Zearalenone(ZEN) is a toxic metabolite produced by Fusarium culmorum, F. graminearum, F. tricinctum, and other fungi, with estrogenic characteristics. Exposure to or ingestion of ZEN during pregnancy can cause reproductive dysfunction, miscarriage, stillbirth, and malformation, and seriously endanger human life and health. The detection methods for ZEN in the Chinese Pharmacopoeia(2020 edition) are liquid chromatography(LC) and liquid chromatography-mass spectrometry(LC-MS), and it is stipulated that ZEN should not exceed 500 μg in 1 000 g of Coicis Semen. Although these detection methods by instruments can achieve the qualitative and quantitative analysis of ZEN in Coicis Semen, their high detection cost and long periods hinder the rapid screening of a large number of samples in the field. In this study, the synthesized ZEN hapten was conjugated with bovine serum albumin(BSA) and ovalbumin(OVA) to obtain the complete ZEN antigen. By virtue of antibody preparation techniques, ZEN monoclonal antibody 4F6 was prepared, which showed 177.5%, 137.1%, and 109.7% cross-reactivity with ZEN structural analogs zearalanol, zearalenone, and α-zearalenol, respectively, and no cross-reactivity with other fungal toxins such as aflatoxin. Direct competitive enzyme-linked immunosorbent assay(dcELISA) based on ZEN monoclonal antibody 4F6 was developed for the determination of ZEN in Coicis Semen with an IC_(50) of 1.3 μg·L~(-1) and a detection range of 0.22-21.92 μg·L~(-1). The recoveries were 83.91%-105.3% and the RSD was 4.4%-8.0%. The established dcELISA method was used to determine the ZEN residuals in nine batches of Coicis Semen samples, and the results were validated by LC-MS. The correlation between the two detection methods was found to be 0.993 9, indicating that the established dcELISA could be used for the rapid qualitative and quantitative detection of ZEN residuals in Coicis Semen.
Humans ; Female ; Pregnancy ; Zearalenone ; Coix ; Enzyme-Linked Immunosorbent Assay ; Mycotoxins ; Antibodies, Monoclonal

Zearalenone is one of the most widely polluted Fusarium toxins in the world, seriously endangering livestock and human health. Zearalenone hydrolase (ZHD) derived from Clonostachys rosea can effectively degrade zearalenone. However, the high temperature environment in feed processing hampers the application of this enzyme. Structure-based rational design may provide guidance for engineering the thermal stability of enzymes. In this paper, we used the multiple structure alignment (MSTA) to screen the structural flexibility regions of ZHD. Subsequently, a candidate mutation library was constructed by sequence conservation scoring and conformational free energy calculation, from which 9 single point mutations based on residues 136 and 220 were obtained. The experiments showed that the thermal melting temperature (T_m) of the 9 mutants increased by 0.4-5.6 ℃. The S220R and S220W mutants showed the best thermal stability, the T_m of which increased by 5.6 ℃ and 4.0 ℃ compared to that of the wild type. Moreover, the thermal half-inactivation time at 45 ℃ were 15.4 times and 3.1 times longer, and the relative activities were 70.6% and 57.3% of the wild type. Molecular dynamics simulation analysis showed that the interaction force at and around the mutation site was enhanced, contributing to the improved thermal stability of ZHD. The probability of 220-K130 hydrogen bond of the mutants S220R and S220W increased by 37.1% and 19.3%, and the probability of K130-D223 salt bridge increased by 30.1% and 12.5%, respectively. This work demonstrated the feasibility of thermal stability engineering strategy where the structural and sequence alignment as well as free energy calculation of natural enzymes were integrated, and obtained ZHD variants with enhanced thermal stability, which may facilitate the industrial application of ZHD.
Humans ; Hydrolases ; Zearalenone ; Trichothecenes ; Gene Library ; Hydrogen Bonding

