Study Of Mycotoxins Toxic Effects And Mechanisms Using A Model Organism Nematode Caenorhabdites Elegans

Mycotoxins are secondary metabolites with strong toxicity produced during the growth of fungi. They can cause cancer, sterility to human health and safety through food or feed. Compared with other model organisms, using Caenorhabditis elegans as a research model could avoid individual differences caused by bioavailability and bioaccessibility for its simple body structure and conserved genetic model. Furthermore, it is beneficial to toxicity evaluation standardization. C. elegans has been the most commonly studied model organism for toxicology recently. The purpose of these experiments is to investigate the toxicological effects of mycotoxins on model organism C. elegans, to find out the best evaluation endpoints and to study the different gene expressions of ZEN-treated worms, with the hope that this may provide new research data on molecular level.1. The major foodborne mycotoxins Aflatoxins B1(AFB1), deoxynivalenol(DON), fumonisin B1(FB1), T-2 toxin(T-2), and zearalenone(ZEN) were used in our research. In the present study, the multiple toxic endpoints of these naturally-occurring mycotoxins were evaluated in C. elegans model for their lethality, toxic effects on growth and reproduction, as well as influence on lifespan. Mycotoxins could reduce worms’ body length, brood size and lifespan. We found that the lethality endpoint was more sensitive for T-2 toxicity with the EC50 at 1.38 mg/L. Also, the growth endpoint was relatively sensitive to AFB1 toxic effects, and the reproduction endpoint was more sensitive for toxicities of AFB1, FB1, and ZEA. Moreover, the lifespan endpoint was sensitive to toxic effects of all five tested mycotoxins.2. After 48 h exposed to ZEN, we determined the morphology of the gonad arm, the vulva, and the oocytes in the uterus using a DIC microscope. And then we calculated the oocytes number. Compared to the control group, the worms in ZEN-treated group were shown with atrophic gonad arms, abnormal vulva structures and smaller oocytes. Oocytes numbers were also found to be inhibited by ZEN.3. ZEN could significantly reduce the stimulation level of C. elegans to heat shock stress and the survival rate was highly significantly decreased. After 48 h ZEN exposure, it could be seen a decrease in both pumping rate and body bends. Above results showed that ZEN caused defects of the resistance to outside stimulation and locomotion behaviors in C. elegans, which indicated that ZEN had neurotoxicity.4. ZEN could cause both developmental and reproductive toxicities in the first generation in C. elegans and most of these toxicities could be transferred from exposed worms to their progeny. In progeny animals, the phenotypic defects could only be partially or very slightly rescued. Moreover, toxic effects for three exposed generations showed the second exposed generation suffered from the most severe defects,instead of the typical pattern for toxicity between generations which contain successive deterioration or recovery.5. Differential gene expression profiles were identified in worms and the data showed 171, 245, 3149 genes were down- or up- regulated(> 2 fold) respectively in 10, 40 and 80 mg/L ZEA dosing groups. Based on the microarray data, Go analysis results showed these different expressed genes focused on the phosphorylation of cellular signaling, metabolism, embryonic development, nerve cell proliferation and differentiation, regulation of life and behavior regulation. Microarray and real-time PCR results also indicated that ZEN affected development and reproduction in C.elegans might go through down regulating Collagen biosynthetic pathway and up regulating DAF-2 insulin signaling pathway and Wnt-β-Catenin signaling pathway. The results above indicated that ZEN has developmental toxicity, reproductive toxicity, cytotoxicity and neurotoxicity.6. Treatment with ZEN resulted in decreasing expression of gene dpy-17, col-121 and hch-1, while it caused an increase in gene dpy-31 and sqt-3 mRNA expression. All of these genes played an important role in cuticle Collagen Biosynthetic Pathway. The DPY-31#GFP mutant worm showed a significantly higher fluorescence expression compared to control. And the mutant worms were much sensitive to ZEN developmental toxicity and reproductive toxicity. All above results confirmed the microarray data that ZEN caused a decrease cuticle collagen biosynthetic pathway expression.7. Studies showed ZEN exposure resulted in ten genes daf-2, age-1, akt-1, akt-2, sgk-1, pdk-1, daf-16, dct-15, daf-21 and ins-11 increased mRNA expression while genes daf-18, hsp-1, hsf-1, dao-5 and egl-4 expresed less than control. The results showed in RT-PCR were consistent by using Microarray chips. And the DAF-16#GFP was distributed throughout the nucleus and the cytoplasm in all tissues. When animals are subjected to heat shock, almost complete nuclear translocation of DAF-16 were determined in control group while ZEN treated worms only appeared an intermediate DAF-16 localization. The stock of translocation from nucleus to cytoplasm was also determined during recovery process. All above the results showed ZEN exposure caused DAF-2 insulin-like signaling pathway increased expression.Taken together, C. elegans is an excellent model organism studying mycotoxins toxic effects for sensitive behaviors. And our studies also demonstrate that disruption of the collagen biosynthetic pathway and DAF-2 insulin signaling pathway is a key mechanism in ZEA-induced reproductive and developmental toxic effects in C. elegans. We suggested that a selection procedure should be set up to determine the major toxicities and molecular toxicology mechanisms on mycotoxins.