Combined Effects Of Predominant Mycotoxins In Feeds On Mice

The aim of this study was to investigate the combined effects of aflatoxin B1(AFB1), zearalenone (ZEA), deoxynivalenol (DON) and fumonisin B1(FB1) on the porcine kidney cell, rat liver cell and Balb/C mice.(1) The combined effects of the four predominant mycotoxins on procine kidney cell (PK15)PK15were incubated with different concentrations of AFB1, ZEA, DON and FB1for12,24and48h, using MTT assay and LDH release assay to evaluate the cytotoxicity of the four mycotoxins. Exposure with mycotoxin for48h, the EC50of AFB1, ZEA and DON was38.8,121.8and3.6μM, and the EC30was26.9,86.6and1.8μM, which were calculated from dose-effect curves. The respones of AFB1, ZEA and DON were in a concentration-and time depended manner, the cytotoxicity was elevated with addition of dose and exposure time. Incubated with250μM FB1, cell viability was above70%, the EC50could not be obtained. With regard to ROS production and cell apoptosis, AFB1and DON significantly induced them in a concentration-dependent manner, but ZEA (10,20,40μM) had no effect on cell apoptosis and ROS production. The cytotoxicity of the four mycotoxins in increasing order:FB1<ZEA<AFB1<DON.EC30of individual mycotoxins as the central point (FB1concentration was set to15μM), binary and ternary mixed was designed. Fitting equation was confirmed by stepwise regression based on the dose and effect, which showed synergetic effect after co-exposure of AFB1+ZEA, or AFB1+DON, or ZEA+DON; Co-exposure of AFB1and FB1, the equation just showed the cytotoxicity of AFB1. However, AFB1and ZEA showed antagonism in the ternary mixture.ZEA could alleviate low concentrations of AFB1(1μM)-induced membrane damage and oxidative damage, but ZEA with the high concentration of AFB1synergistically exacerbate membrane damage and oxidative damage. ZEA weakened AFB1-induced apoptosis, mainly in the early apoptotic; related to slow down the process of apoptosis by bcl-2, caspase-3gene and p53protein expression.(2) The combined effects of the four common mycotoxins on BRLBRL were incubated with different concentrations of AFB1, ZEA, DON and FB1for12,24and48h, using MTT assay and LDH release assay to evaluate the cytotoxicity of the four mycotoxins. Exposure with mycotoxin for48h, the EC50of AFB1, ZEA and DON was37.9,100.3and36.5μM, and the EC30was29.2,91.6and3.27μM, which were calculated from dose-effect curves. The respones of AFB1, ZEA and DON were in a concentration-and time depended manner. Incubated with FB1, cell viability was not affected. With regard to ROS production and cell apoptosis, AFB1and DON significantly induced them (P<0.05), but ZEA (10,20,40μM) had no effect on cell apoptosis and ROS production. The cytotoxicity of the four mycotoxins in increasing order:FB1<ZEA<AFB,<DON.EC30of individual mycotoxins as the central point (FB1concentration was set to15μM), binary and ternary mixed was designed. Fitting equation was confirmed by stepwise regression based on the dose and effect, which showed synergetic effect after co-exposure of AFB1+ZEA, or AFB1+DON; AFB1and DON showed synergism in the ternary mixture. Co-exposure of AFB1and FB1; the equation just showed cytotoxicity of AFB1.AFB1and DON displayed additive or synergistic effects on the cell membrane damage and oxidative stress. Combination of1μM AFB1and2μM DON synergistically reduced bcl-2gene expression levels, and promote box, caspase-3, and p53expression and accelerate the process of apoptosis.(3) The combined effects of the four common mycotoxins on Balb/C miceOne hundred and four4-week-old female Balb/C mice adapted for one week, then were randomly divided into13groups (n=8). Trials were grouped as follows:distilled water group, DMSO group, ethanol group, AFB1(2.5mg/kg.bw), ZEA (5mg/kg.bw), DON (5mg/kg.bw), FB1(8mg/kg.bw), AFB1+ZEA, AFB1+DON, AFB1+FB1, ZEA+DON, ZEA+FB1, and DON+FB1. The dose of combined groups superimposed on the single dose. The mice were administered orally by gavage once daily for14d.There was no difference on the body weight of mice in all groups (P<0.05). FB1significantly increased weight of spleen; the combined group containing FB1could weaken the spleen weight gain caused by FB1. AFB1and DON synergistically increased serum ALT activity and MD A content in the liver, collaborative reduced SOD activity and strengthened liver cell damage and oxidative damage; AFB1and DON could promote apoptosis of liver by synergistic improving P53and caspase-3expression. ZEA and DON not only synergistically increased serum estradiol levels, but also synergistically improved caspase-3gene expression promotes apoptosis. Compared to individual treatment, the combined treatments further enhanced the expression of HSP70in the liver, indicating that the joint could exacerbate the stress in mice. Pro-inflammatory cytokines IL-1β, IL-6and TNF-α mRNA expression of spleen in AZ, AD or ZD mainly significantly affected (P<0.05), which may affected immune function.In a word,(1) the cytotoxicity of mycotoxins on PK15and BRL was consistent, in increasing order FB1<ZEA<AFB1<DON.(2) The combined effects of mycotoxins on the target cell or organ were very complex, combined mode of action of mycotoxin was in dose-dependent manner. ZEA can ease the renal toxicity caused by low doses of AFB1, but presented synergies between ZEA and high-dose AFB1.(3) Binary mixture of AFB1, ZEA and DON decreased the expression of proinflammatory cytokines in spleen which may affect the immune function of mice.(4) AFB1and DON showed synergistic effect on rat hepatocytes and liver cell of mice.