The zearalenone hydrolase (ZHD101) derived from Clonostachys rosea can effectively degrade the mycotoxin zearalenone (ZEN) present in grain by-products and feed. However, the low thermal stability of ZHD101 hampers its applications. High throughput screening of variants using spectrophotometer is challenging because the reaction of hydrolyzing ZEN does not change absorbance. In this study, we used ZHD101 as a model enzyme to perform computation-aided design followed by experimental verification. By comparing the molecular dynamics simulation trajectories of ZHD101 at different temperatures, 32 flexible sites were selected. 608 saturated mutations were introduced into the 32 flexible sites virtually, from which 12 virtual mutants were screened according to the position specific score and enzyme conformation free energy calculation. Three of the mutants N156F, S194T and T259F showed an increase in thermal melting temperature (ΔTm>4 °C), and their enzyme activities were similar to or even higher than that of the wild type (relative enzyme activity 95.8%, 131.6% and 169.0%, respectively). Molecular dynamics simulation analysis showed that the possible mechanisms leading to the improved thermal stability were NH-π force, salt bridge rearrangement, and hole filling on the molecular surface. The three mutants were combined iteratively, and the combination of N156F/S194T showed the highest thermal stability (ΔTm=6.7 °C). This work demonstrated the feasibility of engineering the flexible region to improve enzyme performance by combining virtual computational mutations with experimental verification.
Computer-Aided Design ; Edible Grain ; Enzyme Stability ; Hydrolases/metabolism* ; Hypocreales/enzymology* ; Protein Engineering ; Zearalenone

Zearalenone(ZEN) is a mycotoxin produced by Fusarium, possessing estrogen-like effects, carcinogenicity, and multiple toxicities. To seek more efficient and practical agents for biological detoxification and broaden their application, this study isolated 194 bacterial strains from the moldy tuberous root of Pseudostellaria heterophylla, which were co-cultured with ZEN. An efficient ZEN-degrading strain H4-3-C1 was screened out by HPLC and identified as Acinetobacter calcoaceticus by morphological observation and molecular identification. The effects of culture medium, inoculation dose, culture time, pH, and temperature on the degradation of ZEN by H4-3-C1 strain were investigated. The mechanism of ZEN degradation and the degrading effect in Coicis Semen were discussed. The degradation rate of 5 μg·mL~(-1) ZEN by H4-3-C1 strain was 85.77% in the LB medium(pH 6) at 28 ℃/180 r·min~(-1) for 24 h with the inoculation dose of 1%. The degradation rate of ZEN in the supernatant of strain culture was higher than that in the intracellular fluid and thalli. The strain was inferred to secret extracellular enzymes to degrade ZEN. In addition, the H4-3-C1 strain could also degrade ZEN in Coicis Semen. If the initial content of ZEN in Coicis Semen was reduced from 90 μg·g~(-1) to 40.68 μg·g~(-1), the degradation rate could reach 54.80%. This study is expected to provide a new strain and application technology for the biological detoxification of ZEN in food processing products and Chinese medicinal materials.
Bacteria ; Fusarium ; Mycotoxins ; Temperature ; Zearalenone

OBJECTIVE: Zearalenone (ZEA) is a mycotoxin with potent estrogenic effects. Saffron is an herbal product that has antioxidant activities. The objective of this study was to investigate the protective role of saffron against reproductive toxicity induced by ZEA in female mice. METHODS: Ninety 8-week-old female mice were randomly allocated into three treatment groups. The first group received an intraperitoneal injection of ZEA (2.5 mg/kg) on alternate days. The second group received ZEA (2.5 mg/kg) on alternate days plus oral saffron daily (50 mg/kg). The third group was treated with a vehicle of 1% dimethyl sulfoxide (DMSO) on alternate days, as a control. Ten mice were euthanized from each group at 30, 60, and 90 days of treatment. Serum levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), estradiol (E2), and progesterone (P) were assessed. The uterus and ovaries were examined for changes in size or morphology. RESULTS: Serum levels of LH, FSH, E2, and P in the female mice treated with ZEA plus saffron were significantly higher than in those treated with ZEA alone, and were not significantly different from those treated with 1% DMSO. The female mice treated with ZEA alone showed a reduction in size of the uterus and abnormal architecture of the ovaries. CONCLUSION: The administration of saffron to female mice resulted in a significant reduction in ZEA-induced alterations in reproductive hormone levels, the size of the uterus, and the morphology of the ovaries.
Animals ; Antioxidants ; Crocus* ; Dimethyl Sulfoxide ; Estradiol ; Estrogens ; Female* ; Follicle Stimulating Hormone ; Humans ; Injections, Intraperitoneal ; Luteinizing Hormone ; Mice* ; Ovary ; Progesterone ; Uterus ; Zea mays ; Zearalenone

