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Degradation and ozonolysis pathway elucidation of deoxynivalenol
Toxicon 174 (2020) 13–18
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Toxicon journal homepage: http://www.elsevier.com/locate/toxicon
Degradation and ozonolysis pathway elucidation of deoxynivalenol Dongliang Ren a, b, Enjie Diao b, c, *, Hanxue Hou a, **, Haizhou Dong a a
College of Food Science & Engineering, Shandong Agricultural University, Tai’an, 271018, PR China Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection, Huaiyin Normal University, Huai’an, 223300, PR China c Jiangsu Key Laboratory for Food Safety & Nutrition Function Evaluation, Huaiyin Normal University, Huai’an, 223300, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Deoxynivalenol Criegee mechanism Ozone Ozonolysis products Degradation pathway
To explore the degradation products and ozonolysis pathway of deoxynivalenol (DON), DON (~50 mg/L) in acetonitrile solution was treated by ozone at a concentration of 10.84 g/m3 and a flow rate of 80 mL/min for the times ranging from 0 to 9 min. The results showed that DON concentration rapidly reduced from 51.11 mg/L to 14.97 mg/L within 9 min of ozone exposure with 98.30% of degradation rate, and the ozonolysis of DON fol lowed the first-order kinetic model. Four ozonolysis products of DON were identified based on the analysis of Liquid chromatography–quadrupole time-of-flight mass spectra (LC-QTOF/MS). Their structures were similar to that of DON, while the double bond at C9–C10, 12,13-epoxide ring, and the hydroxyl group at C3 or C7 of DON were all destroyed by ozone. It is deduced that the toxicity of ozonolysis products significantly reduced based on the relationship between structure and toxicity of DON. The ozonolysis pathway of DON followed the Criegee reaction mechanism of ozone according to the chemical structures, accurate mass and molecular formulas of these products.
1. Introduction
Ioi et al., 2017). Ozone, as a powerful antimicrobial agent, has been widely used to detoxify mycotoxins in foods due to its strong detoxification capacity, high safety, low cost, and without toxic residues (Diao et al., 2013; Miller et al., 2013). Many researchers have reported that ozone could effectively degrade DON in agricultural products, and no significant differences in quality between the treated products and the control ones (Alexandre et al., 2017; Li et al., 2015; Sun et al., 2016; Wang et al., 2016a), even the quality of products was improved after being treated by ozone (Wang et al., 2016b). Most reported literatures explored the degradation efficiency of DON and the effects on quality of agricultural products treated by ozone. However, the ozonolysis pathway and intermediate products of DON have not been elucidated in detail, which restricts the application of ozone detoxification technique and safety evaluation of DON after being detoxified by ozone. Therefore, the main aim of this work was designed to isolate and identify the ozonolysis products of DON in acetonitrile solution and propose a detailed degradation pathway of DON based on the analysis of LC-QTOF/MS.
Fusarium molds are a large group of phytopathogenic fungi, which are easy to infect the cereal crops in the field. They often produce type B trichothecenes with potential toxicity to human and animals (Savard et al., 2015). Among the type B trichothecenes, deoxynivalenol (DON) is considered the most potent teratogen, mutagen, and hepatocarcinogen (Pestka, 2010a; Sobrova et al., 2010). DON is often found in many agricultural products with high concentrations, especially in wheat and maize (Ennouari et al., 2013; Sifuentes dos Santos et al., 2013). DON contamination of agricultural products not only causes significant financial losses but also poses a great potential threat to the health of people (Graziani et al., 2015; Pestka, 2010b). Therefore, many physical, chemical, and biological methods have been used to decompose or remove DON in contaminated agricultural products (Alexandre et al., 2017; Bretz et al., 2006; Ikunaga et al., 2011). Compared with the other two kinds of methods, chemical methods have many advantages in detoxification efficiency, low cost, and easy to realize the application of industrialization, so they have been widely studied to degrade myco toxins in agricultural products (Aiko and Mehta, 2015; Diao et al., 2018;
* Corresponding author. Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection, Huaiyin Normal University, Huai’an 223300, PR China. ** Corresponding author. College of Food Science & Engineering, Shandong Agricultural University, Tai’an, 271018, PR China. E-mail address: [email protected] (E. Diao). https://doi.org/10.1016/j.toxicon.2019.11.015 Received 29 July 2019; Received in revised form 26 November 2019; Accepted 28 November 2019 Available online 29 November 2019 0041-0101/© 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
The C18 column was Waters XBridge TM (100 � 4.6 mm i.d., 3.5 μm); mobile phase was acetonitrile-water (6:94, v/v) solution with a flow rate of 0.8 mL/min; the oven temperature was set at 35 � C; the wavelength of UV detector was set at 220 nm; the injection volume was 20 μL. DON was quantified by the external standard method.
2.1. Chemicals and reagents The standard DON (C15H20O6; purity>98%) and high-performance liquid chromatography (HPLC)-grade acetonitrile were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) Standard stock solu tions (0.625 mg/mL) of DON were prepared in HPLC-grade acetonitrile and stored at 20 � C in a refrigerated dark room. The standard DON working solutions (~50 mg/L) were prepared with deionized water.
2.4. Identification of ozonolysis products of DON using LC-QTOF/MS Operational procedures for liquid chromatography-quadrupole timeof-flight mass spectra (LC-QTOF/MS) were as follows: DON solution treated by ozone was firstly separated by a C18 column, and the sepa rated products were ionized by the MS ion source. The ionized products passed through the quadruple and reached the time-of-flight (TOF) by a mass analyzer without collision-induced dissociation (CID). The CID mode was used for MS/MS analysis. The parent ions were fragmented in the cell of CID, and the fragmentation pathways were obtained. Liquid chromatography was performed on Agilent 1260 series equipped with an autoinjector and a quaternary pump. Chromatography was performed on a ZORBAX Extend-C18 column (2.1 � 50 mm, i.d. 1.8 μm). The injection volume was 20 μL. The mobile phase was acetonitrile and deionized water with a ratio of 6:94 (v/v). The total run time was 22 min at a flow rate of 0.6 mL/min. MS was performed with Agilent 6540 accurate-mass QTOF/MS, the optimized conditions were as follows: products were analyzed in positive-ion mode. Capillary and fragmentor voltages were 4000 v and 125 v, respectively; the skimmer voltage was 65.0 v; the flow rate of drying gas was 10.0 L/min, and nebulizer was 40 psi. Nitrogen was used as the collision gas. Mass spectra were acquired in a full-scan analysis within the range of m/z 50–500 using an extended dynamic range and a scan rate of 4 spectra/s and varying the collision energy with mass. The data station operating software used was the Mass Hunter Workstation software (B.07.00).
2.2. Ozone treatment of DON in acetonitrile solution An ozone detoxification reactor (Fig. 1) was used to degrade DON in acetonitrile solution. The gaseous ozone was prepared using an ozone generator (Model DJ Q2020A) by electrolyzing water at low voltage, and the ozone concentration was controlled at about 10 g/m3 and monitored by an ozone gas analyzer (Model IDEAL 2000). The flow rate of ozone was adjusted at 80 mL/min by a gas flowmeter (Model LZB 4). The standard DON working solution (10 mL) was firstly injected into the ozone detoxification reactor using a syringe through the DON solution inlet. And then the gaseous ozone was pressed into the reactor, and was uniformly dispersed into the DON solution by ozone distributor. The exposure times were set at 0, 3, 6, and 9 min at room temperature. The residual ozone passed through the buffer area and went out of the outlet. 2.3. Determination of DON by HPLC-UV Determination of DON was performed using the HPLC-UV method.
