Effects of in vitro exposure to natural levels of zearalenone and its derivatives on chromatin structure stability in equine spermatozoa

The purpose of this study was to assess the natural exposure of male horses (Equus caballus) to the mycotoxin zearalenone (ZEA) by using the ELISA test and to evaluate the effects of in vitro exposure of sperm cells to mycotoxin-containing urine extracts on sperm chromatin structure stability. Because of their occurrence in urine samples, ZEA and its derivatives were tested by sperm chromatin structure assay (SCSA) at natural levels detected by ELISA. Thirty-eight urine extracts of Italian (n = 11) and northeastern European (n = 27) horses were tested on frozen-thawed spermatozoa to evaluate the toxic effect of mycotoxin on their chromatin structure by flow cytometry. Different parameters of the DNA fragmentation index (DFI), such as the mean (), the percentage (%-DFI), and the standard deviation (SD-DFI), were analyzed. Urine samples showed a mean level of 32.3 ng/mL ZEA with significantly higher concentrations in northeastern European samples than in Italian samples, probably in relation to climatic and feeding differences. The toxic effects of ZEA-containing urine samples on SCSA parameters were found at low ZEA concentrations and were mainly observed in Italian samples. By using mycotoxin standards, ZEA, α-zearalenol, and β-zearalenol proved to be more toxic compounds for sperm chromatin stability than other tested derivatives. A nongenomic mechanism of action can be hypothesized.     

Urinary zearalenone measured with ELISA as a biomarker of zearalenone exposure in pigs

The suitability to assess zearalenone (ZEA) exposure in pigs of a commercial ELISA kit for ZEA analysis in urine was tested. A daily dose of 0, 5, 10, 20 and 40 μg synthetic ZEA per kilogram BW was administered via the feed to four gilts per dose group, and after 3 and after 7 days of ZEA intake, urine samples were assayed with the ELISA which has a relative cross-reactivity of 42 % with α-zearalenol. The concentration of urinary ZEA equivalents (ZEA plus 42 % of α-zearalenol present) did not differ between day 4 and day 8 (P?=?0.50) within each dose group. The urinary ZEA equivalent/creatinine ratio was tightly correlated with ZEA intake (r?=?0.95). The urinary ZEA equivalent/creatinine values at 0 and 40 μg/kg BW were distinctly different from those of the intermediate dose levels, whereas there was some overlapping of the individual values at the dose levels 5, 10 and 20 μg/kg BW. The urinary ZEA equivalent/creatinine ratio can be used as a biomarker for ZEA exposure in pigs provided that urine samples of several animals receiving the same diet are assayed, either separately or after pooling.     

Effects of deoxynivalenol (DON), zearalenone (ZEN), and related metabolites on equine peripheral blood mononuclear cells (PBMC) in vitro and background occurrence of these toxins in horses

Both deoxynivalenol (DON), zearalenone (ZEN), and their metabolites are known to modulate immune cells in various species whereby viability and proliferation are influenced. Such effects were rarely examined in horses. Therefore, one aim of the present study was to titrate the inhibitory concentrations of DON, 3-acetyl-DON (3AcDON), de-epoxy-DON (DOM-1), ZEN, and α- and β-zearalenol (ZEL) at which viability and proliferation of equine PBMC were reduced by 50 % (IC₅₀) and 10 % (IC₁₀) in vitro. For evaluation of practical relevance of the in vitro findings, a further aim was to screen horses for the background occurrence of DON, ZEN, and their metabolites in systemic circulation and to relate toxin residues both to the inhibitory toxin concentrations and to hematological and clinical-chemical characteristics.The IC₅₀ (μM) for DON, 3AcDON, β-ZEL, α-ZEL, and ZEN were determined at 3.09, 25.90, 75.44, 97.44, and 98.15 in unstimulated cells, respectively, while in proliferating cells, the corresponding IC₅₀ values were 0.73, 6.89, 45.16, 75.96, and 82.51. Neither viability nor proliferation was influenced by DOM-1 up to a concentration of 100 μM.The in vivo screening (N?=?49) revealed the occurrence of ZEN (N?=?24), α-ZEL (N?=?3), β-ZEL (N?=?37), DON, and DOM-1 (N?=?2). The detected concentrations were much lower than the corresponding IC₅₀ while the IC₁₀ of DON and β-ZEL for proliferating PBMC corresponded to approximately 26 and 35 ng/mL which might be relevant when contaminated diets are fed.Clinical-chemical and hematological traits were not related to mycotoxin residue levels excepting blood urea nitrogen which was positively correlated to the sum of β-ZEL, α-ZEL, and ZEN concentration. Whether this reflects simply the feeding history of the horses or renal failures giving rise to a prolonged half-life of the toxins needs to be clarified further.     

