Metabolism And Signal Transduction Pathway Of T-2Toxin

As one of the primary members of type-A trichothecenes, T-2toxin is produced mainly by Fusarium genus. T-2toxin is the greatest toxic one for animals among trichothecenes, and it could contamiant cereals such as wheat, barley, oats, and maize. T-2toxin is making a great harmful on animals and humans through feed contaimination. Importantly, T-2toxin could also inhibit the sysnthesis of protein, DNA and RNA through conjugating with ribosomes as well as the damage of mitochondria. This toxin could also activate MAPK and JAK/STAT signaling pathways as well as cytokine expression and induce cell apoptosis and immune dysfunction.Metabolism of T-2toxoin has a close relationship with its toxicities. HT-2toxin,3’-OH-T-2, and neosolaniol (NEO) are the major metabolites of T-2toixn in animals. The toxicities change greatly once T-2toxin is biotransformed into different productes. The toxicity of most metabolites such as NEO and T-2triol are decreased. However, the toxicities of some metabolites such as3’-OH-T-2, are not decreased significantly, or even display a slightly higher toxicity. Thus, elucidation of the metabolic pathways and identifying the major metabolites of T-2toxin in animals are prerequisite to reveal its toxicity in animals, and are also crucial for food safety and human health. However, up to date, a global metabolism of T-2toxin in food producing animals is still unclear; especially the metabolic profiles are never compared in different species.T-2toxin can cause damage on ribosomes and mitochondria and ultimately lead to cell apoptosis. MAPK signaling pathway is proved to be an important toxicological pathway of trichothecenes and plays critical roles in toxicity. T-2toxin can active this pathway rapidly and transiently. It is known that animals are normally exposed to trichothecenes slowly. The effects of inflammatory cytokines on cells are performed slowly and continually. For example, IL-6, IL-1β, and TNF-a were found to be activated after12h by trichothecenes in RAW264.7cells. MAPK is proved to be an important target signaling pathway of trichothecenes and could be activated in a rapid and transient way. Thus, a contradiction rises when trying to explain the rapid MAPK activation but slow activation of inflammatory factors. We suspect that some other signal pathways might exist at the downstream to transmit the signals from MAPKs and activate the inflammatory factors. Indeed, it is already proved from our previous work that JAK/STAT pathway are the downstream targets of trichothecenes and played important roles in regulation of proinflammatory cytokines as well as apoptosis. However, the upstream and downstream interrelationship between MAPK and JAK/STAT signal pathways is still unclear. Where do they peform their fuctions once they are activated, in nucleus or cytoplasm? What are the messengers to connect the two pathways and transmite the signals? All these questions need to be answered.It is already known that T-2toxin can be well biotransformed in liver and intestine. Thus, we aimed to study the metabolism of T-2toxin in hepatic subcellular fractions (microsomes and cytosol) of pigs, chickens, rats, and carp (common carp and grass carp), as well as hepatocytes of rats, piglets and chickens. Based on the results, we discussed the metabolic difference of T-2toxin in different species. In addition, we also studied the degradation and metabolism of T-2toxin in pig cecum model. This study combined with the data in liver will further elucidate the metabolic fates of T-2toxin in animals. We also aim to study the cross talk between MAPK and JAK/STAT signaling pathways, which is induced by trichothecene T-2toxin. The cross talk between JNK1and STAT3were especially discussed. Specific inhibitors are useful tools to elucidate the signaling cross talk. In addition to the cross talk, we also monitored the typical ultrastructural changes of RAW264.7cells caused by T-2toxin and found out the potential toxicological targets of T-2toxin. The observation provides crucial visualized evidence for the molecular targets of trichothecenes. Finally, relationship between the toxic profiles and T-2toxin contents in intracellular in RAW264.7cells were also addressed. The results from this study will provide important data for explaning the sensitivity difference to T-2toxin in different animals, and is also important for identifying the residue markers as