Potential Role of Individual and Combined Effects of T-2 Toxin, HT-2 Toxin and Neosolaniol on the Apoptosis of Porcine Leydig Cells.

T-2 toxin usually co-occurs with HT-2 toxin and neosolaniol (NEO) in the grains and feed. Our previous studies found that T-2 toxin and its metabolites' binary or ternary combination exposure to porcine Leydig cells (LCs) displayed synergism in certain range of dosage and cannot be predicted based on individual toxicity. However, the possible mechanism of these mycotoxins' combined exposure to cell lesions remains unknown. Based on 50% cell viability, the mechanism of apoptosis in porcine Leydig cells was investigated after exposure to T-2, HT-2, NEO individual and binary or ternary combinations. Compared with control, the adenosine triphosphate (ATP) content decreased, reactive oxygen species (ROS) level increased, and mitochondrial membrane potential (MMP) decreased in all treated groups. Additionally, the cell apoptosis rates were significantly increased in test groups (p < 0.05), and the B-cell lymphoma 2 (Bcl-2) Associated X (Bax)/Bcl-2 ratio and the expression of caspase 3, caspase 8, cytochrome c (Cytc) in the treated group are all significantly higher than the control group. Moreover, the expression of Cytc and caspase 8 gene in NEO and T-2+NEO groups was significantly higher than that in other individual and combined groups. It can be concluded that the toxicities of T-2, HT-2, and NEO individually and in combination can induce apoptosis related to the oxidative stress and mitochondrial damage, and the synergistic effect between toxins may be greater than a single toxin effect, which is beneficial for assessing the possible risk of the co-occurrences in foodstuffs to human and animal health.

Genistein affects gonadotrophin-releasing hormone secretion in GT1-7 cells via modulating kisspeptin receptor and key regulators.

Epidemiological studies have shown that genistein, an isoflavonoid phytoestrogen from soybean, affects endocrine and reproductive systems and alters pubertal onset. Administration of genistein in mice could impact the electrophysiology of hypothalamic neurons associated with the secretion of gonadotropin-releasing hormone (GnRH), a key component of hypothalamic-pituitary-gonadal (HPG) axis that governs hormone release and reproductive maturation. However, whether genistein could directly influence GnRH secretion in GnRH-specific neurons requires further investigation. Here, mouse hypothalamic GT1-7 neurons were recruited as a GnRH-expressing model to directly evaluate the effect and mechanisms of genistein on GnRH release. Results from this study demonstrated that genistein treatment decreased cell viability, impacted cell cycle distribution, and induced apoptosis of GT1-7 cells. A high concentration of genistein (20 µM) significantly increased GnRH secretion by 122.4% compared to the control. Since GnRH release is regulated by components of the kisspeptin-neurokinin-dynorphin (KNDy) system and regulators including SIRT1, PKCγ, and MKRN3, their transcription and translation were examined. Significant increases were observed for the mRNA and protein levels of the KNDy component kisspeptin receptor (Gpr54/Kissr). Compared to the control, genistein treatment upregulated the level of Sirt1 mRNA level, while it downregulated Prkcg and Mkrn3 expression. Therefore, this study provided direct evidence that genistein treatment could affect GnRH secretion by modulating kisspeptin receptors, SIRT1, PKCγ and MKRN3 in GT1-7 cells.Abbreviations: GnRH: gonadotropin-releasing hormone; HPG: hypothalamic-pituitary-gonadal; KNDy: kisspeptin-neurokinin-dynorphin; LH: luteinizing hormone; FSH: follicle-stimulating hormone; ARC: arcuate nucleus; ER: estrogen receptor; SIRT1: silent information regulator 1; PKCγ: protein kinase c γ: MKRN3: makorin ring finger protein 3; LC: lethal concentration; PI: propidium iodide; ECL: chemiluminescence; BCA: bicinchoninic acid assay; PBS: phosphate-buffered saline; CT: fluorescence reached threshold; PVDF: polyvinylidene difluoride.

Individual and combined cytotoxic effects of T-2 toxin and its four metabolites on porcine Leydig cells.