2.5. Statistical analysis All experiments were conducted in triplicate, and the results were expressed as the means � standard deviation (SD). Analysis of variance (ANOVA) was carried out to determine any significant difference (P < 0.05) among the applied treatments using the SPSS 18.0 software (IBM, Chicago, USA). 3. Results 3.1. Degradation efficacy of DON by ozone As the reported results (Li et al., 2015; Sun et al., 2016), ozone could degrade DON with high efficiency (P < 0.05) (Fig. 2). DON concentra tion was decreased rapidly from (51.11 � 0.52) mg/L to (14.97 � 0.66) mg/L within 3 min of exposure, and reduced by 70.60%. At the 6 min of exposure time, the degradation rate of DON reached to 92.64%, and DON was almost completely degraded at 9 min with 98.30% of degra dation rate. The reduction of DON concentration depended on the exposure times of ozone, and followed a first-order kinetic model (y ¼ 214.49 e 1.368x) with a correlation coefficient (R2) of 0.9983. Fig. 3 is the chromatogram of DON with and without being treated by ozone. Only one big peak (black line) was observed for DON without being treated by ozone and a very small peak (blue line) for the treated one at the same retention time (6.055 min), which obviously indicates that ozone significantly reduced the DON concentration after being exposed for 9 min. 3.2. Identification of ozonolysis products of DON During the ozonolysis process of DON, the peak area of DON was decreased rapidly with the increase of ozone exposure time, while four new peaks gradually appeared and increased within 9 min of exposure
Fig. 1. Ozone detoxification reactor of DON. 14
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calculator based on m/z, a function of Agilent Mass Hunter Qualitative Analysis software (B.07.00). Seen from Table 1, the observed masses of four products are consistent with the calculated ones with the mass differences less than �5.0 ppm and the scores are all 100%. The DBE values (DBE ¼ 7) of ozonolysis products are close to that of DON (DBE ¼ 6), which indicates little structural differences between DON and its ozonolysis products (Fig. 5). TOF MS/MS was used to confirm the structures of the four products based on the fragmentation patterns and their accurate masses. Fig. 4 shows the MS/MS spectra and fragmentation of four ozonolysis prod ucts. According to the accurate masses of the parent ions and their fragments, we deduced the structures of four products (Fig. 5). As shown in Fig. 5, the structures of four products are similar to that of DON. Nevertheless, their double bonds at C9–C10 were all cleaved by ozone and transformed to an aldehyde, ketone or carbonyl acid of DON. The hydroxyl groups at C7 of DON were all oxidized to the carbonyl groups by ozone. The 12,13–epoxide ring of DON was active, which was easily oxidized by ozone to a double bond between C12 and C13. This inter mediate was named “Product 1” (C15H18O7). For the Product 2 (C13H14O10), Product 3 (C13H14O11), and Product 4 (C13H14O12), they were the ozonolysis products of the Product 1. The aldehyde group at the C10 of the Product 1 all transformed to the carboxyl group, and the double bond at the C12–C13 to the carbonyl group. For the Product 3, the single bond at C2–C3 of DON was cleaved, and meanwhile the hy droxyl group was oxidized to a carboxyl group at the C3 by ozone. While for the Product 4, it came from the cleavage of ether bond at the 1 po sition and the formation of carboxyl group at C2 of the Product 3.
Fig. 2. Degradation efficiency of DON treated by ozone at different expo sure times.
3.3. Ozonolysis pathway of DON in acetonitrile solution According to the previous research results, the ozonolysis of unsat urated compounds follows the Criegee reaction mechanism, and forms Criegee intermediates (Vereecken et al., 2014). In this study, the ozo nolysis of DON also abides by the Criegee mechanism (Fig. 6). According to the Criegee mechanism, the double bond at the C9–C10 of DON (C15H20O6) was firstly oxidized to 1,2,3–trioxolane (C15H20O9), and then it transformed to a carbonyl compound spontaneously. The carbonyl compound of DON was very unstable, which was quickly oxidized to a second ozonide (1,2,4–trioxolane, C15H20O9) by 1, 3–dipolar cycloaddition. 1,2,4–trioxolane of DON continued to be oxidized to aldehyde/ketone of DON (C15H20O8) by ozone. It was the real rupture of the double bond at the C9–C10 of DON. While these Criegee intermediates might be all unstable or very few of their residues, and they were not detected by LC-QTOF/MS. After the Criegee reaction of DON, the intermediate aldehyde/ketone of DON (C15H20O8) continued to be oxidized by ozone. The hydroxyl group at the C7 and the 12,13–epoxide ring were oxidized by ozone dues
Fig. 3. Chromatogram of DON after being treated by ozone for 0 min (black line) and 9 min (blue line).
(Fig. 3). We deduced that the four peaks were the ozonolysis products of DON. They were further analyzed using the LC-QTOF/MS, and the re sults are shown in Fig. 4 and Table 1. Table 1 lists the proposed ozo nolysis products of DON, retention times, molecular formulas, observed masses, calculated masses, mass differences between the observed and calculated ones, double bond equivalents (DBE), and scores. Molecular formulas of ozonolysis products were calculated using the formula
Fig. 4. TOF/MS/MS spectra and proposed fragmentation (insets) of ozonolysis products of DON, (A) Product 1, (B) Product 2, (C) Product 3, and (D) Product 4. Blue markers in the structures of four ozonolysis products are the sites of CID. 15
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Table 1 Mass accuracy measurement of DON and ozonolysis products in acetonitrile solution using LC-QTOF/MS. Proposed products of DON
Retention time (min)
Molecular formula
Observed mass (m/z)
Calculated mass (m/z)
Mass difference (ppm)
DBE
Score (%)
Product 1 Product 2 Product 3 Product 4 DON
11.905 13.242 13.872 20.244 6.055
C15H18O7 C13H14O10 C13H14O11 C13H14O12 C15H20O6
311.1129 331.1022 347.0611 363.0555 297.1333
311.1125 331.1024 347.0609 363.0558 297.1333
1.29 0.48 0.61 0.84 0.01
7 7 7 7 6
100 100 100 100 100
Fig. 5. Structures of DON and its ozonolysis products.
Fig. 6. Degradation pathway of DON in acetonitrile solution by ozone.
to its strong oxidative capacity, which were transformed to the carbonyl group and double bond, respectively, and formed the Product 1 (m/z 311.1129, C15H18O7). The aldehyde group at the C10 of Product 1 was also unstable, which was oxidized to carboxyl group and formed an intermediate (C15H18O8). The double bond at the C12–C13 of the in termediate (C15H18O8), which was newly formed in Product 1, was reacted with ozone based on the Criegee mechanism and formed the Product 2 (m/z 331.1022, C13H14O10) by releasing formic acid (HCOOH). The product 2 was transformed to the product 3 (m/z 347.0611, C13H14O11) through an intermediate (C13H14O10), which came from the cleavage of single bond at C2–C3, meanwhile, the oxidation of hydroxyl group at C3 of the Product 2. Due to the open loop at C2–C3 and the existence of carboxyl and carbonyl groups at C10 and C12 of the Product 3, respectively, it was very easy to break the single bond between O1 and C11 at the role of ozone, and formed the Product 4 (m/z 363.0555, C13H14O12) via an intermediate (C13H14O11), an alde hyde at C12 formed from the product 3. Based on the degradation process of DON by ozone, the Criegee re action is the main ozonolysis mechanism of DON. Most of ozonolysis products of DON are ketones and acids, which are relatively stable to ozone, so it is difficult for them to be further degraded by ozone.