well as the prevention of disease. The study of signaling pathways will provide further information for the understanding of toxic mechanism of trichothecenes as well as the interrelationship between MAPK and JAK/STAT signaling pathways.1. A comparation of hepatic metabolism of T-2toxin in pigs, chickens, carp and ratsMetabolic profiles of T-2toxin in the hepatocytes of food producing animals, pigs and chickens; rats-the major experimental model were compared. Metabolic pathways of T-2toxin in liver microsomes and cytosol of pigs, chickens, common carp, grass carp, and rats were also especially concerned. The metabolites were identified by HPLC-MS-IT-TOF.In liver microsomes, five metabolites (MT1-5) were detected and identified. T-2toxin was mainly metabolized to HT-2toxin (MT1), neosolaniol (NEO)(MT2),3’-OH-T-2(MT3),3’-OH-HT-2(MT4), and T-2triol (MT5). T-2triol was only detectable in rat liver microsomes, implying that rat liver microsomes have stronger metabolic capability of T-2toxin than other species. In carp liver microsomes, HT-2toxin, NEO, and3’-OH-T-2were detected. Different with land animals, there was only trance amount of3’-OH-HT-2in carp liver microsomes. In grass carp,3’-OH-HT-2and3’-OH-HT-2, but not HT-2toxin were detected. The relative amount of HT-2toxin in land animals (pig, chicken, and rat) was much higher than that in carp (p<0.05). Hydrolysis to form HT-2toxin was the major metabolic pathway of T-2toxin in land animals (pig, chicken and rat). Different with land animals, HT-2toxin was not found in the liver microsomes of grass carp. In grass carp,3’-OH-T-2showed a relatively high amount, and followed with common carp. But in land animals, the capacity of transforming T-2toxin to3’-OH-T-2was relatively weak.3’-OH-HT-2was not found in liver cytosol and the amount of3’-OH-T-2was also very low. Especially in common carp, the hydroxyl products were not found. Thus, we conclude that the hydroxyl capacity in liver cytosol is weaker than that in liver microsomes.In hepatocytes, HT-2toxin and NEO were detectable. The amount of T-2toxin was much higher than NEO. Pigs produced a higher amount of HT-2toxin, chickens were the weakest. The hydroxyl products and C-8-deisovaleryl metabolites were not detected.Taken together, there was a similarity in the metabolic pathways of T-2toxin among different species, but an interesting and different profile was also monitored. T-2toxin can be metabolized rapidly in liver. Hydrolysis (HT-2, NEO, and T-2triol) and hydroxylation (3’-OH-T-2and3’-OH-HT-2) were the major metabolic pathways in the liver from these species. Hydrolysis and forming HT-2toxin was the same pathway in these land animals. As compared with hydrolysis reaction, the hydroxylation was much weaker in land animals. But carp showed an opposite metabolic characteristic. HT-2, the very common metabolite in land animals, was rarely found in carp, but3’-OH-T-2was the major one. This result implies that the metabolism of T-2toxin in fish has some differences with land animals. The different characteristic of CYP450monooxygenase and carboxylesterase as well as the differece cataltic abilty in hydroxyaiton and hydrolysis actions by these enzymes between carp and land animals are possibly the reasons for the different metabolic profiles of T-2toxin. The findings of this study further improves the metabolic mechanism of T-2toxin and is also important for residue determination, residue marker identification as well as the toxic mechanism of trichothecenes.2. Degradation and metabolism of T-2toxin in pig cecum modelTo further provide the information of metabolism of T-2toxin in animals, a pig cecum model was produced to investigate the fate and degradation of T-2in animal intestines. The data combineding with liver metabolism will better uncover the metabolic pathways and bioavailbility of T-2toxin in animals.T-2toxin was incubated with pig cecum at anaerobic conditions for20min,40min,1h,2h,4h,8h, and24h. Quercetin was used to monitor the activity of the bacterial in the pig cecum. The extraction of incubated samples was performed by a modified QuEChERS method. An Agilent1100series HPLC was linked to an API4000QTrap mass spectrometer. Heated Electrospray Ionization (HESI) coupled with a Thermo HPLC system was used to detect the potential metabolites.Four different cecums were analyzed in comparison to the sterilized control. The degradation by the microbiota in Cecum1-3was much slower compared to