leydig cell apoptosis through activation of mitochondrial pathway. ZEA could also increase the level of autophagy in Leydig cells. Low and high doses of ZEA can activate autophagy and apoptosis in Leydig cells, respectively. The results proved that autophagy can delay apoptosis in Leydig cells. Therefore, autophagy serves an important protective function against avoiding cytotoxicity.