Stability of fumonisin B<Subscript>1</Subscript>, deoxynivalenol,zearalenone, and T-2 toxin during processing of traditional Nigerian beer and spices

The stability of the Fusarium mycotoxins fumonisin B₁, deoxynivalenol, T-2 toxin, and zearalenone during processing of Nigerian traditional spices (dawadawa, okpehe, and ogiri) and beer (burukutu) using artificially contaminated raw materials was investigated. Results revealed the reduction of these toxins in all the final products. Boiling played a significant role (p?<?0.05) in Fusarium mycotoxin reduction in the traditional spices. The highest percentage reduction of deoxynivalenol (76%) and zearalenone (74%) was observed during okpehe processing (boiled for 12 h). Dehulling and fermentation further demonstrated a positive influence on the reduction of these toxins with a total reduction ranging from 85 to 98% for dawadawa, 86 to 100% for okpehe, and 57 to 81% for ogiri. This trend was also observed during the production of traditional beer (burukutu), with malting and brewing playing a major impact in observed reduction. In addition, other metabolites including deoxynivalenol-3-glucoside, 15-acetyl-deoxynivalenol, α-zearalenol, and β-zearalenol which were initially not present in the raw sorghum were detected in the final beer product at the following concentrations 26?±?11, 16?±?7.7, 22?±?18, and 31?±?16 μg/kg, respectively. HT-2 toxin was also detected at a concentration of 36?±?13 μg/kg along the processing chain (milled malted fraction) of the traditional beer. For the traditional spices, HT-2 toxin was detected (12 μg/kg) in ogiri. Although there was a reduction of mycotoxins during processing, appreciable concentrations of these toxins were still detected in the final products. Thus, the use of good quality raw materials significantly reduces mycotoxin contamination in final products.     

Fermentation of wort containing deoxynivalenol and zearalenone

Wort containing deoxynivalenol and zearalenone, each added at a level of 1.9 μg/mL, was fermented by 3 strains ofSaccharomyces cerevisiae for 7 or 9 days to make beer. Analysis showed that deoxynivalenol was stable during this process. The major metabolite of zearalenone was β - zearalenol, which formed in up to 69% of the initial zearalenone concentration, while up to 8.1% of the initial zearalenone was converted to α - zearalenol. The major part of the metabolism of zearalenone occurred by 1 – 2 days. Control experiments, where the yeasts were omitted and deoxynivalenol, zearalenone and α - and β - zearalenol were added, showed good recovery and stability of the mycotoxins over the 7–9 day time period. No deoxynivalenol, zearalenone, α-zearalenol or β-zearalenol was detected in control yeast fermentations where they were not added to the wort.     

Expression profiling of the genes responding to zearalenone and its analogues using estrogen-responsive genes

To compare gene expression profiles in response to estrogen or 17β-estradiol (E₂) and a mycotoxin, zearalenone (ZEA), and its analogues (collectively termed ZEA compounds), breast cancer MCF-7 cells were treated with 10 nM of E₂ or ZEA compounds including ZEA, α-zearalenol, β-zearalenol, zearalanone, α-zearalanol and β-zearalanol. Expression profiles for 120 estrogen-responsive genes were subjected to cluster and statistical analyses using correlation coefficients or R-values. We found that all of the ZEA compounds stimulated the growth of MCF-7 cells, as much as E₂, and showed similar expression profiles to that of E₂ (R-values ranged from 0.82 to 0.96). The effect of ZEA compounds was likely mediated by estrogen-receptor-dependent Erk1/2-signaling. These results provide clues to understand the mechanism of their estrogen-like action.     