Cecum4. In Cecum1-3, the degradation ranged31.1-45.9%of the originally incubated amount of T-2toxin. Only a small increase of the degradation between8and24h incubation time was detectable, e.g., only0.8%of T-2toxin was further degraded between8and24h in Cecum1. However, a very strong degradation was observed in Cecum4. About26±0.6%of T-2toxin were left after incubation for8h. Only3.0±0.1%of T-2toxin was detectable after24h.Besides HT-2toxin, other metabolites, such as deepoxy-HT-2, were not detected. In Cecum4, the formed HT-2toxin was not further metabolized after a further incubation for8h. However, the relatively low recovery after24h incubation might indicate that HT-2toxin was possibly further metabolized to other products to an extent which was lower than the LOD of the HPLC-MS/MS equipment. Thus, we suspect that the toxic effects of T-2toxin in pigs are possibly afforded by the combination of T-2toxin and HT-2toxin.In conclusion, T-2toxin is metabolized to HT-2toxin as the main metabolite by the intestinal microbiota of pigs with large interindividual difference. In one out of four analyzed cecums, T-2was nearly totally metabolized to HT-2toxin, whereas the other three cecums showed a degradation of T-2toxin up to46%. Besides HT-2toxin, no other metabolites were detectable in the incubated samples. For toxicity evaluations of T-2toxin in pigs, the combination of T-2and its major metabolite HT-2has to be considered, as both compounds show a similar cytotoxicity and absorption. The dagradation and metabolism study of T-2toxin in pig cecum further complemented the data of liver metaboism of T-2toxin and enriched the enterohepatic metabolic information; it also provides important references for the in vivo metabolism and toxic mechanism of T-2toxin.3. The cross talk between MAPK and JAK/STAT signaling pathways induced by T-2toxin in RAW264.7cellsIn order to study the cross talk between MAPK and JAK/STAT signaling pathway which is induced by T-2toxin, T-2toxin was incubated with RAW264.7cells for indicated time and the interrelationship between JNK, ERK, p38MAPK and JAK, STAT were investigated using the specific inhibitors. Furthermore, we studied the potential messengers between the two pathways. Besides the cross talk, we also monitored the typical ultrastructural changes of RAW264.7cells, which was caused by T-2toxin, and found out the potential toxicological targets of T-2toxin. This observation provides crucial visualized evidence for the molecular targets of trichothecenes. Moreover, the function of JNK1and STAT3were discussed using the inhibitors, immunoflourenscence and flow cytomery. Finally, the link between the toxic profiles and intracellular T-2toxin contents were addressed.Results showed that the genes of ERK, JNK1and p38MAPK were activated within1-2h. However, they ceased quickly, implying that MAPK is a rapid signaling pathway. JAK2and STAT3mRNA were up regulated significantly, but they were slow and the highest peak was observed at12h. It is very possible that JAK/STAT pathway is the downstream singling of MAPK. IL-6among the studied cytokines showed the relatively highest up-regulation level, which was up regulated30.43-fold at2h and48.47-fold at12h. Thus, we suspected that IL-6possibly plays a very important role in the cross talk between MAPK and JAK/STAT signaling pathways. In addition, K-Ras had an important function in the cross talking, the up regulation after1and12h implying that K-Ras also plays roles in connecting the cross talk between MAPK and JAK/STAT. In order to study the up-and down stream relationships, we used the specific inhibitors and monitored the gene levels and protein phosphorylation. When JNK1gene expression was blocked by its inhibitor SP600125, both the mRNA expression and protein phosphorylation of JAK2, and K-Ras response to T-2toxin were significantly suppressed. The gene expression and protein phosphorylation of STAT3, but not STAT1were significantly decreased. Interestingly, the gene expression of IL-6induced by T-2toxin was suppressed by SP600125at12h but not at2h. These results implies that the activated signals induced by T-2toxin could transfer from JNK1to JAK2/STAT3. Talk from JNK1to STAT3was possibly connected by IL-6. SOCS family is an important negatively regulator of JAK/STAT signaling pathway, we studied the relationships between JNK1and SOCS1,2,3, and CIS through blocking JNK1activity and found out that the mRNA expressions of CIS, SOCS1, and