4. Discussion Ozone as a strong oxidation agent has been widely used to degrade mycotoxins since 1997 (Diao et al., 2013; Mckenzie et al., 1997). As one of mycotoxins, DON can also be degraded by ozone, which has been verified by many researchers in recent years (Alexandre et al., 2017; Li et al., 2015; Sun et al., 2016; Wang et al., 2016a). Wang et al. (2016a) used 100 mg/L of ozone to treat wheat flour for 60 min, and DON concentration decreased from 3.89 mg/kg to 0.83 mg/kg, and the decrease of DON followed the first-order kinetic model. DON solution (1 μg/mL) was reduced by 93.6% after being treated by gaseous ozone (10 mg/L) for 30 s, and it was effective against DON in scabbed wheat having a first-order kinetic relation between the ozone exposure time and degradation rate of DON (Li et al., 2015). Sun et al. (2016) reported that 80 mg/L of gaseous ozone reduced by 83% of DON (initial con centration 10 mg/L) within 7 min (which was fitted for a first-order kinetic equation), and the detoxification rates of DON in contaminated wheat, corn and bran reached to 74.86%, 70.65%, and 76.21% by saturating aqueous ozone (80 mg/L) within 10 min, respectively. In addition, Alexandre et al. (2017) obtained the maximum reduction about 80% of DON in whole wheat flour on a wet milling effluent through ozone processing. While the quality of agricultural products treated by ozone had no significant change according to the results from 16
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the above mentioned literatures (Alexandre et al., 2017; Li et al., 2015; Sun et al., 2016; Wang et al., 2016a), even had been improved in some quality parameters (Wang et al., 2016b). Our results further verified that ozone could quickly degrade DON in acetonitrile solution. The degra dation rate (92.64% within 6 min for 51.11 mg/L of DON) of DON in acetonitrile solution is similar to the one (93.6% within 30 s for 1 mg/L of DON) reported by Li et al. (2015). Generally, the differences in degradation rate of DON by ozone come from the different ozone detoxification conditions, such as ozone concentration, ozone state (gaseous or aqueous), DON concentration, moisture content of treated samples, and ozone exposure time, and so on (Alexandre et al., 2017; Diao et al., 2013; Wang et al., 2016b; Zorlugenç et al., 2008). In addi tion, the degradation rate of DON in pure solution is higher than those in agricultural products due to the medium interferences for the latter. The ozonolysis of DON followed a first-order kinetic model, which was also consistent with the reported results (Li et al., 2015; Wang et al., 2016a). Some literatures have explored the degradation products of DON with different detoxification methods. Bretz et al. (2006) investigated the degradation products of DON during baking and cooking process by NMR and MS experiments, and they found seven main degradation products named norDON A, norDON B, norDON C, norDON D, norDON E, norDON F, and 9–hydroxymethyl DON lactone. Bacterial culture WSN05–2 degraded DON to the metabolite product 3–epi–DON and compound B (unknown structure), which were identified using MS and NMR analysis (Ikunaga et al., 2011). While BBSH 797 bacteria strains decomposed DON to DOM-1, which was identified by GC–MS and LC–PB–MS analysis (Fuchs et al., 2002). Mishra et al. (2014) also found that the degradation product of DON was DOM-1 under high tempera ture and highly acidic conditions. Feng et al. (2012) used UPLC-QTOF/MS to identify the metabolites of DON produced by intes tinal microflora of rats, and they indicated that DON might lose an epoxy atom to form deepoxidized–DON (DOM–1). For the ozonolysis products of DON, lower levels of aqueous ozone could decompose trichothecene mycotoxins to intermediate products, and the degradation begins with attack of ozone at the C9–C10 double bond with the net addition of two atoms of oxygen (Young et al., 2006). Two ozonolysis products of DON were found using UPLC–MS/MS by Sun et al. (2016), and the m/z of the two products were 219 and 214, respectively, while their structures were not elucidated. LC–QTOF/MS was used to isolate and identify the ozonolysis prod ucts of DON in this study. It has been successfully used to analyze and identify metabolites and degradation products of food contaminants by other researchers (Bateman et al., 2010; Mortishire-Smith et al., 2005). Accurate mass measurements from TOF generate the elemental composition of ions (molecules and fragments), moreover, tandem mass spectrometry (MS/MS) provides complementary structural information through in-source fragmentation using CID (Diao et al., 2012; Feng et al., 2012). Many degradation products of DON might be produced during the process of ozone detoxification, while only four products were isolated and identified by LC–QTOF/MS in our study due to the less residues for most of them (Young et al., 2006). Sun et al. (2016) also pointed out that the ozonolysis products of DON were negligible con centrations and ephemeral during the ozone detoxification process. Therefore, it is very difficult to isolate and identify the ozonolysis products of DON. Seen from Fig. 5, the structures of four products are similar to that of DON, which is consistent with the close DBE values of them. The double bond at C9–C10 of DON was firstly attacked by ozone based on the Criegee reaction, and resulted in the cleavage of double bond by forming aldehyde or ketone (Vereecken et al., 2014; Diao et al., 2012). The 12, 13–epoxide ring was destroyed by ozone and transformed to double bond or ketone. In addition, the hydroxyl group at C3 was also oxidized to the carboxyl group for Product 3 and Product 4. It is reported that the double bond between C9–C10 and the 12,13–epoxide ring are essential structural features for the toxicity of trichothecene, and removal of these groups results in a complete loss of toxicity. Additionally, a hydroxyl
group at C3 enhances the toxicity of trichothecene, while its toxicity decreases gradually when the hydroxyl group at C3 is destroyed or substituted with other groups (Nagy et al., 2005; Wu et al., 2013). Therefore, according to the relationship between structure and toxicity of DON, we deduced that the toxicity of the four ozonolysis products significantly reduced, even disappeared. Animal and cell toxicological experiments had also confirmed that the toxicity of DON significantly reduced after being treated by ozone (Bretz et al., 2006; Wang et al., 2017). In previous research reports, only few ozonolysis products of DON were explicated, while the whole ozonolysis pathway of DON has not been clarified in detail. According to the structures and accurate mo lecular masses of ozonolysis products of DON, we deduced the whole ozonolysis pathway of DON in detail (Fig. 6). The Criegee mechanism was the main reaction process between DON and ozone by breaking the double bond at C9–C10. Simultaneously, the hydroxyl groups at C3 and C7, and 12,13–epoxide ring of DON were easily attacked by ozone, which were all beneficial to the reduction of DON toxicity. 