Study On Toxicity Mechanism Of T-2 And HT-2 Toxin And Detoxification Effects In Broilers

T-2 toxin and HT-2 toxin belong to a trichothecene family and were the secondary metabolites produed by F.Poae.T-2 and HT-2 spread wildly in nature could distribute in tissues,and induced toxicity,oxidative stress and apoptosis on humans and animals.Broilers were very sensitive for T-2 which caused damage at a low dose.T-2 could cause decrease in feed intake,mouth lesion,digestive tract lesion and weight reduction,etc.Consequently,during the breeding process of broilers,diets are frequently supplemented with physical adsorbents to protect birds against the toxicity induced by mycotoxins.T-2 also induce cell viability reduction,high ratio of death cells,and oxidative stress and apoptosis.Recently,there are large number of aritcles about toxicity effect on animals,however,no published literature was investigated for the combinative effects of T-2 and HT-2 on animals in vitro and in vivo.As a result,in the present research,we study the combinative toxicity induced by T-2 and HT-2 on broilers and broiler hepatocytes and protective effects.Firstly,a method was developed for the detection of T-2 and HT-2 in broiler tissues,and then we investigated the toxicity induced by T-2 and HT-2 produced by F.poae-comtaminated maize on broilers.Because liver was the target organ of T-2 toxicity in broilers,the hepatocytes were selected to evaluated effects of cytotoxicity,oxidative and apoptosis caused by T-2/HT-2 in vitro.Finally,we investigated protective effects of DL-Selenomethionine in vitro and montmorillonoid in vivo.A method was developed for the determination of T-2 toxin,HT-2 toxin in heart,liver,spleen,lung,kidney,glandular stomach,muscular stomach,small intestine,muscle,bone and brain samples from broilers using liquid chromatography coupled to tandem mass spectrometry(LC-MS/MS).The samples were initially extracted with ethyl acetate before being filtered through a 0.22?m nylon syringe filter and subjected to chromatographic separation on a reversed-phase C18(50×2.1 mm,3 ?m)column.And the mass spectrometer was operated under positive electrospray ionization conditions(ESI+)in the selected reaction monitoring mode.The limit of detection was in the range of 0.02 to 0.05 ng/g,whereas the limit of quantification was in the range of 0.08 to 0.15 ng/g.The extraction recoveries(RE)ranged from 58.5 to 110.5%,and the relative standard deviation(RSD(%))values were less than 17.0%.The matrix effects(ME)ranged from 33.3 to 124.9%,and the RSD(%)was less than 17.1%.The RSD(%)of inter-and intra-day precision and stability were within 14.7%.we study the toxicity experiment in broilers and boiler hepatocytes.T-2 and HT-2 were produced in maize inoculated with F.poae.The broilers were randomly and equally divided into the following 4 groups:control group,low-dose group(1mg·kg-1 T-2),medium-dose group(2mg·kg-1 T-2)and high-dose group(4mg·kg-1 T-2).After 42 d intake of broilers,the physiological index,biochemical index,pathological change,bone index,T-2/HT-2 residue in tissues and expression of mRNA were analyzed.Compared to the control broilers,the broilers treated with T-2/HT-2 were showed significant decreasing of body weight and weight gain and marked increase of food conversion ratio(FCR)(P<0.05);significant increase in activities of alkaline phosphatase(ALP),alanine aminotransferase(ALT)and aspartate aminotransferase(AST)and significant decrease in total protein(TP)in serum(P<0.05);marked decrease of density,stiffness,Young's modulus and Maximum Load in femur and tibia(P<0.05).Different concentrations of T-2 and HT-2 were distributed in tissues of broilers treated with T-2/HT-2.The expression of mRNA related to oxidative stress and apoptosis were significantly up-regulated in toxins group compared with the control group(P<0.05).And in vitro toxicity experiment,primary broiler hepatocytes were treated with different doses of T-2/HT-2 toxins.Cytotoxicity induced by T-2/HT-2 was evaluated by Thiazolyl Blue Tetrazolium Bromide(MTT)assay Hematoxylin,Eosin(HE)staining and ALT/AST activities in medium.T-2/HT-2 also indueced apoptosis