Inducing effect of testosterone on the hepatic reduction of zearalenone in the female prepubertal rat

The prepubertal responsiveness of 3α-hydroxysteroid dehydrogenase and zeara-lenone-reducing activity to either a large subcutaneous dose of testosterone or dietary zearalenone was investigated in female rats. Testosterone induced both the activity of the 3α-hydroxysteroid dehydrogenase, with androsterone as substrate, and zearalenone reduction to α- and (β-zearalenol. Zearalenone had no effect on the activity of 3α-hydroxysteroid dehydrogenase, but had a slight inducing effect on the zearalenone reduction to β-zearalenol. Both testosterone and zearalenone had a growth-promoting effect on uterus.     

The differentiation of α- and β -glucosidase and α- and β-galactosidase isoenzymes from maize and broad bean root tips using disc electrophoresis in polyacrylamide gels

For the separation of α- and β-glucosidase and α- and β-galactosidase isoenzymes fromZea mays L. andVicia fabaL. root tips the system of disc electrophoresis in polyacrylamide gel developed for basic protein separation proved most suitable. The detection was carried out by a simultaneous azocoupling reaction. In maize α-glucosidase was not detected, β-glucosidase gave 3, α-galactosidase 4, and β-galactosidase 3 zones. In broad bean a- and β-glucosidases were absent, α-galactosidase gave 2 and β-galactosidase 3 zones, α- and β-galactosidase activity zones correspond principially to each other in their position. In maize one zone gives a positive reaction for both β-glucosidase and α- and β-galactosidaso.     

The localization and the isoenzymes of α- and β-Galactosidases in root tips

The localization was studied of α- and β-galactosidases in frozen sections of Ca-formol fixed root tips using simultaneous azocoupling reaction. In all species studied (Allium cepa,Cucurbita maxima, Lupinus albus, Pisum sativum, Vicia faba, Zea mays) positive results were obtained, the localization being ubiquitous (according to localization typology given here). InVicia faba andZea mays the isoenzymes of α- and β-galactosidases were revealed by means of acrylamide gel electrophoresis, using authors’ modification of Reisfeld method, in whole root tips, particular growth zones and separately in cortex and central cylinder. No differences were observed comparing stele and cortex. Whereas characteristic isoenzyme patterns were found in individual growth zones in maize, no differences appeared in broad bean. A comparison was made of thein situ localization and of the isoenzyme patterns of α- and β-galactosidases with α- and β-glucosidases. In the case of galactosidases, positive results appear with both α- and β-galactoside. The rising of pH to neutrality leads to considerable decrease in the activity of both galactosidases.     

Insulin analogues with modifications in the β-turn of the B-chain

The β-turn formed by the amino acid residues 20–23 of the B-chain of insulin has been implicated as an important structural feature of the molecule. In other biologically active peptides, stabilization of β-turns has resulted in increases in activity. We have synthesized three insulin analogues containing modifications which would be expected to increase the stability of the β-turn. In two analogues, we have substituted α-aminoisobutyric acid (Aib) for the Glu residue normally present in position B21 or for the Arg residue normally present in position B22; in a third compound, we have replaced the Glu residue with its D-isomer. Biological evaluation of these compounds showed that [B21 Aib]insulin displays a potencyca. one-fourth that of natural insulin, while [B22 Aib]insulin is less than 10% as potent. In contrast, [B21 D-Glu]insulin is equipotent with natural insulin. We conclude that the β-turn region of the insulin molecule normally possesses considerable flexibility, which may be necessary for it to assume a conformation commensurate with high biological activity. If this is the case, [B21 D-Glu]insulin may exhibit a stabilized geometry similar to that of natural insulin when bound to the insulin receptor.     