5. Conclusions Ozone could effectively degrade DON in acetonitrile solution, and the ozonolysis pathway of DON followed the Criegee reaction process. Many degradation products of DON might be produced during the process of ozone detoxification, while only four ozonolysis products of DON were isolated and identified by LC-QTOF/MS. It was deduced that their toxicity was reduced or even disappeared based on the relationship between structure and toxicity of DON. Conflict of interest The authors declare no competing financial interest. Ethical statement I certify that this manuscript is original and has not been published and will not be submitted elsewhere for publication while being considered by Toxicon. And the study is not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipu lated (including images) to support our conclusions. No data, text, or theories of others are presented as if they were our own. The submission has been received explicitly from all co-authors. And authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results. This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was ob tained from all individual participants included in the study. Author contributions section Dongliang Ren and Enjie Diao designed and carried out the experi ments; Dongliang Ren and Hanxue Hou analyzed the experimental re sults; Enjie Diao and Haizhou Dong wrote the manuscript. Acknowledgements This work was financially supported by National Key Research & Development Program of China (2017YFC1600904), Major Projects of Natural Science Research in Colleges and Universities, Jiangsu Province, China (19KJA430012), Young Talents Project for Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection (HSXT2-312), and Huai’an Municipal Science & Technology Project (HABZ201703). 17
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References
Miller Fatima, A., Silva Cristina, L.M., Brandao Teresa, R.S., 2013. A review on ozonebased treatments for fruit and vegetables preservation. Food Eng. Rev. 5 (2), 77–106. Mishra, S., Dixit, S., Dwivedi, P.D., Pandey, H.P., Das, M., 2014. Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product. Food Addit. Contam. A 31 (1), 121–131. Mortishire-Smith, R.J., Desmond, O.C., Castro-Perez, J.M., Jane, K., 2005. Accelerated throughput metabolic route screening in early drug discovery using high-resolution liquid chromatography/quadrupole time-of-flight mass spectrometry and automated data analysis. Rapid Commun. Mass Spectrom. 19 (18), 2659–2670. Nagy, C.M., Fejer, S.N., Berek, L., Molnar, J., Viskolcz, B., 2005. Hydrogen bondings in deoxynivalenol (DON) conformations—a density functional study. J. Mol. Struc. Theochem 726 (1 3), 55–59. Pestka, J.J., 2010. Deoxynivalenol-induced proinflammatory gene expression: mechanisms and pathological sequelae. Toxins 2 (6), 1300–1317. Pestka, J.J., 2010. Deoxynivalenol: mechanisms of action, human exposure, and toxicological relevance. Arch. Toxicol. 84 (9), 663–679. Savard, C., Provost, C., Alvarez, F., Pinilla, V., Music, N., Jacques, M., Gagnon, C.A., Chorfi, Y., 2015. Effect of deoxynivalenol (DON) mycotoxin on in vivo and in vitro porcine circovirus type 2 infections. Vet. Microbiol. 176 (3 4), 257–267. Sifuentes dos Santos, J., Souza, T.M., Ono, E.Y., Hashimoto, E.H., Bassoi, M.C., de Miranda, M.Z., Itano, E.N., Kawamura, O., Hirooka, E.Y., 2013. Natural occurrence of deoxynivalenol in wheat from Paran� a State, Brazil and estimated daily intake by wheat products. Food Chem. 138 (1), 90–95. Sobrova, P., Adam, V., Vasatkova, A., Beklova, M., Zeman, L., Kizek, R., 2010. Deoxynivalenol and its toxicity. Interdiscip. Toxicol. 3 (3), 94–99. Sun, C., Ji, J., Wu, S., Sun, C., Pi, F.W., Zhang, Y.Z., Tang, L.L., Sun, X., 2016. Saturated aqueous ozone degradation of deoxynivalenol and its application in contaminated grains. Food Control 69, 185–190. Vereecken, L., Harder, H., Novelli, A., 2014. The reactions of Criegee intermediates with alkenes, ozone, and carbonyl oxides. Phys. Chem. Chem. Phys. 16, 4039–4049. Wang, L., Luo, Y.P., Luo, X.H., Wang, R., Li, Y.F., Li, Y.N., Shao, H.L., Chen, Z.X., 2016. Effect of deoxynivalenol by ozone treatment in wheat grains. Food Control 66, 137–144. Wang, L., Shao, H.L., Luo, X.H., Wang, R., Li, Y.H., Li, Y.N., Luo, Y.P., Chen, Z.X., 2016. Effect of ozone treatment on deoxynivalenol and wheat quality. PLoS One 11 (1). https://doi.org/10.1371/journal.pone.0147613. Wang, L., Wang, Y., Shao, H.L., Luo, X.H., Wang, R., Li, Y.F., Li, Y.N., Luo, Y.P., Zhang, D. J., Chen, Z.X., 2017. In vivo toxicity assessment of deoxynivalenol-contaminated wheat after ozone degradation. Food Addit. Contam. A 34 (1), 103–112. Wu, Q.H., Dohnal, V., Kuca, K., Yuan, Z.H., 2013. Trichothecenes: structure-toxic activity relationships. Curr. Drug Metabol. 14 (6), 641–660. Young, J.C., Zhu, H.H., Zhou, T., 2006. Degradation of trichothecene mycotoxins by aqueous ozone. Food Chem. Toxicol. 44 (3), 417–424. € Zorlugenç, B., Zorlugenç, F.K., Oztekin, S., Evliya, I.B., 2008. The influence of gaseous ozone and ozonated water on microbial flora and degradation of aflatoxin B1 in dried figs. Food Chem. Toxicol. 46 (12), 3593–3597.
Aiko, V., Mehta, A., 2015. Occurrence, detection and detoxification of mycotoxins. J. Biosci. 40 (5), 943–954. Alexandre, A.P.S., Castanha, N., Calori-Domingues, M.A., Augusto, P.E.D., 2017. Ozonation of whole wheat flour and wet milling effluent: degradation of deoxynivalenol (DON) and rheological properties. J. Environ. Health B 52 (7), 516–524. Bateman, K.P., Castro-Perez, J., Wrona, M., Shockcor, J.P., Yu, K., Oballa, R., NicollGriffith, D.A., 2010. MSE with mass defect filtering for in vitro and in vivo metabolite identification. Rapid Commun. Mass Spectrom. 21 (9), 1485–1496. Bretz, M., Beyer, M., Cramer, B., Knecht, A., Humpf, H.U., 2006. Thermal degradation of the Fusarium mycotoxin deoxynivalenol. J. Agric. Food Chem. 54 (17), 6445–6451. Diao, E.J., Hou, H.H., Dong, H.Z., 2013. Ozonolysis mechanism and influencing factors of aflatoxin B1: a review. Trends Food Sci. Technol. 33, 21–26. Diao, E.J., Hou, H.H., Hu, W.C., Dong, H.Z., Li, X.Y., 2018. Removing and detoxifying methods of patulin: a review. Trends Food Sci. Technol. 81, 139–145. Diao, E.J., Shan, C.P., Hou, H.H., Wang, S.S., Li, M.H., Dong, H.Z., 2012. Structures of the ozonolysis products and ozonolysis pathway of aflatoxin B1 in acetonitrile solution. J. Agric. Food Chem. 60, 9364–9370. Ennouari, A., Sanchis, V., Marín, S., Rahouti, M., Zinedine, A., 2013. Occurrence of deoxynivalenol in durum wheat from Morocco. Food Control 32 (1), 115–118. Feng, P.S., Shen, J.Z., Wu, H.X., Li, L.X., Dai, Y.Y., Liu, K.L., Wang, Y., Zhang, S.X., 2012. In vitro biotransformation of deoxynivalenol by intestinal microflora of rats. Chin. J. Anal. Chem. 40 (1), 119–1123. Fuchs, E., Binder, E.M., Heidler, D., Krska, R., 2002. Structural characterization of metabolites after the microbial degradation of type A trichothecenes by the bacterial strain BBSH 797. Food Addit. Contam. 19 (4), 379–386. Graziani, F., Pujol, A., Nicoletti, C., Pinton, P., Armand, L., Di-Pasquale, E., Oswald, I.P., Perrier, J., Maresca, M., 2015. The food-associated ribotoxin deoxynivalenol modulates inducible NO synthase in human intestinal cell model. Toxicol. Sci. 145 (2), 372–382. Ikunaga, Y., Sato, I., Grond, S., Numaziri, N., Yoshida, S., Yamaya, H., Hiradate, S., Hasegawa, M., Toshima, H., Koitabashi, M., Ito, M., Karlovsky, P., Tsushima, S., 2011. Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epi-deoxynivalenol. Appl. Microbiol. Biotechnol. 89 (2), 419–427. Ioi, J.D., Zhou, T., Tsao, R., Marcone, M.F., 2017. Mitigation of patulin in fresh and processed foods and beverages. Toxins 9 (5). https://doi.org/10.3390/ toxins9050157. Li, M.M., Guan, E.Q., Bian, K., 2015. Effect of ozone treatment on deoxynivalenol and quality evaluation of ozonized wheat. Food Addit. Contam. A 32 (4), 544–553. Mckenzie, K.S., Sarr, A.B., Mayura, K., Bailey, R.H., Miller, D.R., Rogers, T.D., Norred, W. P., Voss, K.A., Plattner, R.D., Kubena, L.F., Phillips, T.D., 1997. Oxidative degradation and detoxification of mycotoxins using a novel source of ozone. Food Chem. Toxicol. 35, 807–820.
