Rapid detection of zearalenone in Coicis Semen based on ELISA / 中国中药杂志

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.

Structure-guided engineering for improving the thermal stability of zearalenone hydrolase / 生物工程学报

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 (Tm) of the 9 mutants increased by 0.4-5.6 ℃. The S220R and S220W mutants showed the best thermal stability, the Tm 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.

Does the mycotoxin ingestion by beef heifers on feedlot change the productive parameters?

In intensive beef cattle production systems, silage, corn, soy bean, and their coproducts are commonly used as feed. However, these ingredients are highly susceptible to contamination by fungi and mycotoxins, which may lead to immunological challenges and reduce animal production. The aim of the present study was to evaluate the effects of mycotoxin contamination of diet on intake, digestibility, and performance of heifers. Twenty non-pregnant (Nellore) heifers (age, >18 months; initial body weight, 348±30 kg) were used and randomly distributed in two treatments: (1) control (non-contaminated diet) and (2) zearalenone-contaminated diet (300 ppb). The diet comprised 70% corn silage and 30% concentrate. Individual dry matter intake and digestibility were estimated using external and internal markers. Heifer body weight was evaluated every week without fasting to calculate performance. The experimental design was completely randomized. Each animal was considered one experimental unit. Assumptions were tested for variance analyses (error normality, independence of errors, and homogeneity of variances) (p<0.05). There were no differences in dry matter intake (p=0.96) and digestibility (p=0.62). Performance (kg/day) did not vary as a function of zearalenone ingestion (p=0.68). Therefore, contamination of diet with 300 ppb zearalenone did not affect the intake, digestibility, and performance of feedlot-finished heifers.

Screening of zearalenone-degrading bacteria and analysis of degradation conditions / 中国中药杂志

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.

Computation-aided design of the flexible region of zearalenone hydrolase improves its thermal stability / 生物工程学报

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.

Influência da qualidade micotoxicológica e nutricional de híbridos de milho no custo da ração de frangos de corte / Influence of mycotoxicological and nutritional quality of maize hybrids on broiler chickens feed cost

Objetivou-se avaliar as variáveis micotoxicológicas e nutricionais de híbridos de milho com diferentes características que influenciam no custo da ração para frangos de corte. Foram avaliados 26 híbridos de milho geneticamente modificados nas safrinhas de 2016 e 2017, com diferentes germoplasmas, textura de endosperma e duração do ciclo. Nos híbridos, foram avaliados grãos avariados, fumonisinas (B1+B2) (FUM), aflatoxinas (B1+B2+G1+G2) (AFLA), zearalenona (ZEA), deoxinivalenol (DON), umidade, proteína bruta (PB), energia metabolizável aparente corrigida para balanço de nitrogênio (EMAn), aminoácidos digestíveis para aves (lisina, metionina, cistina e treonina) e o respectivo custo da ração inicial para frangos de corte, que foi calculada pelo custo mínimo. A prevalência de FUM, AFLA, ZEA e DON foi de 90, 17, 33 e 0%, com médias de 3067, 1, 38 e 0µg/kg nos dois anos, respectivamente. A média de EMAn e PB foi de 3264kcal/kg e 8,02%, respectivamente, e diferiu (P<0,05) nos dois anos. O custo da ração foi influenciado significativamente (P<0,05) por FUM, PB, EMAn nos dois anos. Híbridos com tecnologia Viptera apresentam menor concentração por FUM e menor custo da ração. Híbridos de ciclo precoce têm menor concentração de FUM, maiores percentuais de PB e de aminoácidos digestíveis e menor custo da ração.(AU)

