Mechanism Of T-2 Toxin And Its Major Metabolites On Combined Toxicity Of Porcine Leydig Cells

T-2 toxin belongs to the Type A trichothecene family produced by various Fusarium fungi,which are prevalent mycotoxins in moldy corn,moldy wheat,cereal grains or related products.Its harmful to human and animals through the food chain.And T-2 toxin is usually contaminated with its metabolites,can also be metabolized in cells,tissues and other organisms.Therefore,at present study we chose porcine Leydig cells(LCs)as the model to investigate the combined toxic effects and mechanisms of T-2 toxin and its main metabolites.Aiming to provide a reference for reproductive risks of co-contamination of type A trichothecenes in food or animal feed ingredients.(1)In order to assess 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 test,toxicological interactions were investigated using Combination index method.The cytotoxic potency of both five tested mycotoxins and their mixtures all showed with a dose-dependent manner.In view of IC₅₀ values of T-2 toxin and its metabolites exposed to porcine Leydig cells,decreasing cytotoxicity was in the order:T-2(0.0209?M)>HT-2(0.0401?M)>T-2 triol(0.5547?M)>NEO(0.2300?M)>T-2 tetraol(1.5199?M).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 T-2+HT-2+NEO ternary combination revealed adverse situation from antagonism to synergism.However,all binary and ternary combinations of T-2 toxin,T-2 triol,and T-2 tetraol showed antagonistic interactions.Our results suggest that co-occurrence of T-2 toxin and its metabolites might pose a more slight threat to reproductive health than the mycotoxin alone due to antagonistic interactions.However,the observed synergy should be taken into account depending on the doses co-occurrence of mycotoxins in the diet.(2)Synergistic means that the toxic effect of the mycotoxin in combination is greater than that of the mycotoxin alone.Therefore,further studies on the mechanism of apoptosis of porcine Leydig cells after exposure to T-2,HT-2,NEO alone and binary or ternary combinations were carried out.The results showed that compared with the control group,the ATP content of all test groups decreased,ROS content increased,and mitochondrial membrane potential(??m)decreased(P<0.05);while the ATP,ROS,??m of binary and ternary combinations were lower or higher(P<0.05 or P>0.05)than the individual mycotoxin in the combination.And at 50%cell viability,the results were not completely different between the individual and combined mycotoxins(P<0.05 or P>0.05),which indicating that T-2,HT-2,NEO alone or in combinations will cause various degrees of damage.Compared with the control group,the cell apoptosis rates of all test groups were significantly increased(P<0.05).Meanwhile,cell apoptosis rates of all combined groups were higher than mycotoxins in the combinations except T-2+NEO group.And at 50%cell viability,the apoptosis rate of combined groups were also higher than individual mycotoxins.The results of RT-q PCR and Western blot examinations of the expression levels of apoptosis-related genes and proteins in cells showed that Bax,Bcl-2,Caspase-3,and CYCs genes and their proteins induced to up/down,which associated with the mitochondrial apoptotic pathway in all test groups.Besides,the Caspase-8 gene and its protein related to the death receptor pathway had been changed too.It can be concluded that the toxicities of T-2,HT-2 and NEO in combinations are greater than themselves.Additionally,they are not solely dependent on the mitochondrial pathway to induce apoptosis,but the death receptor pathway is also involved.(3)The metabolomic study was conducted on the combined toxicity of T-2+HT-2 at50%cell viability and the individual mycotoxin in the combination to further investigate the synergy.Multivariate statistical analysis and univariate statistical analysis were used to screen the differential metabolites in porcine Leydig cells which exposure to T-2,HT-2 and T-2+HT-2.The differential metabolites were clustered based on the heat map and the Wayne chart,then the metabolic pathways were enriched by the bubble chart.Results showed that T-2 alone caused changes in central carbon metabolism in cancer pathway and protein digestion and absorption pathways by affecting amino acid metabolism,TCA cycle,glycolysis/gluconeogenesis mainly to interfere with the cell metabolism.When HT-2 alone,it caused changes in choline metabolism in cancer pathway and central carbon metabolism in cancer pathway by affecting the choline metabolism phosphorylation pathway,amino acid metabolism,TCA cycle,glycolysis/gluconeogenesis mainly to interfere with the cell metabolism.When T-2 combined with HT-2,the central carbon metabolism in cancer pathway and choline metabolism in cancer pathway were affected a lot through choline metabolism phosphorylation pathway,amino acid metabolism,and TCA cycle mainly to interfere with the cell metabolism.Moreover,the metabolic pathways of T-2+HT-2 overlap with the main metabolic pathways of the T-2 and HT-2,separately.It indicating that the two mycotoxins in the combination caused the metabolic toxicity together,showing a synergistic effect.