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Toxicon journal homepage: http://www.elsevier.com/locate/toxicon
Degradation and ozonolysis pathway elucidation of deoxynivalenol Dongliang Ren a, b, Enjie Diao b, c, *, Hanxue Hou a, **, Haizhou Dong a a
College of Food Science & Engineering, Shandong Agricultural University, Tai’an, 271018, PR China Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection, Huaiyin Normal University, Huai’an, 223300, PR China c Jiangsu Key Laboratory for Food Safety & Nutrition Function Evaluation, Huaiyin Normal University, Huai’an, 223300, PR China b
A R T I C L E I N F O
A B S T R A C T
Keywords: Deoxynivalenol Criegee mechanism Ozone Ozonolysis products Degradation pathway
To explore the degradation products and ozonolysis pathway of deoxynivalenol (DON), DON (~50 mg/L) in acetonitrile solution was treated by ozone at a concentration of 10.84 g/m3 and a flow rate of 80 mL/min for the times ranging from 0 to 9 min. The results showed that DON concentration rapidly reduced from 51.11 mg/L to 14.97 mg/L within 9 min of ozone exposure with 98.30% of degradation rate, and the ozonolysis of DON fol lowed the first-order kinetic model. Four ozonolysis products of DON were identified based on the analysis of Liquid chromatography–quadrupole time-of-flight mass spectra (LC-QTOF/MS). Their structures were similar to that of DON, while the double bond at C9–C10, 12,13-epoxide ring, and the hydroxyl group at C3 or C7 of DON were all destroyed by ozone. It is deduced that the toxicity of ozonolysis products significantly reduced based on the relationship between structure and toxicity of DON. The ozonolysis pathway of DON followed the Criegee reaction mechanism of ozone according to the chemical structures, accurate mass and molecular formulas of these products.
1. Introduction
Ioi et al., 2017). Ozone, as a powerful antimicrobial agent, has been widely used to detoxify mycotoxins in foods due to its strong detoxification capacity, high safety, low cost, and without toxic residues (Diao et al., 2013; Miller et al., 2013). Many researchers have reported that ozone could effectively degrade DON in agricultural products, and no significant differences in quality between the treated products and the control ones (Alexandre et al., 2017; Li et al., 2015; Sun et al., 2016; Wang et al., 2016a), even the quality of products was improved after being treated by ozone (Wang et al., 2016b). Most reported literatures explored the degradation efficiency of DON and the effects on quality of agricultural products treated by ozone. However, the ozonolysis pathway and intermediate products of DON have not been elucidated in detail, which restricts the application of ozone detoxification technique and safety evaluation of DON after being detoxified by ozone. Therefore, the main aim of this work was designed to isolate and identify the ozonolysis products of DON in acetonitrile solution and propose a detailed degradation pathway of DON based on the analysis of LC-QTOF/MS.
Fusarium molds are a large group of phytopathogenic fungi, which are easy to infect the cereal crops in the field. They often produce type B trichothecenes with potential toxicity to human and animals (Savard et al., 2015). Among the type B trichothecenes, deoxynivalenol (DON) is considered the most potent teratogen, mutagen, and hepatocarcinogen (Pestka, 2010a; Sobrova et al., 2010). DON is often found in many agricultural products with high concentrations, especially in wheat and maize (Ennouari et al., 2013; Sifuentes dos Santos et al., 2013). DON contamination of agricultural products not only causes significant financial losses but also poses a great potential threat to the health of people (Graziani et al., 2015; Pestka, 2010b). Therefore, many physical, chemical, and biological methods have been used to decompose or remove DON in contaminated agricultural products (Alexandre et al., 2017; Bretz et al., 2006; Ikunaga et al., 2011). Compared with the other two kinds of methods, chemical methods have many advantages in detoxification efficiency, low cost, and easy to realize the application of industrialization, so they have been widely studied to degrade myco toxins in agricultural products (Aiko and Mehta, 2015; Diao et al., 2018;
* Corresponding author. Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection, Huaiyin Normal University, Huai’an 223300, PR China. ** Corresponding author. College of Food Science & Engineering, Shandong Agricultural University, Tai’an, 271018, PR China. E-mail address: [email protected] (E. Diao). https://doi.org/10.1016/j.toxicon.2019.11.015 Received 29 July 2019; Received in revised form 26 November 2019; Accepted 28 November 2019 Available online 29 November 2019 0041-0101/© 2019 Elsevier Ltd. All rights reserved.
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2. Materials and methods
The C18 column was Waters XBridge TM (100 � 4.6 mm i.d., 3.5 μm); mobile phase was acetonitrile-water (6:94, v/v) solution with a flow rate of 0.8 mL/min; the oven temperature was set at 35 � C; the wavelength of UV detector was set at 220 nm; the injection volume was 20 μL. DON was quantified by the external standard method.
2.1. Chemicals and reagents The standard DON (C15H20O6; purity>98%) and high-performance liquid chromatography (HPLC)-grade acetonitrile were purchased from Sangon Biotech Co., Ltd. (Shanghai, China) Standard stock solu tions (0.625 mg/mL) of DON were prepared in HPLC-grade acetonitrile and stored at 20 � C in a refrigerated dark room. The standard DON working solutions (~50 mg/L) were prepared with deionized water.
2.4. Identification of ozonolysis products of DON using LC-QTOF/MS Operational procedures for liquid chromatography-quadrupole timeof-flight mass spectra (LC-QTOF/MS) were as follows: DON solution treated by ozone was firstly separated by a C18 column, and the sepa rated products were ionized by the MS ion source. The ionized products passed through the quadruple and reached the time-of-flight (TOF) by a mass analyzer without collision-induced dissociation (CID). The CID mode was used for MS/MS analysis. The parent ions were fragmented in the cell of CID, and the fragmentation pathways were obtained. Liquid chromatography was performed on Agilent 1260 series equipped with an autoinjector and a quaternary pump. Chromatography was performed on a ZORBAX Extend-C18 column (2.1 � 50 mm, i.d. 1.8 μm). The injection volume was 20 μL. The mobile phase was acetonitrile and deionized water with a ratio of 6:94 (v/v). The total run time was 22 min at a flow rate of 0.6 mL/min. MS was performed with Agilent 6540 accurate-mass QTOF/MS, the optimized conditions were as follows: products were analyzed in positive-ion mode. Capillary and fragmentor voltages were 4000 v and 125 v, respectively; the skimmer voltage was 65.0 v; the flow rate of drying gas was 10.0 L/min, and nebulizer was 40 psi. Nitrogen was used as the collision gas. Mass spectra were acquired in a full-scan analysis within the range of m/z 50–500 using an extended dynamic range and a scan rate of 4 spectra/s and varying the collision energy with mass. The data station operating software used was the Mass Hunter Workstation software (B.07.00).
2.2. Ozone treatment of DON in acetonitrile solution An ozone detoxification reactor (Fig. 1) was used to degrade DON in acetonitrile solution. The gaseous ozone was prepared using an ozone generator (Model DJ Q2020A) by electrolyzing water at low voltage, and the ozone concentration was controlled at about 10 g/m3 and monitored by an ozone gas analyzer (Model IDEAL 2000). The flow rate of ozone was adjusted at 80 mL/min by a gas flowmeter (Model LZB 4). The standard DON working solution (10 mL) was firstly injected into the ozone detoxification reactor using a syringe through the DON solution inlet. And then the gaseous ozone was pressed into the reactor, and was uniformly dispersed into the DON solution by ozone distributor. The exposure times were set at 0, 3, 6, and 9 min at room temperature. The residual ozone passed through the buffer area and went out of the outlet. 2.3. Determination of DON by HPLC-UV Determination of DON was performed using the HPLC-UV method.