The objective of this study was to evaluate the mycotoxicological and nutritional variables of maize hybrids with different characteristics that influence the broiler chicken's feed costs. In 2016 and 2017 winter crops, 26 genetically modified hybrids of maize with different germplasm, endosperm texture and cycle duration were evaluated. The analyzed variables were damaged grains, fumonisins (B 1 +B 2 ) (FUM), aflatoxins (B 1 +B 2 +G 1 +G 2 ) (AFLA), zearalenone (ZEA), deoxynivalenol (DON), moisture, crude protein (CP), apparent metabolizable energy corrected for nitrogen balance (AMEn), digestible amino acids for poultry (lysine, methionine, cystine and threonine) and the respective cost of the initial feed for broiler chickens calculated at the minimum cost. The prevalence of FUM, AFLA, ZEA and DON was 90, 17, 33 and 0%, with means of 3067, 1, 38 and 0µg/kg in the two years, respectively. The mean of AMEn and CP was 3264kcal/kg and 8.02%, respectively, and differed (P< 0.05) in the two years. The feed cost was significantly influenced (P<0.05) by FUM, PB, AMEn in two years. Hybrids with Viptera technology show lower concentration per FUM and lower feed cost. Early cycle hybrids have lower concentrations of FUM, higher percentages of CP and digestible amino acids, and lower feed costs.(AU)

Progress in bio-degradation of mycotoxin zearalenone / 生物工程学报

Zearalenone (ZEN) and its derivatives are non-steroidal estrogenic mycotoxins mainly produced by Fusarium species. They are widely distributed in grain feeds originated from maize, barley, wheat and sorghum, causing serious harm to animal and human health. Currently, there is a pressing need of an efficient technology for ZEN degradation and detoxification. Because traditional physical and chemical methods could not effectively detoxify ZEN in grains, and might also affect the grain nutrients and food taste, and even result in secondary pollution, the biological technologies are developed to detoxify ZEN and its derivatives. In this paper, we reviewed the structure of ZEN and its derivatives, the fungi and bacteria species with ability of degradation of ZEN. In addition, the characterization, protein sequences and conformation of currently identified ZEN degrading enzymes, the only solved ZHD structure from Clonostachys rose were analyzed and compared, and the enzymes heterologous expression and application were also reviewed. This review will provide reference for reducing the cost of ZEN degrading enzymes by biological technologies such as enzyme engineering and fermentation engineering.

Dual flow immunochromatographic assay for rapid and simultaneous quantitative detection of ochratoxin A and zearalenone in corn, wheat, and feed samples / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

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.

Protective effects of saffron against zearalenone-induced alterations in reproductive hormones in female mice (Mus musculus) / 대한생식의학회지

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.

Zearalenone adsorption capacity of lactic acid bacteria isolated from pigs

ABSTRACT The ability to adsorb zearalenone by five strain of lactic acid bacteria was evaluated: four strains of Lactobacillus spp. isolated from pig rectal swabs and one commercial strain (Lactobacillus rhamnosus). Several factors affecting the adsorption capacity were evaluated in order to improve the adsorption of the mycotoxin by bacteria. The stability of the zearalenone-bacteria complex was analyzed. In every case, bacterial adsorption capacity was higher than 40.0%. The strain showing the highest adsorption (68.2%) was selected for the following steps of this research. The adsorption percentages obtained after processing 6.5 and 7.5 mL MRS broth were 57.40% + 3.53 and 64.46% + 0.76, respectively. The stability of zearalenone-bacteria complex was evaluated by successively rinsing. In the first rinsing step 42.26% + 0.414 was still bound. In the second rinsing step 25.12% + 0.664 was still bound, whereas 15.82% + 0.675 remained in the pellet after the third rinse. Results obtained demonstrated that Lactic Acid Bacteria has capacity to adsorb zearalenone. Finally adsorption was increased using a higher volume of initial broth. These results could be used to design a new lyophilized powder for detoxification, using lactic acid bacteria as potential zearalenone adsorbents.