<Emphasis Type="Italic">Fusarium</Emphasis> toxin-contaminated maize in diets of growing bulls: effects on performance,slaughtering characteristics,and transfer into physiological liquids

The present feeding study was carried out to examine the effects of Fusarium toxin-contaminated diets on performance and slaughtering characteristics and on the transfer of the Fusarium toxins zearalenone (ZEN), deoxynivalenol (DON) and their metabolites into physiological matrices. A total of 61 bulls (483?±?46 kg) were fed with graded proportions of Fusarium toxin-contaminated feed over a period of 10 weeks. The total mixed rations (TMR) consisted of 47 % grass silage, 20 % press pulp silage, and 33 % concentrate on dry matter (DM) basis. Increasing toxin concentrations were achieved by the exchange of control maize with Fusarium toxin-contaminated maize in the concentrates. Thus, dietary toxin concentrations between 0.08 and 0.69 mg ZEN and 0.36 and 8.31 mg DON per kg DM were covered by the four feeding groups. Based on increasing DM intake with increasing mycotoxin contaminations of the diet, the live weight gain and energy intake differed significantly between the groups. No effects were observed on slaughtering characteristics and organ weights. ZEN, α-zeralenol, β-zeralenol (β-ZEL), zeralanone, α-zearalanol, β-zearalanol, DON, and de-deepoxy-DON (de-DON) were simultaneously determined in urine, plasma, and liquor whereby quantifiable concentrations of ZEN, β-ZEL, DON, and de-DON were found in urine, of DON and de-DON in plasma, and solely of de-DON in liquor. Based on overall results it can be concluded that current EU-guidance values for critical concentrations of DON and ZEN can be regarded as safe levels also for growing bulls. Urine and blood toxin residue levels can be used to assess exposure of bulls.     

Role of Zearalenone Lactonase in Protection of Gliocladium roseum from Fungitoxic Effects of the Mycotoxin Zearalenone

Zearalenone is a mycotoxin with estrogenic effects on mammals that is produced by several species of Fusarium. We found that zearalenone and its derivatives inhibit the growth of filamentous fungi on solid media at concentrations of ≤10 μg/ml. The fungitoxic effect declined in the order zearalenone > α-zearalenol > β-zearalenol. The mycoparasitic fungus Gliocladium roseum produces a zearalenone-specific lactonase which catalyzes the hydrolysis of zearalenone, followed by a spontaneous decarboxylation. The growth of G. roseum was not inhibited by zearalenone, and the lactonase may protect G. roseum from the toxic effects of this mycotoxin. We inactivated zes2, the gene encoding zearalenone lactonase in G. roseum, by inserting a hygromycin resistance cassette into the coding sequence of the gene by means of Agrobacterium tumefaciens-mediated genetic transformation. The zes2 disruption mutants could not hydrolyze the lactone bond of zearalenone and were more sensitive to zearalenone. These data are consistent with a hypothesis that resorcylic acid lactones exemplified by zearalenone act to reduce growth competition by preventing competing fungi from colonizing substrates occupied by zearalenone producers and suggest that they may play a role in fungal defense against mycoparasites.     

Sulfation of agarose from subantarctic Ahnfeltia plicata (Ahnfeltiales,Rhodophyta): studies of its antioxidant and anticoagulant properties in vitro and its copolymerization with acrylamide

Aqueous extraction of Ahnfeltia plicata collected in the Magellan ecoregion afforded agarose devoid of sulfate groups. This neutral agarose was subjected to sulfation with SO₃-pyridine complex, giving an aqueous soluble derivative with 35.5 % sulfate groups. Analysis by Fourier transform infrared spectroscopy (FT-IR) and by ¹H and ¹³C NMR spectroscopy indicated that this derivative was sulfated at positions C-6 of the β-galactopyranosyl residue and C-2 of the α-3,6-anhydrogalactopyranosyl residue and partially sulfated at position C-2 of the β residue. The antioxidant capacity of sulfated agarose was evaluated by the oxygen radical absorbance capacity (ORAC) method, ABTS radical cation, hydroxyl radicals, and chelating assays. This capacity of sulfated agarose toward peroxyl radicals was higher than that of commercial λ-carrageenan, while native agarose presented good activity, with an ORAC value similar to that of commercial κ-carrageenan. Sulfated agarose presented good antioxidant capacity toward other radicals. Copolymerization of sulfated agarose with acrylamide was achieved using ceric ammonium nitrate as initiator. NMR spectroscopy indicated grafting of polyacrylamide at position C-4 of β-galactopyranosyl residues.     