A one-step dual flow immunochromatographic assay (DICGA), based on a competitive format, was developed for simultaneous quantification of ochratoxin A (OTA) and zearalenone (ZEN) in corn, wheat, and feed samples. The limit of detection for OTA was 0.32 ng/ml with a detection range of 0.53‒12.16 ng/ml, while for ZEN it was 0.58 ng/ml with a detection range of 1.06‒39.72 ng/ml. The recovery rates in corn, wheat, and feed samples ranged from 77.3% to 106.3% with the coefficient of variation lower than 15%. Naturally contaminated corn, wheat, and feed samples were analyzed using both DICGA and liquid chromatography-tandem mass spectrometry (LC-MS/MS) and the correlation between the two methods was evaluated using a regression analysis. The DICGA method shows great potential for simple, rapid, sensitive, and cost-effective quantitative detection of OTA and ZEN in food safety control.
Animal Feed ; Calibration ; Chromatography, Affinity ; Chromatography, Liquid ; Colloids ; Food Contamination/analysis* ; Food Safety ; Gold ; Immunoassay/methods* ; Inhibitory Concentration 50 ; Limit of Detection ; Metal Nanoparticles ; Ochratoxins/analysis* ; Regression Analysis ; Reproducibility of Results ; Sensitivity and Specificity ; Triticum ; Zea mays ; Zearalenone/analysis*

OBJECTIVETo elucidate the dietary exposure to deoxynivalenol (DON) and zearalenone (ZEN) from cereal-based products in Chinese populations using the probabilistic assessment approach.

METHODSA total of 292 wheat flours and 347 corn-based products were collected from sampling sites of 107 supermarkets or farmers markets, which were randomly selected from 44 cities of 13 provinces in 2009 by the stratified cluster random sampling method. Then, DON and ZEN contamination levels in these samples above analyzed by UPLC-MS/MS in combination with the food consumption data of 68 959 respondents, who were divided into group 1 aged 3 to 13 years old, and group 2 aged 14 and over 14 years old (≥14 years old), obtained by China National Nutrition and Health Survey in 2002 were investigated. A probabilistic assessment model using Monte Carlo simulation was applied to derive the intake distribution of P(1)-P(99) percentile of dietary exposure to DON and ZEN. Meanwhile, all parameters related to dietary exposure to both toxins were compared with either the provisional maximum tolerable daily intake (PMTDI) of 1 µg·kg(-1)·d(-1) for DON, or the tolerable daily intake (TDI) of 0.25 µg·kg(-1)·d(-1) for ZEN in order to evaluate the risk of dietary intake of two toxins and find the minimum percentile of dietary exposure to these two toxins. The statistical differences of dietary exposure to these two toxins between two groups were achieved by t test.