Impact of zearalenone on male fertility: An update / 中华男科学杂志

Zearalenone (ZEA) is a nonsteroidal estrogen-like mycotoxin widely distributed in maize, wheat, rice and other cereals with its derivants. It also presents in meat or dairy products or even in the aquatic ecosystem via rain, and thus can affect human health. ZEA affects the body function in various ways. On the one hand, it can disturb the synthesis of estrogen and its combination with the receptor, influence the reproductive ability via the estrogen signaling pathway, and cause the dysfunction of the reproductive systems. On the other hand, it can disturb the synthesis of DNA and proteins and result in lipid peroxidation and cytotoxicity by inducing the apoptosis of germ cells. It is known that exposure to different doses of ZEA can affect the female reproductive system by increasing the apoptosis of germ cells and inducing germ cell prematurity, sexual precocity, endocrine disorder, reproductive cycle disorder, and so on. But studies of its influence on the male reproductive system are relatively rare, especially about its unique male-related action mechanisms. This review presents an overview of the studies on the mechanisms of ZEA affecting male fertility and the phenotype changes in the male reproductive system after exposure to ZEA, hoping to provide some new ideas for the protection of human fertility.

Preparation and Application of Immunoaffinity Column for Multi-mycotoxins / 分析化学

The three-in-one immunoaffinity column ( IAC ) for the determination of aflatoxin B1 ( AFB1 )-zearalenone (ZEN)-deoxynivalenol (DON) was prepared with rProtein A-sepharose 4B as the column matrix. The comprehensive performance ( such as nonspecific adsorption, column blank, column capacity, column efficiency and sample standard addition recovery rate) was evaluated and investigated. The results showed that, the column capacities of AFB1 , ZEN, DON were 295 ng per 0. 25 mL gel, 905 ng per 0. 25 mL gel, 2342 ng per 0. 25 mL gel, respectively, and the column blank was 0. The average recoveries of AFB1 , ZEN and DON were 97. 4%, 98. 0% and 98. 4%, respectively. By optimizing conditions, the samples were extracted using the mixture of methanol and water (80:20, V/V), and diluted with phosphate buffered saline (contain 0. 1% Tween-20, PBST). The detection results of FAPAS (Food Analysis Performance Assessment Scheme) by different batch three-in-one columns were close to the target value. The prepared three-in-one immunoaffinity column which could take the place of conventional single immunoaffinity column was able to meet the requirement for treatment of food and feed samples, and lay a foundation for one step enrichment, purification and detection of multi-mycotoxins.

Correlation of zearalenone pollution with precocious puberty girls / 中华实用儿科临床杂志

Objective To explore the relationship of zearalenone (ZEA) and precocious puberty in girls.Methods The peripheral serums from 71 cases of precocious puberty girls and 50 cases of healthy girls were collected respectively and concentrations of ZEA were detected by high performance liquid chromatography.Bone age,body mass index (BMI),volume of uterus and ovaries,concentrations of estradiol (E2),luteinizing hormone (LH) and folliclestimulating hormone (FSH) were detected,and the residence of each subject was recorded as well.Results (1) In 71 patients,52 patients were diagnosed as idiopathic central precocious puberty,others were diagnosed as premature thelarche.(2) In 71 patients,serum ZEA was detected from 51 patients,and undetected in 20 patients.(3)Concentration of ZEA in precocious puberty girls [(318 ±34) ng/L]was significantly increased than that in healthy girls [(143 ± 35) ng/L,P =0.002],but no distinct difference existed between ICPP group and PT group(P =0.326).(4)Compared with uterus volume of ZEA in the undetected patients (1.975 ±0.150) cm3,the uterus volume of ZEA detected patients (2.972 ±0.180) cm3,which was significantly enlarged,there was significant difference (P =0.01) ; Percentage of overweight girls in ZEA detected patients (31.4％,16/51 cases) was lower than which in ZEA undetected patients (65.0％,13/20 cases,P =0.01) ; Although there was no statistical differences in the breast diameter,bone age,value of E2,LH and FSH between ZEA detected patients and undetected patients (all P ＞ 0.05),but the increased tendency in ZEA detected patients existed.(5) ZEA in serum was detected in 53.3％ (16/30 cases) patients living in the cities,and the rate was obviously lower than 82.9％ of the patients (34/41 cases) living in the countryside,there was significant difference (P =0.007).Conclusions ZEA is correlated with precocious puberty in girls.ZEA pollution might be one of reasons for precocious puberty occurrence.

