Elucidation Of Zearalenone Ozonolytic Products And Safety Evaluation Of Pre-and Post-treated Zearalenone

Zearalenone is a secondary metabolite produced by several Fusarium species with a strong estrogen-like response in mammals. It has been reported that the contamination of ZEN is widely spread in corn, barley, oats, rye, sorghum and wheat, and it is one of the most widely spread contaminated mycotoxins. Owing to accumulation of ZEN in the food chain, the health of human beings and animals are on the hazard. Thus, an efficient and safe method of ZEN detoxification was focused. This paper aims to study the effects of ozone on zearalenone degradation, investigate the degradation mechanism between ozone and ZEN, and evaluate the pre- and post-treated ZEN in vitro cytotoxicity and in vivo toxicity.1. The structure of ZEN ozonolytic products is elucidated by UPLC-Q-TOF-MS. Four ZEN ozonolytic products were observed, which 20 mg/L ozone treated ZEN for 1 min, with the [M+H]+ 335.1841,351.1907,321.1864 and 367.1752, respectively; the ozonolytic products were degradaed under detectable levels when treated for 5 min. The accurate mass and in-source fragmentation acquired by UPLC-Q-TOF-MS contributed to structural elucidation of ozonolytic products thus: C₁₈H₂₁O₆, C₁₈H₂₁O₇, C₁₇H₁₉O₆ and C₁₈H₂₁O₈. In comparison with ZEN and structural elucidation of ozonolytic products, ZEN is transformed ozonolytic products with the addition reaction of double bond by the Criegge mechanism.2. The evaluation of ZEN in vitro cytotoxicity, which contained two stages sections（10 μg/m L ZEN degraded by ozone with ozonolytic products for 1 min and without ozonolytic products for 10 min）, was conducted with Hep G2, MCF-7 and Ames tests.（1） The results of in vitro cytotoxicity tests showed that 10.5 μg/m L ZEN induced a 50% inhibition ratio in HepG2 cell growth, a significant increase of concentration of Ca²⁺ and ROS levels as well as the apoptosis rate（P<0.05）. However, ozonolytic products in ZEN treated for 1 min reduced the effects of ZEN on the above cell parameters, still existed continued toxicity; from another aspect, the cell parameters of ozonolytic products completely degraded group showed no significant difference with the control group（P>0.05）.（2） The results of E-Screen tests showed that ZEN can generate estrogenicity, which fact was approved that less estrogenicity was confirmed in the estrogenicity assessment under the treatment of ozone.（3） The results in Ames tests indicated pre- and post-treated ZEN were negative compared to the control group.3. The evaluation of ZEN was conducted with BALB/c mice in vivo toxicity. Compared with the control group, the weight of mice, lymphocyte levels of ZEN group were significantly decreased and the leukocytes levels in blood, alanine aminotransferase, aspartate aminotransferase, total bilirubin, urea levels in serum were significantly increased, resulting in the damaging liver and kidney（P<0.05）; the blood parameters ozonolytic products group tended to a normal level of control group; the blood parameters of completely degraded ozonolytic products group showed no siginificant difference with the control group（P>0.05）. Thus, it is indicated that the post-treated ZEN showed less toxicity in vivo toxicity.4. Based on the continuous toxicity of ozonolytic products, a quantitative detection method of main ZEN ozonolytic products was indispensable in the process of detoxifying contaminated by ozonation. Two main ozonolytic products（Compound 2 and Compound 4） were isolated through the semi-preparative HPLC. An efficient and sensible UPLC-MS/MS method was established to detect ZEN, Compound 2 and Compound 4 and ZEN biological metabolites（α-ZEN and β-ZEN）, which both ozonolytic products occurred in practical process model for detoxifying contaminated corn flour by ozonation. The kinetic equation of ZEN degradation process was calculated: Ct=A2+（A1-A2）/[1+（t/t0）p], A1=308.31, A2=-57.76,t0=1.09925×10-4, p=0.12024.