RESULTSThe detection frequencies of DON in wheat flours and corn-based products were 100% (292/292) and 97.4% (338/347), respectively. A total of 21 out of 639 samples (wheat flours: 5/292, corn-based products: 16/347) were positive for DON at the levels exceeding the Chinese regulatory limit of 1 000 µg/kg for DON. And the detection frequencies of ZEN in wheat flours and corn-based products were 53.4% (156/292) and 87.6% (304/347), respectively.54 out of 347 corn-based products and no wheat flours were positive for ZEN at the levels exceeding the Chinese regulatory limit of 60 µg/kg for ZEN. Meanwhile, the mean values (95% CI) of the P(50), P(75), P(90), P(95), P(97.5) and P(99) percentile of dietary exposure to DON in populations of 3 to 13 years old were 0.170 (0.170-0.171), 0.762 (0.759-0.765), 2.066 (2.038-2.069), 3.515 (3.501-3.530), 5.342 (5.314-5.372), and 9.220 (9.155-9.279) µg · kg(-1)·d(-1), which were higher than those in populations of ≥14 years old (0.131 (0.130-0.131), 0.500 (0.498-0.501), 1.280 (1.276-1.285), 2.138 (2.128-2.14), 3.510 (3.494-3.527), and 5.512 (5.474-5.546) µg·kg(-1)·d(-1)), with t values of 87.19, 163.87, 164.66, 157.78, 105.47 and 96.31, and all P values less than 0.001. And the mean values (95% CI) of the P(50), P(75), P(90), P(95), P(97.5) and P(99) percentile of dietary exposure to ZEN in populations of 3 to 13 years old were 0.001 (0.001-0.001), 0.006 (0.006-0.006), 0.039 (0.038-0.039), 0.101 (0.100-0.101), 0.195 (0.194-0.197) and 0.378 (0.374-0.381) µg · kg(-1)·d(-1), which were also higher than those in populations of ≥14 years old (0.001 (0.001-0.001), 0.004 (0.004-0.004), 0.026 (0.026-0.026), 0.061 (0.060-0.061), 0.115 (0.115-0.116) and 0.232 (0.231-0.235) µg·kg(-1)·d(-1)) with T-values of 151.11, 73.80, 96.81, 100.81, 91.93 and 76.13, and all P values less than 0.001. Besides, the minimum percentile of dietary exposure to DON in populations of 3 to 13 years old and ≥14 years old exceeded the corresponding PMTDI of 1 µg·kg(-1)·d(-1) was found in the probability distribution of P(76) (99% percentile = 1.03 µg·kg(-1)·d(-1)) and P(84) (95% percentile = 1.01 µg·kg(-1)·d(-1)) percentile, respectively. And the minimum percentile of dietary exposure to ZEN in populations of 3 to 13 years old and ≥14 years old exceeded the corresponding TDI of 0.25 µg·kg(-1)·d(-1) was found in the probability distribution of P(97) (95% percentile = 0.25 µg·kg(-1)·d(-1)) and P(98) (90% percentile = 0.26 µg·kg(-1)·d(-1)) percentile, respectively.

CONCLUSIONThe contamination levels of DON and ZEN in wheat flours and corn-based products and the risk of dietary exposure to both DON and ZEN in populations in Chinese populations were at relatively low levels. The dietary exposure to both DON and ZEN in populations of 3 to 13 years old was higher than those in populations of ≥14 years old . Populations of 3 to 13 years old were the populations at the high risk of dietary exposure to both mycotoxins.

Adolescent ; Adult ; Child ; Child, Preschool ; China ; Diet ; Edible Grain ; Food Contamination ; Humans ; Middle Aged ; Mycotoxins ; Tandem Mass Spectrometry ; Trichothecenes ; Triticum ; Zea mays ; Zearalenone

Zearalenone (ZEN) is a non-steroidal estrogenic mycotoxin produced by several species of Fusarium that are found in cereals and agricultural products. ZEN has been implicated in mycotoxicosis in farm animals and in humans. The toxic effects of ZEN are well known, but the ability of an alkaline Comet assay to assess ZEN-induced oxidative DNA damage in Chang liver cells has not been established. The first aim of this study was to evaluate the Comet assay for the determination of cytotoxicity and extent of DNA damage induced by ZEN toxin, and the second aim was to investigate the ability of N-acetylcysteine amide (NACA) to protect cells from ZEN-induced toxicity. In the Comet assay, DNA damage was assessed by quantifying the tail extent moment (TEM; arbitrary unit) and tail length (TL; arbitrary unit), which are used as indicators of DNA strand breaks in SCGE. The cytotoxic effects of ZEN in Chang liver cells were mediated by inhibition of cell proliferation and induction of oxidative DNA damage. Increasing the concentration of ZEN increased the extent of DNA damage. The extent of DNA migration, and percentage of cells with tails were significantly increased in a concentration-dependent manner following treatment with ZEN toxin (p < 0.05). Treatment with a low concentration of ZEN toxin (25 microM) induced a relatively low level of DNA damage, compared to treatment of cells with a high concentration of ZEN toxin (250 microM). Oxidative DNA damage appeared to be a key determinant of ZEN-induced toxicity in Chang liver cells. Significant reductions in cytolethality and oxidative DNA damage were observed when cells were pretreated with NACA prior to exposure to any concentration of ZEN. Our data suggest that ZEN induces DNA damage in Chang liver cells, and that the antioxidant activity of NACA may contribute to the reduction of ZEN-induced DNA damage and cytotoxicity via elimination of oxidative stress.
Acetylcysteine ; Animals, Domestic ; Cell Proliferation ; Edible Grain ; Comet Assay ; DNA ; DNA Damage ; Electrophoresis ; Estrogens ; Fusarium ; Humans ; Liver ; Mycotoxicosis ; Oxidative Stress ; Zearalenone