Study progress of relativity between zearalenone pollution and precocious puberty in children / 中华实用儿科临床杂志

As one of endocrine dysfunction related diseases among children,precocious puberty has trend for increased rates in recent years.Widespread children exposure to known or suspected endocrine disrupting chemicals has been documented in worldwide,in which exogenous estrogen chemicals trigger precocious puberty onset have been attracting more and more attentions.Zearalenone widely contaminates foods such as grain,milk,meat and eggs,which displayed its estrogen effect to disrupt organism development.In this review,Zearalenone is suspected as triggering factor for precocious pubertal to be discussed,will lead to people pay more attentions to food safety and the prevention of precocious puberty.

Occurrence of zearalenone in wheat- and corn-based products commercialized in the State of Paraná, Brazil

The productivity of wheat and corn crops depends on climatic conditions and resistance against phytopathogenic fungi such as those of the genus Fusarium. Some species of this genus produce zearalenone (ZEA), a mycotoxin with hyperestrogenic effects. The objective of this study was to investigate the presence of ZEA in samples of cracked wheat (n = 109), popcorn (n = 51) and corn grits (n = 50) commercialized in the State of Paraná, Brazil. Commercial samples of each crop were collected between September 2007 and June 2008 and analyzed by thin-layer chromatography. The method used for detection of the mycotoxin in wheat and corn derivatives presented a recovery rate of 94.5% and 99.5%, respectively, detection limit of 40 μg.kg-1 and quantification limit of 55 μg.kg-1. No contamination with ZEA was detected in cracked wheat samples. Among the corn derivatives, only one cracked corn sample was contaminated with ZEA (64 μg.kg-1). Despite the low contamination observed, monitoring the occurrence of mycotoxins in foods is important to ensure safety.

Evaluation of Oxidative DNA Damage Using an Alkaline Single Cell Gel Electrophoresis (SCGE) Comet Assay, and the Protective Effects of N-Acetylcysteine Amide on Zearalenone-induced Cytotoxicity in Chang Liver Cells

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.

Effect of Zearalenone administrated at late gestation days on fertility of the F-1 offspring in Swiss webster (Mus musculus) / Efecto de la Zearalenona administrada los días finales de gestación sobre la fertilidad de las crías F-1 en ratones Swiss Webster (Mus musculus)

Zearalenonesolution was given to dams micesubcutaneouslyat a dose of30 mg/kg body weightat the age ofpregnancy 13 to 18 days.Control micewere given only sesameoil. Furthermore, damsmice were allowed todeliver their litter and pups were weanedon 21 days of age. The live birth index andviability of the F-1 offspring were recorded. Determination of fertility of the F-1 offspring were undertaken by mating interlitters. On days 18 of gestation, the F-1 offspring were killed by cervical dislocation. The observation was performed on the number of live or dead fetus, embryo resorption, the number of implantation and the percentage of gestation loss. The result revealed that administration of zearalenone on days 13 to 18 of gestation caused a significant decreasein the number of implantation as the result of mating between control male with treated female and treated male with tretaed female of the F-1 offspring. It could be concluded that in the F-1 offspring, zearalenone interfered the process of ovarian develoment, stimulated the differentation of the uterus, decreased the fertility of the female and the effect of zearalenon was more persistent in the female than in the male...