SOCS2were increased significantly, whereas the mRNA expression of SOCS3was decreased markedly. This observation implies that JNK1could regulate CIS, SOCS1, and SOCS2negatively, but positively regulate SOCS3.Once induced by T-2toxin, signal of JNK1could transmit to JAK2, STAT3, ERK, and p38. Moreover, these activated genes can reversely regulate JNK1activity. Blocking STAT3activity, IL-6gene expression at12was suppressed remarkedly. Thus, we suspected that STAT3would further activate IL-6after the activation of JAK2/STAT3by IL-6and join in the next circulations.JNK1was phosphorylated rapidly and imported into the nucleus, but the nucleus translocation is in a dynamic way and will later export to cytoplasm. When JNK1activity was blocked, the phosphorylation and nucleus translocation of STAT3were inhibited, but STAT1was not affected. This observation implies that phosphorylation and nucleus translocation of STAT3is JNK1-dependent.When examined by transmission electron microscopy, mitochondrial swelling and the rough endoplasmic reticulum dilation were clearly visible when treated with T-2toxin at the level of14nM for12h. Once the toxin was increased to28nM, more serious morphological changes were observed. Besides the swelling mitochondria and the dilation of rough endoplasmic reticulum, polysomes on rough endoplasmic reticulum were also breakdown and degranulated. In addition, condensation and marginalization of chromatin aggregation were also monitored, which suggests that cells are induced to apoptosis by T-2toxin.Interestingly, when the gene expression of JNK1was blocked, the apoptotic ratio was increased significantly than the blank or toxin treated group (p<0.05). This result demonstrates that JNK1induced by T-2toxin has an anti-apoptotic function in RAW264.7cells. The ratios of Bcl-xL/Bax and Bcl-2/Bax were both decreased after blocking JNK1or STAT3activity. The mRNA expression of Caspase-3and Caspas-9were markedly increased (p<0.05) when blocking JNK1and STAT3activities. These results further prove that the apoptosis induced by T-2toxin is a mitochondria-dependent caspase pathway. JNK1-STAT3pathway could inhibit apoptosis and its function is very possibly mediated by regulating the function of mitochondria. JNK1-STAT3is newly proved to be a cell survival pathway, and T-2toxin is shown to have a Janus face. This study adds to our further understanding of the toxic mechanism of trichothecenes.Finally, the intracellular content of T-2toxin in different time points was detected. The content of T-2toxin was increased significantly at12h than at2h, indicating that intracellular content of T-2toxin can be accumulated with time increase. Moreover, T-2toxin can be biotransformed to HT-2toxin and3’-OH-T-2toxin at2h, whereas3’-OH-T-2toxin was the sole metabolite at12h in RAW264.7cells. But at the two time points, T-2toxin was the major product to perform the toxic effects and induced gene expression.Taken together, a complicated cross talk between MAPK and JAK/STAT signaling pathways mediated by T-2toxin is reported for the first time. K-Ras and IL-6are proved to be critical messengers for the cross talk between JNK1and STAT3. Importantly, T-2toxin has a Janus face, which not only induces cell apoptosis, cell survival/defense pathways, such as JNK1-STAT3, could also be activated simultaneously. However, the cell defense signaling is possibly not strong and was swamped in the cell death signals, which makes it easily to be ignored. On the other hand, this study also reveals that cells could up regulate some defense signaling pathways when they are exposed to dangerous environment. This work also showes that ribosome and mitochondria are two major toxic targets of T-2toxin. The findings add to our further understanding of the toxic mechanisms of trichothecenes and the cross talk between MAPK and JAK/STAT signaling.In summary, we first investigated the metabolism of T-2toxin in liver and intestine, and dentified5metabolites as well as the major metabolic pathways, also the metabolic profiles in different speices were compared. This study consummated the metabolic mechanism of T-2toxin, and provided crucial information for residue mornitoring and determination of residue marker of T-2toxin. The cross talk between MAPK and JAK/STAT signaling pathway further uncovered the toxic mechanism of T-2toxin and provide an important referece for toxin controlling, disease prevention, cancer treatment and drug development.