2.5. Statistical analysis All experiments were conducted in triplicate, and the results were expressed as the means � standard deviation (SD). Analysis of variance (ANOVA) was carried out to determine any significant difference (P < 0.05) among the applied treatments using the SPSS 18.0 software (IBM, Chicago, USA). 3. Results 3.1. Degradation efficacy of DON by ozone As the reported results (Li et al., 2015; Sun et al., 2016), ozone could degrade DON with high efficiency (P < 0.05) (Fig. 2). DON concentra tion was decreased rapidly from (51.11 � 0.52) mg/L to (14.97 � 0.66) mg/L within 3 min of exposure, and reduced by 70.60%. At the 6 min of exposure time, the degradation rate of DON reached to 92.64%, and DON was almost completely degraded at 9 min with 98.30% of degra dation rate. The reduction of DON concentration depended on the exposure times of ozone, and followed a first-order kinetic model (y ¼ 214.49 e 1.368x) with a correlation coefficient (R2) of 0.9983. Fig. 3 is the chromatogram of DON with and without being treated by ozone. Only one big peak (black line) was observed for DON without being treated by ozone and a very small peak (blue line) for the treated one at the same retention time (6.055 min), which obviously indicates that ozone significantly reduced the DON concentration after being exposed for 9 min. 3.2. Identification of ozonolysis products of DON During the ozonolysis process of DON, the peak area of DON was decreased rapidly with the increase of ozone exposure time, while four new peaks gradually appeared and increased within 9 min of exposure
Fig. 1. Ozone detoxification reactor of DON. 14
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calculator based on m/z, a function of Agilent Mass Hunter Qualitative Analysis software (B.07.00). Seen from Table 1, the observed masses of four products are consistent with the calculated ones with the mass differences less than �5.0 ppm and the scores are all 100%. The DBE values (DBE ¼ 7) of ozonolysis products are close to that of DON (DBE ¼ 6), which indicates little structural differences between DON and its ozonolysis products (Fig. 5). TOF MS/MS was used to confirm the structures of the four products based on the fragmentation patterns and their accurate masses. Fig. 4 shows the MS/MS spectra and fragmentation of four ozonolysis prod ucts. According to the accurate masses of the parent ions and their fragments, we deduced the structures of four products (Fig. 5). As shown in Fig. 5, the structures of four products are similar to that of DON. Nevertheless, their double bonds at C9–C10 were all cleaved by ozone and transformed to an aldehyde, ketone or carbonyl acid of DON. The hydroxyl groups at C7 of DON were all oxidized to the carbonyl groups by ozone. The 12,13–epoxide ring of DON was active, which was easily oxidized by ozone to a double bond between C12 and C13. This inter mediate was named “Product 1” (C15H18O7). For the Product 2 (C13H14O10), Product 3 (C13H14O11), and Product 4 (C13H14O12), they were the ozonolysis products of the Product 1. The aldehyde group at the C10 of the Product 1 all transformed to the carboxyl group, and the double bond at the C12–C13 to the carbonyl group. For the Product 3, the single bond at C2–C3 of DON was cleaved, and meanwhile the hy droxyl group was oxidized to a carboxyl group at the C3 by ozone. While for the Product 4, it came from the cleavage of ether bond at the 1 po sition and the formation of carboxyl group at C2 of the Product 3.
Fig. 2. Degradation efficiency of DON treated by ozone at different expo sure times.
3.3. Ozonolysis pathway of DON in acetonitrile solution According to the previous research results, the ozonolysis of unsat urated compounds follows the Criegee reaction mechanism, and forms Criegee intermediates (Vereecken et al., 2014). In this study, the ozo nolysis of DON also abides by the Criegee mechanism (Fig. 6). According to the Criegee mechanism, the double bond at the C9–C10 of DON (C15H20O6) was firstly oxidized to 1,2,3–trioxolane (C15H20O9), and then it transformed to a carbonyl compound spontaneously. The carbonyl compound of DON was very unstable, which was quickly oxidized to a second ozonide (1,2,4–trioxolane, C15H20O9) by 1, 3–dipolar cycloaddition. 1,2,4–trioxolane of DON continued to be oxidized to aldehyde/ketone of DON (C15H20O8) by ozone. It was the real rupture of the double bond at the C9–C10 of DON. While these Criegee intermediates might be all unstable or very few of their residues, and they were not detected by LC-QTOF/MS. After the Criegee reaction of DON, the intermediate aldehyde/ketone of DON (C15H20O8) continued to be oxidized by ozone. The hydroxyl group at the C7 and the 12,13–epoxide ring were oxidized by ozone dues
Fig. 3. Chromatogram of DON after being treated by ozone for 0 min (black line) and 9 min (blue line).
(Fig. 3). We deduced that the four peaks were the ozonolysis products of DON. They were further analyzed using the LC-QTOF/MS, and the re sults are shown in Fig. 4 and Table 1. Table 1 lists the proposed ozo nolysis products of DON, retention times, molecular formulas, observed masses, calculated masses, mass differences between the observed and calculated ones, double bond equivalents (DBE), and scores. Molecular formulas of ozonolysis products were calculated using the formula
Fig. 4. TOF/MS/MS spectra and proposed fragmentation (insets) of ozonolysis products of DON, (A) Product 1, (B) Product 2, (C) Product 3, and (D) Product 4. Blue markers in the structures of four ozonolysis products are the sites of CID. 15
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Table 1 Mass accuracy measurement of DON and ozonolysis products in acetonitrile solution using LC-QTOF/MS. Proposed products of DON
Retention time (min)
Molecular formula
Observed mass (m/z)
Calculated mass (m/z)
Mass difference (ppm)
DBE
Score (%)
Product 1 Product 2 Product 3 Product 4 DON
11.905 13.242 13.872 20.244 6.055
C15H18O7 C13H14O10 C13H14O11 C13H14O12 C15H20O6
311.1129 331.1022 347.0611 363.0555 297.1333
311.1125 331.1024 347.0609 363.0558 297.1333
1.29 0.48 0.61 0.84 0.01
7 7 7 7 6
100 100 100 100 100
Fig. 5. Structures of DON and its ozonolysis products.
Fig. 6. Degradation pathway of DON in acetonitrile solution by ozone.
to its strong oxidative capacity, which were transformed to the carbonyl group and double bond, respectively, and formed the Product 1 (m/z 311.1129, C15H18O7). The aldehyde group at the C10 of Product 1 was also unstable, which was oxidized to carboxyl group and formed an intermediate (C15H18O8). The double bond at the C12–C13 of the in termediate (C15H18O8), which was newly formed in Product 1, was reacted with ozone based on the Criegee mechanism and formed the Product 2 (m/z 331.1022, C13H14O10) by releasing formic acid (HCOOH). The product 2 was transformed to the product 3 (m/z 347.0611, C13H14O11) through an intermediate (C13H14O10), which came from the cleavage of single bond at C2–C3, meanwhile, the oxidation of hydroxyl group at C3 of the Product 2. Due to the open loop at C2–C3 and the existence of carboxyl and carbonyl groups at C10 and C12 of the Product 3, respectively, it was very easy to break the single bond between O1 and C11 at the role of ozone, and formed the Product 4 (m/z 363.0555, C13H14O12) via an intermediate (C13H14O11), an alde hyde at C12 formed from the product 3. Based on the degradation process of DON by ozone, the Criegee re action is the main ozonolysis mechanism of DON. Most of ozonolysis products of DON are ketones and acids, which are relatively stable to ozone, so it is difficult for them to be further degraded by ozone.