Se administró una solución subcutánea de zearalenona en una dosis de 30 mg/kg de peso corporal en ratonas preñadas entre 13 a 18 días de gestación. Los ratones control recibieron sólo aceite de sésamo. Además, a las ratonas preñadas se les permitió amamantar a sus crías para ser destetadas a los 21 días de edad. El índice de nacidos vivos y viabilidad de la descendencia F-1 fuer registrado. La determinación de la fertilidad de la descendencia F-1 se llevó a cabo por apareamiento intercrías. El día 18 de gestación, la descendencia F-1 se sacrificó por dislocación cervical. Se observó el número de fetos vivos y muertos, reabsorción del embrión, número de implantaciones y porcentaje de pérdida de gestación. El resultado reveló que la administración de la zearalenona entre los días 13-18 de gestación causó un significativo descenso en el número de implantaciones, como resultado del apareamiento entre machos controles con hembras tratadas y machos tratados con hembras tratadas (F-1 crías). Se puede concluir que en la descendencia F-1, la zearalenona interfiere en el proceso de desarrollo ovárico, estimulado la diferenciación del útero, disminuyendo la fertilidad de la hembra; además el efecto del zearalenon fue más persistente en la hembra que en el macho...

Hepatic hyperplasia and damages induces by zearalenone Fusarium mycotoxins in BALB/c mice / Hiperplasia e danos hepáticos induzidos por micotoxinas do gênero Fusarium-zearalenone em camundongos BAB/c

CONTEXT: Zearalenone is a mycoestrogen and considered a mycotoxin. OBJECTIVE: To establish whether zearalenone produced hepatotoxicity via oral administration. METHODS: Zearalenone was orally administered at a dose of 50 mg, 100 mg and 200 mg ZEN/body weight/daily, respectively, for 14 days to three groups of BALB/c mice. Diagnostic modalities used to evaluate hepatic damage and impaired hepatic function pre- and post zearalenone administration included hepatic marker enzyme activity, pentobarbital sleeping time, cytochrome P-450 activities and histopathologic evaluation of liver. RESULTS: Significant histopathologic changes viz. sinusoidal congestion, cytoplasmic vacuolization, hepatocellular necrosis and neutrophil infiltration were observed after evaluating of liver section from each group after accumulated zearalenone exposure. Further, zearalenone exposure increased activities of alanine transaminase, aspartate transaminase and lipid peroxides whereas activities of tissue glutathione and cytochrome P450 were decreased as compared to control mice. Zearalenone also increased the sleeping time and decreased sleeping latency after pentobarbital through intraperitoneal route as compared to control mice which indicates that the impairment of hepatic metabolizing enzymes by zearalenone. CONCLUSION: Zearalenone is a potential hepatotoxin by oral route.

CONTEXTO: Zearalenone é um micoestrógeno e considerado como micotoxina. OBJETIVO: Avaliar se o Zearalenone produz hepatotoxicidade por administração via oral. MÉTODOS: Zearalenone foi administrada por via oral em doses de 50 µg, 100 µg e 200 µg/peso corporal/dia/14 dias, respectivamente, para três grupos de camundongos BAB/C. Modalidades diagnósticas usadas para avaliar o dano hepático e comprometimento da função hepática pré- e pós-administração de Zearalenone incluíram atividade enzimática de marcadores hepáticos, tempo de sono por pentobarbital, atividade do citocromo P-450 e avaliação histopatológica hepática. RESULTADOS: Alterações histopatológicas significantes como congestão sinusoidal, vacuolização citoplasmática, necrose hepatocelular e infiltração neutrofílica foram observadas após avaliação histológica de cada grupo após exposição acumulada de Zearalenone. Além disto, a exposição à Zearalenone incrementou a atividade das enzimas alanina transaminase e aspartato transaminase e peróxidos lipídicos, ao passo que as atividades teciduais de glutationa e citocromo P-450 diminuiram, quando comparadas com camundongos-controle. Zearalenone também aumentou o tempo de sono e diminuiu a latência do sono após a administração de pentobarbital por via intra-abdominal, quando comparados com camundongos-controle, o que indica o comprometimento das enzimas do metabolismo hepático por ela. CONCLUSÃO: Zearalenone é uma potente hepatotoxina quando administrada por via oral.

A novel mycotoxin purification system using magnetic nanoparticles for the recovery of aflatoxin B1 and zearalenone from feed

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 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.

Production of a highly group-specific monoclonal antibody against zearalenone and its application in an enzyme-linked immunosorbent assay

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.