Interactions of zearalenone and its reduced metabolites α-zearalenol and β-zearalenol with serum albumins: species differences,binding sites,and thermodynamics

Zearalenone (ZEN) is a mycotoxin produced by Fusarium species. ZEN mainly appears in cereals and related foodstuffs, causing reproductive disorders in animals, due to its xenoestrogenic effects. The main reduced metabolites of ZEN are α-zearalenol (α-ZEL) and β-zearalenol (β-ZEL). Similarly to ZEN, ZELs can also activate estrogen receptors; moreover, α-ZEL is the most potent endocrine disruptor among these three compounds. Serum albumin is the most abundant plasma protein in the circulation; it affects the tissue distribution and elimination of several drugs and xenobiotics. Although ZEN binds to albumin with high affinity, albumin-binding of α-ZEL and β-ZEL has not been investigated. In this study, the complex formation of ZEN, α-ZEL, and β-ZEL with human (HSA), bovine (BSA), porcine (PSA), and rat serum albumins (RSA) was investigated by fluorescence spectroscopy, affinity chromatography, thermodynamic studies, and molecular modeling. Our main observations are as follows: (1) ZEN binds with higher affinity to albumins than α-ZEL and β-ZEL. (2) The low binding affinity of β-ZEL toward albumin may result from its different binding position or binding site. (3) The binding constants of the mycotoxin-albumin complexes significantly vary with the species. (4) From the thermodynamic point of view, the formation of ZEN-HSA and ZEN-RSA complexes are similar, while the formation of ZEN-BSA and ZEN-PSA complexes are markedly different. These results suggest that the toxicological relevance of ZEN-albumin and ZEL-albumin interactions may also be species-dependent.     

Influence of organically or conventionally produced wheat on health,performance and mycotoxin residues in tissues and bile of growing pigs

From 1999?–?2001 three different varieties of wheat [Contur (susceptible to Fusarium), Batis and Petrus (less susceptible to Fusarium)] were cultivated under organic and conventional conditions in order to determine mycotoxin burden. Soil quality, preceding crop and weather conditions were comparable in the different production systems. The wheat batches were analysed for moulds, and the contents of zearalenone (ZEN) and deoxynivalenol (DON). Feeding trials were carried out with growing pigs (n?=?96; average initial live weight 22.2 ±?1.5?kg [mean?±?SD]) to examine a possible influence on the animal performance and on mycotoxin residues. The data recorded were clinical conditions, performance, biochemical and hematological data. Residues of ZEN, α- and β-zearalenol (ZEL) and of DON were determined in bile, liver and muscle after slaughtering. Conventionally cultivated wheat was more frequently contaminated with Fusarium and contained more frequently ZEN and DON in higher concentrations than the organically produced wheat. Hematological and biochemical parameters of pigs fed with organically cultivated diets were not different from those of conventionally fed pigs. Pigs fed with organically produced wheat showed a slightly higher daily weight gain, but a lower carcass yield than the conventionally fed animals. The highest residues of DON and total-ZEN (ZEN + α-ZEL + β-ZEL) were found in bile. Bile samples of organically fed pigs contained lower concentrations of total-ZEN than those of conventionally fed pigs. Altogether, these data suggest that wheat from an organic farming does not have higher mycotoxin-contamination than wheat from the conventional farming system.     

On the distribution and metabolism of Fusarium-toxins along the gastrointestinal tract of endotoxaemic pigs

