Metabolism Of Fusarial Toxins And Their Masked Forms

Fusarium toxins, a catalog of secondary metabolites produced by the fungus Fusarium, are harmaful to plants and animals. It is widely present in grain and feed, which causes potential harm for animals and our human being. Toxins would experience wide metabolism in vivo and produce numerous metabolites once absorbed into the body. To make comprehensive evaluation of Fusarium toxins, it is meaningful to conduct the metabolism of Fusarium toxins in livestock and human, clarify the metabolic pathways, identify the major metabolites. In present study, based on ultra performance liquid chromatography tandem quadrupole/time of flight mass spectrometry (UPLC-Q/TOF), the metabolism of a single-ended trichothecenes Class A and Class B toxin, zearalenone and its relative toxins in liver microsomes of rats, chickens, swine, goat, cow and human was investigated to compare the metabolic species difference in vitro. In addition, in vivo metabolism of T-2 toxin, DON, DAS, ZEN, T-2-3G and ZEN-14G in rats and chicken was conducted.Liver microsomes of rats, chickens, swine, goats, and cow were prepared according the differential centrifugation. Based on the obtained microsomes, phase I and phase II metabolism of T-2 toxin, HT-2 and DAS were conducted. As a result, a total of 7,11 and 12 kinds of metabolites were discovered, respectively. The results showed that hydrolysis, hydroxylation and combination to glucuronide (GlcA) were the three major metabolic pathways and significant metabolic differences among species were observed. Hydrolysis of an acetyl group at C-4 was the major metabolic pathway of T-2 toxin and DAS, with the main metabolite of HT-2 and 15-MAS, respectively. Hydroxylation at C-3’was the main reaction for HT-2. For phase Ⅱ reaction, combination to GlcA was based on the hydroxylated position of C-3. In addition, phase Ⅱ metabolism of DON and NIV was carried out,3 and 1 kind of GlcA conjugates were identified, and the binding reaction occurred mainly at the C-3-hydroxyl group but at C-15-hydroxyl group in human. Comparative phase I and phase Ⅱ metabolism of ZEN, a-ZEL and β-ZEL in liver microsomes of livestock and human were investigated. As a result, a total of 20 kinds of phase I metabolites and 4 kinds of GlcA conjugate were found in ZEN while a-ZEL was the major reduction product of ZEN, and hydroxy reaction could occur at the postion of C-2,3,4,5,6,8,9,10,11,13 and 15 with significant difference, whereas the hydroxylated metabolite at C-13/15 was the major one. In addition, ZEN, a-ZEL and β-ZEL could be transformed into each other in liver microsomes. GlcA binding to ZEN occurred mainly at the C-14 position.8 and 7 kinds of GlcA conjugate were found in Phase II metabolism of a-ZEL and p-ZEL, while C-14 and C-7 were the main binding sites. Moreover, a serious of double GlcA combination metabolites were identified.Based on the results of in vitro metabolism, further in vivo metabolic studies of T-2 toxin, DAS, DON and ZEN in rats and chickens were conducted. A total of 19 and 18 kinds of T-2 toxin metabolites were identified in rats and chickens, respectively. The metabolic difference was significant between the two species:hydrolysis, hydroxylation, de-epoxidation and rearrangement were the major metabolic pathways of T-2 toxin in rats with significant differences between male and female ones, while hydrolysis, hydroxylation, acid and sulfate conjugates in chickens.4 and 2 kinds of metabolites for DAS were identified in rats and chickens with the main metabolic pathway of hydrolysis and hydroxylation. De-epoxy and sulfate conjugates were metabolic pathway of DON in rats and chickens, respectively. ZEN was extensively metabolized in rats and chickens, and 23 and 6 kinds of metabolites were found. Reduction, hydroxylation and binding to GlcA were major metabolic pathway for ZEN in rats, while reduction and sulfate conjugates in chickens.Three kinds of masked mycotoxins, ZEN-14G, a-ZEL-14G and β-ZEL-14G, were successfully synthesized and their phase I and phase II metabolism in microsomes were conducted. The results indicated that glycosidic bond of all the three toxins could be hydrolyzed to be their prototype, ZEN, α-ZEL and β-ZEL in microsomes. In addition, hydroxylation, hydrogenation/dehydrogenation and binding to GlcA were three major metabolic pathways, the hydroxylation mainly occurred at C-4、5、6 and C-9, while binding to GlcA at C-16 and C-7 for α-ZEL-14G and β-ZEL-14G. Moreover, in vivo metabolism of ZEN-14G in rats was carried out, suggesting that hydrolysis of glycosidic bond, reduction, hydroxylation, binding to GlcA and sulfate conjugates were the main metabolic pathway. In addition, in vitro metabolism of T-2-3G was carried out and five kinds of metabolites were identified, the results showed that hydroxylation and hydrolysis were the major metabolic pathways with the main metabolite of 3’-OH-T-2-3G. In vivo metabolism of T-2-3G in rats indicated that it could not be absorbed in gastrointestinal, and its degradation product, T-2 toxin, was only found in feces.