on hepatocytes.Apoptosis of hepatocytes was evaluated with flurescence microscope using Hoechst 33342 fluorescent staining and TUNEL assay.And then we detected apoptosis ratio and age by Annexin V-FITC and PI staining.The relative expressions of mRNA and protein related to apoptosis were determinated by real-time PCR and western-blot.Compared to the control,hepatocytes treated with T-2/HT-2 showed significant decreases in cell viability and marked increases in ALT/AST activites(P<0.05).T-2/HT-2 significantly increased apoptosis cells and apoptosis ratio(P<0.05)in a dose dependent manner.50 nM T-2/HT-2 induced apoptosis on hepatocytes in the early stage,and the mRNA and proteins related to apoptosis played roles in apoptosis process between 0 h and 24 h.Compared to the mRNA expression at 0 h,the relative expressions(Bax,Caspases-3,Caspases-7,Caspases-9 and P53)were significantly up-regulated at 8 to 24 h(P<0.05),however,Bcl-2 mRNA expression showed no significant difference(P>0.05).Expression of all the protein,except for Bcl-2 protein,related to apoptosis were up-regulated significantly from 0 h to 24 h in time dependent manner(P<0.05).The primary hepatocytes were treated with different concentration of T-2/HT-2 and DL-Selenomethionine for 24 h.The protective effects of DL-Selenomethionine against cytotoxicity were investigated by MTT assay and lacate dehydrogenase(LDH)leakage.Next,oxidative stress and protective efficiency of DL-Selenomethionine were evaluated by reactive oxygen species(ROS)levels,malondialdehyde(MDA)and Glutathione(GSH)concentration,the activities of glutathione peroxidase(GSH-PX),Superoxide Dismutase(SOD),Catalase(CAT),and the relative expression of Glutathione S-transferase(GST),GSH-PX,SOD and CAT mRNA.Compared to the control group,hepatocytes treated with T-2/HT-2 showed significant decreases in intracellular GSH concentration,markedly increased LDH leakage,increased activities of ALT,AST,intracellular ROS,GSH-PX,MDA and CAT,and significantly up-regulated expression of mRNA related to oxidative stress in line with exposure levels of the toxins(P<0.05).Compared to the toxins group,DL-Selenomethionine addition into hepatocytes treated with T-2/HT-2 indicated significantly increased cells viabilities and markedly decreased in LDH leakage,and significantly decreased in intracellular ROS and MDA,significantly increased in levels of mRNA expression and activities in GSH,GSH-PX,SOD and CAT(P<0.05).In the protective experiment in vivo,T-2/HT-2 were also produced in contamianted maize.Mont,the strongest adsorbent based on in vitro adsorption ratios,was added to the contaminated diet.The broilers were randomly and equally divided into the following 4 groups:control group,detoxicant-control group,toxins group and detoxicant group.The experiment lasted for 42 days.Compared to the toxins group,the detoxification group showed significant increasing of body weight,weight gain,feed intake,bone density,stiffness,Young's modulus and Maximum Load(P<0.05).Whearas,the detoxification group showed significantly lower levels of FCR,alkaline phosphatase(ALP),AST,ALT,T-2/HT-2 residues in the tissues and the expression of mRNA related to oxidative stress and apoptosis(P<0.05).Finally,for determination of T-2/HT-2 in broiler tissues,a quick,efficiency method was developed,and this method could be successfully applied to the analysis of T-2 toxin and HT-2 toxin in the real samples.T-2/HT-2 produced by F.poae casused toxicity on broilers,distributed in different tissues and affected expression of mRNA related to oxidative stress and apoptosis.T-2/HT-2 induced cytotoxicty and apoptosis in broiler hepatocytes,and mRNA and protein related to apoptosis play roles in apoptosis process.T-2/HT-2 could also induced oxidative stress.DL-Selenomethionine supplementation in medium provided protective effects on hepatocytes against cytotoxicity and oxidative stress caused by T-2/HT-2.Whearas,Mont supplementation in feed decreased toxicity effects casued by T-2/HT-2,residues in tissues and the levels of oxidative stress and apoptosis.Mont is an effective and safety detoxification agent for T-2/HT-2 in feed for broilers.