4. Discussion Ozone as a strong oxidation agent has been widely used to degrade mycotoxins since 1997 (Diao et al., 2013; Mckenzie et al., 1997). As one of mycotoxins, DON can also be degraded by ozone, which has been verified by many researchers in recent years (Alexandre et al., 2017; Li et al., 2015; Sun et al., 2016; Wang et al., 2016a). Wang et al. (2016a) used 100 mg/L of ozone to treat wheat flour for 60 min, and DON concentration decreased from 3.89 mg/kg to 0.83 mg/kg, and the decrease of DON followed the first-order kinetic model. DON solution (1 μg/mL) was reduced by 93.6% after being treated by gaseous ozone (10 mg/L) for 30 s, and it was effective against DON in scabbed wheat having a first-order kinetic relation between the ozone exposure time and degradation rate of DON (Li et al., 2015). Sun et al. (2016) reported that 80 mg/L of gaseous ozone reduced by 83% of DON (initial con centration 10 mg/L) within 7 min (which was fitted for a first-order kinetic equation), and the detoxification rates of DON in contaminated wheat, corn and bran reached to 74.86%, 70.65%, and 76.21% by saturating aqueous ozone (80 mg/L) within 10 min, respectively. In addition, Alexandre et al. (2017) obtained the maximum reduction about 80% of DON in whole wheat flour on a wet milling effluent through ozone processing. While the quality of agricultural products treated by ozone had no significant change according to the results from 16
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the above mentioned literatures (Alexandre et al., 2017; Li et al., 2015; Sun et al., 2016; Wang et al., 2016a), even had been improved in some quality parameters (Wang et al., 2016b). Our results further verified that ozone could quickly degrade DON in acetonitrile solution. The degra dation rate (92.64% within 6 min for 51.11 mg/L of DON) of DON in acetonitrile solution is similar to the one (93.6% within 30 s for 1 mg/L of DON) reported by Li et al. (2015). Generally, the differences in degradation rate of DON by ozone come from the different ozone detoxification conditions, such as ozone concentration, ozone state (gaseous or aqueous), DON concentration, moisture content of treated samples, and ozone exposure time, and so on (Alexandre et al., 2017; Diao et al., 2013; Wang et al., 2016b; Zorlugenç et al., 2008). In addi tion, the degradation rate of DON in pure solution is higher than those in agricultural products due to the medium interferences for the latter. The ozonolysis of DON followed a first-order kinetic model, which was also consistent with the reported results (Li et al., 2015; Wang et al., 2016a). Some literatures have explored the degradation products of DON with different detoxification methods. Bretz et al. (2006) investigated the degradation products of DON during baking and cooking process by NMR and MS experiments, and they found seven main degradation products named norDON A, norDON B, norDON C, norDON D, norDON E, norDON F, and 9–hydroxymethyl DON lactone. Bacterial culture WSN05–2 degraded DON to the metabolite product 3–epi–DON and compound B (unknown structure), which were identified using MS and NMR analysis (Ikunaga et al., 2011). While BBSH 797 bacteria strains decomposed DON to DOM-1, which was identified by GC–MS and LC–PB–MS analysis (Fuchs et al., 2002). Mishra et al. (2014) also found that the degradation product of DON was DOM-1 under high tempera ture and highly acidic conditions. Feng et al. (2012) used UPLC-QTOF/MS to identify the metabolites of DON produced by intes tinal microflora of rats, and they indicated that DON might lose an epoxy atom to form deepoxidized–DON (DOM–1). For the ozonolysis products of DON, lower levels of aqueous ozone could decompose trichothecene mycotoxins to intermediate products, and the degradation begins with attack of ozone at the C9–C10 double bond with the net addition of two atoms of oxygen (Young et al., 2006). Two ozonolysis products of DON were found using UPLC–MS/MS by Sun et al. (2016), and the m/z of the two products were 219 and 214, respectively, while their structures were not elucidated. LC–QTOF/MS was used to isolate and identify the ozonolysis prod ucts of DON in this study. It has been successfully used to analyze and identify metabolites and degradation products of food contaminants by other researchers (Bateman et al., 2010; Mortishire-Smith et al., 2005). Accurate mass measurements from TOF generate the elemental composition of ions (molecules and fragments), moreover, tandem mass spectrometry (MS/MS) provides complementary structural information through in-source fragmentation using CID (Diao et al., 2012; Feng et al., 2012). Many degradation products of DON might be produced during the process of ozone detoxification, while only four products were isolated and identified by LC–QTOF/MS in our study due to the less residues for most of them (Young et al., 2006). Sun et al. (2016) also pointed out that the ozonolysis products of DON were negligible con centrations and ephemeral during the ozone detoxification process. Therefore, it is very difficult to isolate and identify the ozonolysis products of DON. Seen from Fig. 5, the structures of four products are similar to that of DON, which is consistent with the close DBE values of them. The double bond at C9–C10 of DON was firstly attacked by ozone based on the Criegee reaction, and resulted in the cleavage of double bond by forming aldehyde or ketone (Vereecken et al., 2014; Diao et al., 2012). The 12, 13–epoxide ring was destroyed by ozone and transformed to double bond or ketone. In addition, the hydroxyl group at C3 was also oxidized to the carboxyl group for Product 3 and Product 4. It is reported that the double bond between C9–C10 and the 12,13–epoxide ring are essential structural features for the toxicity of trichothecene, and removal of these groups results in a complete loss of toxicity. Additionally, a hydroxyl
group at C3 enhances the toxicity of trichothecene, while its toxicity decreases gradually when the hydroxyl group at C3 is destroyed or substituted with other groups (Nagy et al., 2005; Wu et al., 2013). Therefore, according to the relationship between structure and toxicity of DON, we deduced that the toxicity of the four ozonolysis products significantly reduced, even disappeared. Animal and cell toxicological experiments had also confirmed that the toxicity of DON significantly reduced after being treated by ozone (Bretz et al., 2006; Wang et al., 2017). In previous research reports, only few ozonolysis products of DON were explicated, while the whole ozonolysis pathway of DON has not been clarified in detail. According to the structures and accurate mo lecular masses of ozonolysis products of DON, we deduced the whole ozonolysis pathway of DON in detail (Fig. 6). The Criegee mechanism was the main reaction process between DON and ozone by breaking the double bond at C9–C10. Simultaneously, the hydroxyl groups at C3 and C7, and 12,13–epoxide ring of DON were easily attacked by ozone, which were all beneficial to the reduction of DON toxicity. 