T-2 toxin, one of the most toxic mycotoxins, is commonly presented along with its metabolites, HT-2 toxin, neosolaniol (NEO), T-2 triol, and T-2 tetraol in foodstuff and feed. The aim of this study was to evaluate the cytotoxic effects of T-2 toxin alone and in combination with its metabolites on porcine Leydig cells. Based on the determination of cell viability with CCK-8, toxicological interactions were investigated using Combination Index method. The cytotoxic potency of five tested mycotoxins individual and their mixtures all showed with a dose-dependent manner. In view of IC50 values, the decreasing cytotoxicity of mycotoxins was ranking: T-2 toxin > HT-2 toxin > T-2 triol > NEO > T-2 tetraol. Combinations of T-2+HT-2, T-2+NEO, and HT-2+NEO displayed synergism at low doses but antagonism at high doses, while the ternary combination of T-2+HT-2+NEO revealed adverse situation from antagonism to synergism. All binary and ternary combinations of T-2 toxin, T-2 triol, and T-2 tetraol exhibited antagonistic interactions. Our results suggest that the co-occurrence of T-2 toxin and its metabolites might pose a slight threat to reproductive health due to antagonistic interactions. However, the synergy observed should be not ignored especially at low doses of mycotoxins co-occurrence in the diet.

Toxic Effects and Possible Mechanisms of Deoxynivalenol Exposure on Sperm and Testicular Damage in BALB/c Mice.

Deoxynivalenol (DON, vomitoxin) is the most common mycotoxin in cereals and grains. DON contamination can cause a serious health threat to humans and farm animals. DON has been reported to exert significant toxicity effects on the male reproductive system. However, the causes and mechanisms underlying efforts of DON on sperm and testicular damage remain largely unclear. In the present study, we thoroughly investigated this issue. Eighty male BALB/c mice were randomly divided into a control group ( n = 40) and DON treatment group (2.4 mg/kg of body weight, n = 40). The ratio of testes and seminal vesicle to body, sperm survival and motility, and morphology of sperm and testis were observed in DON-treated and control mice. In addition, the concentrations of reactive oxygen species (ROS) and malondialdehyde (MDA), the activities of superoxide dismutase (SOD) and glutathione (GSH), and also the expression levels of JNK/c-Jun signaling and apoptotic factors such as caspase-3, caspase-8, caspase-9, Bim, and Bid were analyzed and compared between the two groups. The results demonstrated that a single topical application of DON significantly increased the percentage of abnormal sperm and decreased the motility of sperm, indicating the sperms are damaged by DON. Additionally, the reduced relative body weight of testis and severe destruction of testicular morphology were observed. Moreover, the increased levels of ROS and MDA levels and decreased activities of SOD and GSH were found in testicular tissues, suggesting that oxidative stress is induced by DON treatment. Furthermore, DON upregulated the expression of stress-induced JNK/c-Jun signaling pathway proteins as well as JNK/c-Jun phosphorylation proteins. In addition, DON could enhance testicular apoptosis by increasing expression levels of apoptotic genes including Bim, cytochrome c, caspase 3, caspase 8, and caspase 9. These results suggest that DON exposure can cause sperm damage, oxidative stress, testicular apoptosis, and phosphorylation of JNK/c-Jun signaling pathway. The underlying mechanisms may be that DON induces sperm damage by exacerbating oxidative stress-mediated testicular apoptosis via JNK/c-Jun signaling pathway.

[Simultaneous determination of six aflatoxins and sterigmatocystin in livestock and poultry tissues by QuEChERS-ultra high performance liquid chromatography-tandem mass spectrometry].

A method for the simultaneous determination of six aflatoxins (AFTs) and sterigmatocystin (SMC) in livestock and poultry tissues by QuEChERS-ultra high performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed and validated. The samples were extracted with 10 mL acetonitrile-water (84:16, v/v) after enzymatic hydrolysis with 20 µL ß-glucosidase. The extracts were cleaned with 1.0 g each of sodium chloride and anhydrous magnesium sulfate, and then degreased with 5 mL hexane. The residues were re-dissolved in 1 mL acetonitrile-water (80:20, v/v) containing 5 mmol/L ammonium acetate. Methanol and 5 mmol/L ammonium acetate were used as the mobile phases. Six AFTs and SMC were analyzed by multiple reaction monitoring in the positive ion mode. Matrix-matched calibration curves and external standards were used for the accurate quantification of the six AFTs and SMC. Good linear relationships were obtained, the correlation coefficients (R2) were greater than 0.99. The limits of detection (LODs, S/N=3) and the limits of quantification (LOQs, S/N=10) ranged from 0.007 to 0.30 µg/kg, and 0.02 to 0.91 µg/kg, respectively. The recoveries at different spiked levels were satisfactory, and ranged from 77.3% to 118.5%. This method is simple, easy and sensitive, and is suitable for the rapid determination of the six AFTs and SMC in different pork, pork liver and chicken samples.