The aim of this study was to investigate the potential modulatory effect of E. coli lipopolysaccharides (LPS) on residues of deoxynivalenol (DON), de-epoxy-deoxynivalenol (DOM-1), zearalenone (ZEN) and its metabolites α-zearalenol (α-ZEL), β-zearalenol (β-ZEL), zearalanone (ZAN), α-zearalanol (α-ZAL) and β-zearalanol (β-ZAL) after pre- or post-hepatic administration along the gastrointestinal axis. Fifteen barrows were exposed to a naturally mycotoxin contaminated diet (4.59 mg DON/kg feed and 0.22 mg ZEN/kg feed) and equipped with jugular (ju) and portal (po) catheters. On sampling day (day 29), the barrows were infused with LPS or a control fluid (LPS, 7.5 µg/kg body weight; control, 0.9% NaCl) either pre- or post-hepatically, resulting in three infusion groups: CON_ju-CON_po, CON_ju-LPS_po and LPS_ju-CON_po. At 195 min relative to infusion start (210 min post-feeding), pigs were sacrificed and content of stomach and small intestine (proximal, medial and distal part) as well as faeces were collected. In all LPS-infused animals, higher amounts of dry matter were recovered irrespective of LPS entry site suggesting a reduced gastric emptying and a decreased gastrointestinal motility under endotoxaemic conditions. DON metabolism in the gastrointestinal tract (GIT) remained unaltered by treatments and included an increase in the proportion of DOM-1 along the GIT, particularly from distal small intestine to faeces. Variables describing ZEN metabolism suggest a stimulated biliary release of ZEN and its metabolites in LPS-infused groups, particularly in the LPS_ju-CON_po group. In conclusion, the GIT metabolism of ZEN was markedly influenced in endotoxaemic pigs whereby a jugular induction of an acute phase reaction was more effective than portal LPS infusion hinting at a strong hepatic first-pass effect.     

Different Biomarkers in Non-Small Cell Lung Cancer in Blood Vessel Invasion

In clinical settings, lung cancer is divided into small cell lung cancer and non-small cell lung cancer, and chemotherapy is depended on the difference. Using the same chemotherapy treatment, different effects and prognosis can be seen among squamous-cell carcinoma and adenocarcinoma. These differences indicate that there may be various methods of invasion and immunity between squamous-cell carcinoma and adenocarcinoma. Blood vessel invasion and tumor immune escape play very important roles in the progression and metastasis of cancer, and CD105 and integrins are novel therapeutic targets. We assessed the possible association of CD105 expression and integrins with TNM classification in patients with two types of NSCLC. A total of 72 patients with resected Non-Small Cell Lung Cancer (NSCLC) were reviewed retrospectively. Integrin β1, β2, β3, and α5β1 are assayed by immunofluorescence and integrin α5β1 using immunoblot. Intratumoral microvessel density was determined with an anti-CD34 mAb and an anti-CD105 mAb. Invasive ability was assayed with MMP2 and MMP9 using immunofluorescence. The expressions of all integrins, CD105, and CD34 are low in the normal lung tissue and highly expressed in the cancer niche compared to the adjacent tissues. CD105 is highly expressed in the adenocarcinoma niche compared to the squamous-cell carcinoma in NSCLC. The expressions of both MMP2 and MMP9 are low in the normal lung tissue and highly expressed in adjacent tissues. This study shows that blood vessel invasion appears to be an independent negative prognosticator in surgically managed types of NSCLC. However, adequately designed large prospective studies are warranted to confirm the present findings.     

Development of a liquid chromatography tandem mass spectrometry method for the simultaneous determination of zearalenone,deoxynivalenol and their metabolites in pig serum

A sensitive and selective liquid chromatography tandem mass spectrometry method using negative electrospray ionisation (LC-ESI-MS/MS) was developed for the simultaneous determination of zearalenone (ZEN), deoxynivalenol (DON) and their metabolites α-zearalenol, β-zearalenol, zearalanone, α-zearalanol, β-zearalanol and de-epoxy-deoxynivalenol in pig serum. For method development, different sample preparation columns were tested for their suitability for extraction and clean up. Finally, preparation of serum samples was carried out using Oasis? HLB solid-phase extraction (SPE) columns. The analyte concentrations were determined by the use of isotopically labelled internal standards (IS). The method was in-house validated for all analytes. Calibration graphs (0.3–480 ng/ml) were prepared and high degree of linearity was achieved (r?≥?0.99). Results for method precision ranged between 2.7 and 21.5 % for inter-day and between 1.1 and 11.1 % for intra-day. The recoveries were in the range of 82–131 %. Limits of detection and quantification ranged 0.03–0.71 and 0.08–2.37 ng/ml, respectively. The method has been successfully used for quantitative determination of ZEN, DON and their metabolites in pig serum from a feeding trial with practically relevant ZEN and DON concentrations. This method is precise and reproducible and can be used as a multi-biomarker method to assess animal exposure to these mycotoxins and for diagnosis of intoxications.