5. Conclusions Ozone could effectively degrade DON in acetonitrile solution, and the ozonolysis pathway of DON followed the Criegee reaction process. Many degradation products of DON might be produced during the process of ozone detoxification, while only four ozonolysis products of DON were isolated and identified by LC-QTOF/MS. It was deduced that their toxicity was reduced or even disappeared based on the relationship between structure and toxicity of DON. Conflict of interest The authors declare no competing financial interest. Ethical statement I certify that this manuscript is original and has not been published and will not be submitted elsewhere for publication while being considered by Toxicon. And the study is not split up into several parts to increase the quantity of submissions and submitted to various journals or to one journal over time. No data have been fabricated or manipu lated (including images) to support our conclusions. No data, text, or theories of others are presented as if they were our own. The submission has been received explicitly from all co-authors. And authors whose names appear on the submission have contributed sufficiently to the scientific work and therefore share collective responsibility and accountability for the results. This article does not contain any studies with human participants or animals performed by any of the authors. Informed consent was ob tained from all individual participants included in the study. Author contributions section Dongliang Ren and Enjie Diao designed and carried out the experi ments; Dongliang Ren and Hanxue Hou analyzed the experimental re sults; Enjie Diao and Haizhou Dong wrote the manuscript. Acknowledgements This work was financially supported by National Key Research & Development Program of China (2017YFC1600904), Major Projects of Natural Science Research in Colleges and Universities, Jiangsu Province, China (19KJA430012), Young Talents Project for Jiangsu Collaborative Innovation Center of Regional Modern Agriculture & Environmental Protection (HSXT2-312), and Huai’an Municipal Science & Technology Project (HABZ201703). 17
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References
Miller Fatima, A., Silva Cristina, L.M., Brandao Teresa, R.S., 2013. A review on ozonebased treatments for fruit and vegetables preservation. Food Eng. Rev. 5 (2), 77–106. Mishra, S., Dixit, S., Dwivedi, P.D., Pandey, H.P., Das, M., 2014. Influence of temperature and pH on the degradation of deoxynivalenol (DON) in aqueous medium: comparative cytotoxicity of DON and degraded product. Food Addit. Contam. A 31 (1), 121–131. Mortishire-Smith, R.J., Desmond, O.C., Castro-Perez, J.M., Jane, K., 2005. Accelerated throughput metabolic route screening in early drug discovery using high-resolution liquid chromatography/quadrupole time-of-flight mass spectrometry and automated data analysis. Rapid Commun. Mass Spectrom. 19 (18), 2659–2670. Nagy, C.M., Fejer, S.N., Berek, L., Molnar, J., Viskolcz, B., 2005. Hydrogen bondings in deoxynivalenol (DON) conformations—a density functional study. J. Mol. Struc. Theochem 726 (1 3), 55–59. Pestka, J.J., 2010. Deoxynivalenol-induced proinflammatory gene expression: mechanisms and pathological sequelae. Toxins 2 (6), 1300–1317. Pestka, J.J., 2010. Deoxynivalenol: mechanisms of action, human exposure, and toxicological relevance. Arch. Toxicol. 84 (9), 663–679. Savard, C., Provost, C., Alvarez, F., Pinilla, V., Music, N., Jacques, M., Gagnon, C.A., Chorfi, Y., 2015. Effect of deoxynivalenol (DON) mycotoxin on in vivo and in vitro porcine circovirus type 2 infections. Vet. Microbiol. 176 (3 4), 257–267. Sifuentes dos Santos, J., Souza, T.M., Ono, E.Y., Hashimoto, E.H., Bassoi, M.C., de Miranda, M.Z., Itano, E.N., Kawamura, O., Hirooka, E.Y., 2013. Natural occurrence of deoxynivalenol in wheat from Paran� a State, Brazil and estimated daily intake by wheat products. Food Chem. 138 (1), 90–95. Sobrova, P., Adam, V., Vasatkova, A., Beklova, M., Zeman, L., Kizek, R., 2010. Deoxynivalenol and its toxicity. Interdiscip. Toxicol. 3 (3), 94–99. Sun, C., Ji, J., Wu, S., Sun, C., Pi, F.W., Zhang, Y.Z., Tang, L.L., Sun, X., 2016. Saturated aqueous ozone degradation of deoxynivalenol and its application in contaminated grains. Food Control 69, 185–190. Vereecken, L., Harder, H., Novelli, A., 2014. The reactions of Criegee intermediates with alkenes, ozone, and carbonyl oxides. Phys. Chem. Chem. Phys. 16, 4039–4049. Wang, L., Luo, Y.P., Luo, X.H., Wang, R., Li, Y.F., Li, Y.N., Shao, H.L., Chen, Z.X., 2016. Effect of deoxynivalenol by ozone treatment in wheat grains. Food Control 66, 137–144. Wang, L., Shao, H.L., Luo, X.H., Wang, R., Li, Y.H., Li, Y.N., Luo, Y.P., Chen, Z.X., 2016. Effect of ozone treatment on deoxynivalenol and wheat quality. PLoS One 11 (1). https://doi.org/10.1371/journal.pone.0147613. Wang, L., Wang, Y., Shao, H.L., Luo, X.H., Wang, R., Li, Y.F., Li, Y.N., Luo, Y.P., Zhang, D. J., Chen, Z.X., 2017. In vivo toxicity assessment of deoxynivalenol-contaminated wheat after ozone degradation. Food Addit. Contam. A 34 (1), 103–112. Wu, Q.H., Dohnal, V., Kuca, K., Yuan, Z.H., 2013. Trichothecenes: structure-toxic activity relationships. Curr. Drug Metabol. 14 (6), 641–660. Young, J.C., Zhu, H.H., Zhou, T., 2006. Degradation of trichothecene mycotoxins by aqueous ozone. Food Chem. Toxicol. 44 (3), 417–424. € Zorlugenç, B., Zorlugenç, F.K., Oztekin, S., Evliya, I.B., 2008. The influence of gaseous ozone and ozonated water on microbial flora and degradation of aflatoxin B1 in dried figs. Food Chem. Toxicol. 46 (12), 3593–3597.
Aiko, V., Mehta, A., 2015. Occurrence, detection and detoxification of mycotoxins. J. Biosci. 40 (5), 943–954. Alexandre, A.P.S., Castanha, N., Calori-Domingues, M.A., Augusto, P.E.D., 2017. Ozonation of whole wheat flour and wet milling effluent: degradation of deoxynivalenol (DON) and rheological properties. J. Environ. Health B 52 (7), 516–524. Bateman, K.P., Castro-Perez, J., Wrona, M., Shockcor, J.P., Yu, K., Oballa, R., NicollGriffith, D.A., 2010. MSE with mass defect filtering for in vitro and in vivo metabolite identification. Rapid Commun. Mass Spectrom. 21 (9), 1485–1496. Bretz, M., Beyer, M., Cramer, B., Knecht, A., Humpf, H.U., 2006. Thermal degradation of the Fusarium mycotoxin deoxynivalenol. J. Agric. Food Chem. 54 (17), 6445–6451. Diao, E.J., Hou, H.H., Dong, H.Z., 2013. Ozonolysis mechanism and influencing factors of aflatoxin B1: a review. Trends Food Sci. Technol. 33, 21–26. Diao, E.J., Hou, H.H., Hu, W.C., Dong, H.Z., Li, X.Y., 2018. Removing and detoxifying methods of patulin: a review. Trends Food Sci. Technol. 81, 139–145. Diao, E.J., Shan, C.P., Hou, H.H., Wang, S.S., Li, M.H., Dong, H.Z., 2012. Structures of the ozonolysis products and ozonolysis pathway of aflatoxin B1 in acetonitrile solution. J. Agric. Food Chem. 60, 9364–9370. Ennouari, A., Sanchis, V., Marín, S., Rahouti, M., Zinedine, A., 2013. Occurrence of deoxynivalenol in durum wheat from Morocco. Food Control 32 (1), 115–118. Feng, P.S., Shen, J.Z., Wu, H.X., Li, L.X., Dai, Y.Y., Liu, K.L., Wang, Y., Zhang, S.X., 2012. In vitro biotransformation of deoxynivalenol by intestinal microflora of rats. Chin. J. Anal. Chem. 40 (1), 119–1123. Fuchs, E., Binder, E.M., Heidler, D., Krska, R., 2002. Structural characterization of metabolites after the microbial degradation of type A trichothecenes by the bacterial strain BBSH 797. Food Addit. Contam. 19 (4), 379–386. Graziani, F., Pujol, A., Nicoletti, C., Pinton, P., Armand, L., Di-Pasquale, E., Oswald, I.P., Perrier, J., Maresca, M., 2015. The food-associated ribotoxin deoxynivalenol modulates inducible NO synthase in human intestinal cell model. Toxicol. Sci. 145 (2), 372–382. Ikunaga, Y., Sato, I., Grond, S., Numaziri, N., Yoshida, S., Yamaya, H., Hiradate, S., Hasegawa, M., Toshima, H., Koitabashi, M., Ito, M., Karlovsky, P., Tsushima, S., 2011. Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epi-deoxynivalenol. Appl. Microbiol. Biotechnol. 89 (2), 419–427. Ioi, J.D., Zhou, T., Tsao, R., Marcone, M.F., 2017. Mitigation of patulin in fresh and processed foods and beverages. Toxins 9 (5). https://doi.org/10.3390/ toxins9050157. Li, M.M., Guan, E.Q., Bian, K., 2015. Effect of ozone treatment on deoxynivalenol and quality evaluation of ozonized wheat. Food Addit. Contam. A 32 (4), 544–553. Mckenzie, K.S., Sarr, A.B., Mayura, K., Bailey, R.H., Miller, D.R., Rogers, T.D., Norred, W. P., Voss, K.A., Plattner, R.D., Kubena, L.F., Phillips, T.D., 1997. Oxidative degradation and detoxification of mycotoxins using a novel source of ozone. Food Chem. Toxicol. 35, 807–820.
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