Molecular Mechanism Underlying The Combined Toxic Effects Of Deoxynivalenol And Zearalenone

Mycotoxins produced by certain fungi are common contaminants in food and grain feeds,posing a global threat to human and animal health.Among them,deoxynivalenol(DON)and zearalenone(ZEA)are two kinds of mycotoxins that exist widely in nature.They are frequently detected simultaneously in various cereals and foods,and thus lead to serious food contamination.Studies have shown that the coexistence of multiple mycotoxins may cause stronger toxic effects than a single mycotoxin.Although some studies have been carried out to investigate the interactive toxic effects between DON and ZEA,the molecular mechanisms underlying their synergistic action have not been fully understood yet.Recently,the micro RNAs(mi RNAs),a class of short non-coding RNAs,have been increasingly recognized as new biomarkers for various biological processes,since they are closely related to the occurrence and development of various diseases and toxicity responses.However,it remains unclear whether mi RNAs are involved in the combined toxicity of DON and ZEA.In addition,it is evidenced that maternal exposure to DON or ZEA could cause toxic effects on offspring in different degrees.Therefore,it is of practical significance to clarify whether long-term co-exposure of DON and ZEA can induce toxic effects on offspring.Accordingly,this study was set up to evaluate the combined toxic effects of DON and ZEA by using zebrafish as an in vivo model based on multiple life stages of zebrafish embryo,adult,and offspring.Furthermore,the possible role of mi RNA in mediating synergistic toxicity of DON and ZEA was analyzed using Hep G2 cell model.The main results are summarized as follows:(1)The combined toxic effects of DON and ZEA on zebrafish embryos.DON and ZEA at their non-toxic or low-toxic concentrations were used for co-exposure to zebrafish embryos.Results from 96 hpf survival rate assay showed that there was no significant(P>0.05)difference in the survival rate and hatching rate of zebrafish embryos after co-exposure of both mycotoxins compared with the control group.The hatching rate of zebrafish embryos was also not influenced.However,the co-exposure of DON and ZEA significantly(P<0.05)reduced the autonomic movement times at 24 hpf and the heart rate at 48 hpf of zebrafish embryos compared with the control group.Among three co-exposure groups,the heart rate showed a more significant downward trend in the co-exposure group of DON and ZEA at relatively high concentration(Z0.4+D4 group and Z0.8+D4 group)than that of the corresponding single exposure group,suggesting that the co-exposure of DON and ZEA had negative effects on embryo development.In addition,co-exposure of DON and ZEA increased the ROS levels and MDA contents of zebrafish embryos,changed the activities of antioxidant enzymes(SOD,CAT,and GPX)and the expressions of antioxidant enzymes-related genes,resulting in oxidative damage to zebrafish embryos.Further analysis showed that co-exposure of DON and ZEA changed the expressions of marker genes related to liver toxicity,up-regulated the expressions of pro-inflammatory genes(IL-1?,IL-6,and TNF-?)and pro-apoptotic genes p53,Caspase-9,and Caspase-3,and reduced the expression of anti-apoptotic gene Bcl-2.These results suggested that the co-exposure of DON and ZEA could trigger oxidative stress,promote the inflammatory response,and induce apoptosis,thus leading to potential combined toxic effects on zebrafish embryos.