zearalenone and 4-phenylbutyric-acid

Zearalenone (ZEA) is a common mycotoxin produced by fungi within the genus Fusarium. However, few studies have examined the direct effects of the toxin on the mammary glands. In the present study, the effects of ZEA treatment on bovine mammary epithelial cells (MAC-T) from dairy cows were investigated. The cells were treated with different concentrations of ZEA to evaluate the effect of the toxin on cell viability, intracellular reactive oxygen species (ROS) concentrations, mitochondrial membrane potential, endoplasmic reticulum (ER) stress, and the expression of apoptosis-related genes. The results indicated that different concentrations (5, 10, 15, 20, 25, 30, 50, 60, or 100 μM) of ZEA were able to inhibit growth of MAC-T cells. After exposing the MAC-T cells to 30 μM ZEA, compared with the control group, ROS levels increased, mitochondrial membrane potential decreased, and mRNA expression of the ER-specific stress-related genes GRP78, HSP70, ATF6, EIF2A, ASK1, and CHOP was upregulated in the ZEA-treated group. Further, we analyzed the increase in apoptotic rate by flow cytometry. At the mRNA level, compared with the control group, the expression of the apoptosis-promoting gene BAX was increased in the ZEA-treated group, the expression of the inhibitory gene BCL2 decreased, and the expression of the gene CASP3 increased. We observed a significant increase in caspase-3 activity in ZEA-treated MAC-T cells. Furthermore, the apoptotic rate of the cells in the ZEA group treated with 4-phenylbutyric acid (ER stress inhibitor) decreased and the mRNA expression levels of ER stress markers GRP78 and CHOP decreased. Compared with the ZEA treatment group, the mRNA expression level of the apoptosis-related gene BAX was decreased and the expression level of BCL2 was increased in the ZEA + 4-phenylbutyric acid cotreatment group. These findings indicate that ZEA-induced ER stress increases apoptosis in MAC-T cells. The treatment of MAC-T cells with ZEA reduced cell viability, increased ROS content, decreased mitochondrial membrane potential, increased ER stress marker expression, and induced apoptosis.

Topics: Animals; Apoptosis; Caspase 3; Cattle; Cell Line; Cell Survival; Endoplasmic Reticulum Stress; Epithelial Cells; Female; Flow Cytometry; Gene Expression; Mammary Glands, Animal; Membrane Potential, Mitochondrial; Phenylbutyrates; Reactive Oxygen Species; Real-Time Polymerase Chain Reaction; RNA, Messenger; Zearalenone

Zearalenone (ZEN) is a Fusarium mycotoxin that causes several reproductive disorders and genotoxic effects. This study demonstrated the involvement of endoplasmic reticulum (ER) stress in ZEN-induced mouse Leydig cell death. Our study showed that ZEN reduced cell proliferation in a murine Leydig tumour cell line in a dose-dependent manner. The involvement of apoptosis as a major cause of ZEN-induced cell death was further confirmed by the results of a caspase-3 activity assay, which showed a ZEN dose-dependent increase in cell death. Treatment of MLTC-1 and primary mouse Leydig cells with ZEN upregulated the expression of the ER stress-typical markers GRP78, CHOP and caspase-12 protein. Further, pre-treating the cells with 4-phenylbutyrate or knocking down GRP78 using lentivirus-encoded shRNA significantly diminished ZEN-induced apoptosis and inhibited the expression of CHOP and caspase-12. In summary, these results suggest that the activation of an ER stress pathway plays a key role in ZEN-induced apoptosis in the mouse Leydig cells.

Topics: Animals; Apoptosis; Caspase 3; Cell Line; Cell Proliferation; Endoplasmic Reticulum; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Enzyme Activation; Heat-Shock Proteins; Leydig Cells; Male; Mice; Phenylbutyrates; RNA, Small Interfering; Signal Transduction; Testosterone; Zearalenone

Zearalenone (ZEA) is a fungal mycotoxin that causes cell apoptosis and necrosis. However, little is known about the molecular mechanisms of ZEA toxicity. The objective of this study was to explore the effects of ZEA on the proliferation and apoptosis of RAW 264.7 macrophages and to uncover the signaling pathway underlying the cytotoxicity of ZEA in RAW 264.7 macrophages. This study demonstrates that the endoplasmic reticulum (ER) stress pathway cooperated in ZEA-induced cell death of the RAW 264.7 macrophages. Our results show that ZEA treatment reduced the viability of RAW 264.7 macrophages in a dose- and time-dependent manner as shown by the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay (MTT) and flow cytometry assay. Western blots analysis revealed that ZEA increased the expression of glucose-regulated protein 78 (GRP78) and CCAAT/enhancer binding protein homologous protein (CHOP), two ER stress-related marker genes. Furthermore, treating the cells with the ER stress inhibitors 4-phenylbutyrate (4-PBA) or knocking down CHOP, using lentivirus encoded short hairpin interfering RNAs (shRNAs), significantly diminished the ZEA-induced increases in GRP78 and CHOP, and cell death. In summary, our results suggest that ZEA induces the apoptosis and necrosis of RAW 264.7 macrophages in a dose- and time-dependent manner via the ER stress pathway in which the activation of CHOP plays a critical role.

Topics: Animals; Apoptosis; Cell Line; Cell Proliferation; Cell Survival; Dose-Response Relationship, Drug; Endoplasmic Reticulum Chaperone BiP; Endoplasmic Reticulum Stress; Gene Expression Regulation; Heat-Shock Proteins; Macrophages; Mice; Phenylbutyrates; Transcription Factor CHOP; Zearalenone