A monoclonal antibody (mAb) against zearalenone (ZEN) was produced using ZEN-carboxymethoxylamine and -BSA conjugates. Antibody produced by one clone showing a very high binding ability was selected and found to have a higher affinity for ZEN compared to a commerciall ZEN antibody. We developed two direct competitive ELISA systems using the selected antibody (ZEN-coated and anti-ZEN antibody-coated ELISA). Quantitative ranges for the anti-ZEN antibody-coated ELISA and ZEN-coated ELISA were from 25 to 750 ppb and from 12.5 to 100 ppb, respectively. The detection limit of both methods as measured with standard solutions was 10 ppb. The intra-plate and inter-well variation of both ELISAs were less than 10%. The IC50 values for alpha-zearalenol, beta-zearalenol, alpha-zearalanol, and beta-zearalanol compared to ZEN were 108.1, 119.3, 114.1, and 130.3% for the ZEN-coated ELISA. These values were 100.7, 120.7, 121.6, and 151.6% for the anti-ZEN antibody-coated ELISA. According to the anti-ZEN antibody-coated ELISA, the average recovery rates of ZEN from spiked animal feed containing 150 to 600 ng/mL of ZEN ranged from 106.07 to 123.00% with 0.93 to 2.28% coefficients of variation. Our results demonstrate that the mAb developed in this study could be used to simultaneously screen for ZEN and its metabolites in feed.
Aminooxyacetic Acid/*chemistry ; Animals ; Antibodies, Monoclonal/*immunology ; Enzyme-Linked Immunosorbent Assay/*methods ; Female ; Inhibitory Concentration 50 ; Mice ; Mice, Inbred BALB C ; Reproducibility of Results ; Serum Albumin, Bovine/chemistry ; Zearalenone/*immunology

In this study, we developed a novel tool for purifying two mycotoxins, aflatoxin B1 (AFB1) and zearalenone (ZEN), in feed. This system utilized monoclonal antibodies (mAbs) against AFB1 and ZEN, and magnetic nanoparticles (MNPs). Among ten MNPs with different diameters and functional groups, a 100-nm diameter MNP (fMA) conjugated to an amine group (-NH2) was found to be optimum for coupling with mAbs. The optimal mAb concentrations for coupling to the fMA along with mycotoxin purification capacities of the fMA-mAb conjugates (fMA-AFB1 and fMA-ZEN) were determined. A comparison of mean recovery rates (from corn and product X feed) between the fMA-mAb conjugates and immunoaffinity columns (IAC-AFB1 and IAC-ZEN) showed that the rate for fMA-AFB1 (90~92% and 81~88%) was higher (p > 0.05) than that of IAC-AFB1 (81~84% and 72~78%) for AFB1 (5, 10, 15 ng/mL), and the rate for fMA-ZEN (99~100% and 92~94%) was significantly higher (p < 0.01) than that of IAC-ZEN (86~88% and 81~88%) for ZEN (10, 25, 50 ng/mL) except at a concentration of 10 ng/mL, demonstrating the remarkable purification efficiency of the novel fMA-mAb method. Additionally, mycotoxin purification was much faster using our novel method (approx. 5 min) than the IAC-based technique (> 30 min). This study suggests that the novel purification system we developed would be a useful tool for monitoring and regulating mycotoxin contamination in feed, and replace IAC methods.
Aflatoxin B1 ; Antibodies, Monoclonal ; Magnetics ; Magnets ; Mycotoxins ; Nanoparticles ; Zea mays ; Zearalenone