(2)The toxic effects of DON and ZEA at environmentally relevant concentrations on the liver of zebrafish.There were no significant differences(P>0.05)in the body weight of zebrafish among all treatment groups after the co-exposure of DON(2?g/L)and ZEA(2?g/L)to zebrafish for 28 days.However,the weight gains of zebrafish were lower in the co-treatment group than in the control group,suggesting that long-term exposure to mycotoxins could influence the normal growth of zebrafish.The co-exposure of DON and ZEA increased ROS levels and MDA contents of zebrafish liver.At the same time,the activities of antioxidant enzyme and the expression levels of antioxidant enzyme-related genes were influenced,indicating that the co-exposure of DON and ZEA caused oxidative stress in zebrafish liver.The expressions of marker genes related to liver toxicity were changed in the co-exposure group,indicative of the potential toxic effects on the zebrafish liver.Compared with the single exposure of DON and ZEA,the combination of 2?g/L DON and 2?g/L ZEA promoted the expressions of inflammatory genes and proteins through activating the TLR4/My D88/NF-?Bp65 signaling pathway,resulting in a significant inflammatory response.The ratio of Bax/Bcl-2 and Caspase-3 gene were increased,whilst Bax and Caspase-9 protein were up-regulated,suggesting that the co-exposure of DON and ZEA has the potential to induce apoptosis.(3)The toxic effects of DON and ZEA at environmentally relevant concentrations on zebrafish offspring.Zebrafish were co-treated with 2?g/L DON and 2?g/L ZEA for 28 days.Compared with the control group,the survival rate of zebrafish offspring at 120 hpf and spontaneous movement times at 24 hpf significantly(P<0.05)were reduced,and the heart rate at 48 hpf was significantly(P<0.05)increased in the single exposure group and co-exposure group of DON and ZEA,suggesting that the development of embryos was influenced.At the same time,compared with the control group,the single exposure or the co-exposure of DON and ZEA significantly(P<0.05)decreased the expressions of genes related to the antioxidant enzyme but significantly(P<0.05)increased the expressions of pro-inflammatory genes(IL-1?,IL-8,and TNF-?).These findings suggest that the single exposure or the co-exposure of DON and ZEA could induce oxidative stress and inflammatory response in zebrafish offspring.However,no significant apoptosis was induced.(4)The in vitro model construction and the molecular mechanism underlying the synergistic hepatotoxicity induced by DON and ZEA.Hep G2 cells were used as the toxicity model.The results showed that cells treated with DON(0.01-5?M)or ZEA(1-80?M)for 24h had significantly(P<0.05)lower relative cell viability and higher ROS level compared with the control,and these effects were dose-dependent.The IC₅₀ values of DON and ZEA were 1.62?M and 30.95?M,respectively.Since the IC₅₀ ratio of ZEA and DON was 20:1,a series of combination concentrations of ZEA and DON at their non-toxic or low-toxic concentrations were used to treat Hep G2 cells.Compared with the single treatment of ZEA(4?M)or DON(0.2?M),the combination of 2?M ZEA and 0.1?M DON significantly(P<0.05)decreased cell viability.The synergistic index CI value was 0.608 by the analysis of Calcu Syn software,indicative of a significant synergistic toxicity effect.Subsequently,the biological changes upon this co-treatment concentration were determined.The co-treatment of DON and ZEA increased ROS level,MDA content,GPX activity,and reduced SOD activity in Hep G2 cells,while no significant change in CAT activity,suggesting that the oxidative damage was induced.The expression levels of pro-inflammatory genes IL-1?,IL-6,and TNF-?were significantly(P<0.05)up-regulated,suggesting that the inflammatory response was induced.Results from RT-q PCR and Western blot revealed that the co-treatment of DON and ZEA significantly(P<0.05)increased the gene and protein expression of PTEN,Bax,and Caspase-3,and significantly(P<0.05)reduced the phosphorylation level of AKT protein.These results indicated that PTEN/PI3K/AKT signaling pathway plays an important regulatory role in the synergistic toxicity.Bioinformatics prediction and RT-q PCR verification showed that mi R-221 as an upstream target negatively regulated the PTEN gene.The protein expressions in PTEN/PI3K/AKT signaling pathway induced by DON and ZEA were significantly changed after mi R-221 was over-expressed.Meanwhile,overexpression of mi R-221 significantly(P<0.05)increased cell survival rate and reduced ROS level.An opposite trend was observed after mi R-221 was inhibited.These results suggested that the synergistic toxic effects caused by DON and ZEA could be regulated by mi R-221-mediated PTEN/PI3K/AKT signaling pathway.