Selenium And N-acetylcysteine Alleviate The Joint Toxicities Of AFB₁ And OTA In Porcine Alveolar Macrophages

Aflatoxin and Ochratoxin are mycotoxins produced by a variety of Aspergillus and Penicillium species that persist as secondary metabolites and continue to contaminate food and animal feeds.Aflatoxin has been identified as a detrimental carcinogen by the International Agency for Research on Cancer.Aflatoxin B1(AFB1)is considered as the first carcinogen and its target organ is the liver.Ochratoxin A(OTA)is a potential carcinogen of human Balkan endemic nephropathy,a potential carcinogen of type 2B,and the target organ of OTA is the kidney.Both AFBi and OTA can cause nephrotoxicity,hepatotoxicity and immunotoxicity,and the co-contamination of AFB1 and OTA has been a worldwide problem.However,there is a few study on the joint toxins and its mechanisms.Selenium is a necessary trace element in various life forms and an essential component of glutathione peroxidase.Selenomethionine(SeMet),as a kind of organic selenium,can promote the absorption efficiency and bioavailability of selenium in the human body,and eliminate oxidizing free radicals and so on.N-acetylcysteine(NAC)is a precursor of intracellular reductive glutathione(GSH),which is a recognized antioxidant.But GSH can not be directly added into the body.As a result,NAC,a precursor of reducing glutathione,is applied to the body to exert an antioxidant effect.Researches have reported that Selenomethionine(SeMet)and NAC can resist apoptosis and oxidative stress.Although there are many reports on selenium and NAC,there is no research on the combined protective effects of selenomethionine and NAC when multiple toxins are exposed to porcine immune cells.In this dissertation,we mainly studied the effects of AFB1 and OTA on the oxidative stress and immunotoxicity of porcine alveolar macrophages and the protective effects of SeMet and NAC and discussed the mechanism of them.Experiment I The combined toxicities and mechanism of AFB1 and OTA on the porcine alveolar macrophagesThe main purpose of this study was to investigate the combined toxic effects of AFB1 and OTA on porcine alveolar macrophages(3D4/21)and its mechanism.Porcine alveolar macrophages 3D4/21 was used as a model in this study.3D4/21 cells were cultured in 96-well plates,12-well plates and 6-well plates at a density of 8×103 cells/well,1×105 cells/well and 2×105 cells/well respectively and incubated at 37℃ in a humidified atmosphere of 5%C02 incubator.The median lethal dose(IC50)of cells co-cultured with AFB1 and OTA for 48 h were determined by MTT assay.The half lethal dose of 10%-50%was determined simultaneously.Laser confocal microscope and flow cytometry were used to observe and determine the apoptosis and ROS level after exposure to toxins.Antioxidant NAC could reverse the combined toxins-induced oxidative stress and the release of pro-inflammatory cytokines.At the same time,macrophage phagocytic index and mRNA expression of TNF-α and IL-6 were determined by neutral red and real-time PCR.Western blotting was used to determine the protein expression of IκBα,p65 and p-p65 in order to study the signaling pathways involved.The results showed that the median lethal doses of AFB1 and OTA were 0.8 μg/mL and 2 μg/mL,respectively.The combination of AFB1 and OTA at 20%concentrations of respective IC50(AFB1 0.16 μg/mL and OTA 0.4 p.g/mL)could significantly reduce cell viability,increase LDH activity and induce cell apoptosis,significantly reduce the phagocytic capacity of 3D4/21 cells,increase the release of TNF-αand IL-6,and induce oxidative stress including increasing ROS levels and decreasing GSH production.The supplement of antioxidant NAC significantly reversed toxins-induced oxidative stress and immunotoxicity.In addition,western blotting and laser confocal microscope showed that the combined of AFB1 and OTA could significantly promote the degradation of IκBα,the phosphorylation of NFκB(p65)and the translocation of NFκB.BAY 11-7082,an inhibitor of NFκB,was able to reverse the above effects at the concentrations that did not significantly toxic to cells.From this it can be concluded that the combination of AFB1 and OTA aggravates the immunotoxicity by activating the NFκB signaling pathway.Experiment II Effects of selenomethionine and N-acetylcysteine on oxidative stress and immunotoxicity induced by aflatoxin B1 and ochratoxin A and its mechanismSelenomethionine(SeMet)and N-acetylcysteine(NAC)have anti-inflammatory and antioxidant activities.Our previous study showed that aflatoxin B1(AFB1)and ochratoxin A(OTA)can induce immunotoxicity and oxidative stress.The purpose of this study was to determine the joint protection and potential mechanisms of SeMet and NAC.In this study,SeMet and NAC significantly attenuated the decrease of cell viability,glutathione and phagocytosis induced by the combination of AFBi and OTA in porcine alveolar macrophages.The addition of SeMet and NAC reduced the up-regulation of lactate dehydrogenase(LDH)activity,apoptosis,caspase3 protein expression level,reactive oxygen species(ROS)and expression of pro-inflammatory cytokines(TLR4,TNF-α and IL-6).The results showed that SeMet and NAC can reduce the oxidative stress and immunotoxicity induced by the combination of AFBi and OTA,and the joint protection effects of low dose of SeMet and NAC was significantly more effective than that of single use.In order to analyze the activation of phosphorylation of different MAPKs after the effective joint protection concentration of SeMet and NAC was determined,the proteins expression of p38,p-p38,ERK1/2,p-ERK1/2,JNK and p-JNK were determined by western blotting.The results showed that the combination of SeMet and NAC significantly reduced the activation of ERK MAPK pathway proteins induced by AFB1 and OTA.The use of activators of ERK1/2,p-38 and JNK demonstrated that the combined protection of SeMet and NAC was achieved through the ERK MAPK signaling pathway,independent of the p-38 and JNK signaling pathways.Taken together,NAC combined with SeMet attenuated AFBi and OTA-induced combined immunotoxicity and oxidative stress in porcine alveolar macrophages and it was associated with ERK MAPK pathway.