Bacterial Lactonases ZenA with Noncanonical Structural Features Hydrolyze the Mycotoxin Zearalenone.

Zearalenone (ZEN) is a mycoestrogenic polyketide produced by Fusarium graminearum and other phytopathogenic members of the genus Fusarium. Contamination of cereals with ZEN is frequent, and hydrolytic detoxification with fungal lactonases has been explored. Here, we report the isolation of a bacterial strain, Rhodococcus erythropolis PFA D8-1, with ZEN hydrolyzing activity, cloning of the gene encoding α/ß hydrolase ZenA encoded on the linear megaplasmid pSFRL1, and biochemical characterization of nine homologues. Furthermore, we report site-directed mutagenesis as well as structural analysis of the dimeric ZenARe of R. erythropolis and the more thermostable, tetrameric ZenAScfl of Streptomyces coelicoflavus with and without bound ligands. The X-ray crystal structures not only revealed canonical features of α/ß hydrolases with a cap domain including a Ser-His-Asp catalytic triad but also unusual features including an uncommon oxyanion hole motif and a peripheral, short antiparallel ß-sheet involved in tetramer interactions. Presteady-state kinetic analyses for ZenARe and ZenAScfl identified balanced rate-limiting steps of the reaction cycle, which can change depending on temperature. Some new bacterial ZEN lactonases have lower KM and higher kcat than the known fungal ZEN lactonases and may lend themselves to enzyme technology development for the degradation of ZEN in feed or food.

An In Silico Target Fishing Approach to Identify Novel Ochratoxin A Hydrolyzing Enzyme.

Ochratoxin A (OTA), a mycotoxin that is of utmost concern in food and feed safety, is produced by fungal species that mainly belong to the Aspergillus and Penicillium genera. The development of mitigation strategies to reduce OTA content along the supply chains is key to ensuring safer production of food and feed. Enzyme-based strategies are among the most promising methods due to their specificity, efficacy, and multi-situ applicability. In particular, some enzymes are already known for hydrolyzing OTA into ochratoxin alpha (OTα) and phenylalanine (Phe), eventually resulting in detoxification action. Therefore, the discovery of novel OTA hydrolyzing enzymes, along with the advancement of an innovative approach for their identification, could provide a broader basis to develop more effective mitigating strategies in the future. In the present study, a hybrid in silico/in vitro workflow coupling virtual screening with enzymatic assays was applied in order to identify novel OTA hydrolyzing enzymes. Among the various hits, porcine carboxypeptidase B was identified for the first time as an effective OTA hydrolyzing enzyme. The successful experimental endorsement of findings of the workflow confirms that the presented strategy is suitable for identifying novel OTA hydrolyzing enzymes, and it might be relevant for the discovery of other mycotoxin- mitigating enzymes.

Reduced toxicity of 3-epi-deoxynivalenol and de-epoxy-deoxynivalenol through deoxynivalenol bacterial biotransformation: In vivo analysis in piglets.

Ingestion of deoxynivalenol (DON), one of the most common mycotoxin contaminants of cereals, leads to adverse effects for animal and human health. Bacterial biotransformation is a strategy to mitigate the toxicity of this mycotoxin. The present study aims to evaluate the toxicity of two bacterial biotranformation products of DON: 3-epi-deoxynivalenol (3-epi-DON) and de-epoxy-deoxynivalenol (DOM-1) through zootechnical, hematological, histological and immunological assays. Twenty-four 4-weeks-old piglets received a control diet or a diet contaminated with 3 mg kg-1 DON, DOM-1, or 3-epi-DON for 7 days. Sample tissues were collected for histomorphometrical analysis, expression of cytokines and cell protein junctions. The zootechnical and hematological parameters were not modulated by any treatment. Ingestion of DON induced histological alterations in the intestine, liver and lymphoid organs, as well as an overexpression of pro-inflammatory cytokines, E-cadherin and occludin. These changes were not observed in piglets receiving the DOM-1 and 3-epi-DON contaminated diets. Pigs fed 3-epi-DON contaminated diet showed an increase in IgM levels in comparison with other diets, while no change was observed in IgA and IgG levels among the diets. Our results indicate that DOM-1 and 3-epi-DON are not toxic for piglets; thus bacterial biotransformation seems to be a sustainable alternative to reduce mycotoxin toxicity.

Detoxification of the Fumonisin Mycotoxins in Maize: An Enzymatic Approach.

Enzymatic detoxification has become a promising approach for control of mycotoxins postharvest in grains through modification of chemical structures determining their toxicity. In the present study fumonisin esterase FumD (EC 3.1.1.87) (FUMzyme®; BIOMIN, Tulln, Austria), hydrolysing fumonisin (FB) mycotoxins by de-esterification, was utilised to develop an enzymatic reduction method in a maize kernel enzyme incubation mixture. Efficacy of the FumD FB reduction method in "low" and "high" FB contaminated home-grown maize was compared by monitoring FB1 hydrolysis to the hydrolysed FB1 (HFB1) product utilising a validated LC-MS/MS analytical method. The method was further evaluated in terms of enzyme activity and treatment duration by assessing enzyme kinetic parameters and the relative distribution of HFB1 between maize kernels and the residual aqueous environment. FumD treatments resulted in significant reduction (≥80%) in "low" (≥1000 U/L, p < 0.05) and "high" (100 U/L, p < 0.05; ≥1000 U/L, p < 0.0001) FB contaminated maize after 1 h respectively, with an approximate 1:1 µmol conversion ratio of FB1 into the formation of HFB1. Enzyme kinetic parameters indicated that, depending on the activity of FumD utilised, a significantly (p < 0.05) higher FB1 conversion rate was noticed in "high" FB contaminated maize. The FumD FB reduction method in maize could find application in commercial maize-based practices as well as in communities utilising home-grown maize as a main dietary staple and known to be exposed above the tolerable daily intake levels.

Biotransformation of the Mycotoxin Zearalenone to its Metabolites Hydrolyzed Zearalenone (HZEN) and Decarboxylated Hydrolyzed Zearalenone (DHZEN) Diminishes its Estrogenicity In Vitro and In Vivo.

Zearalenone (ZEN)-degrading enzymes are a promising strategy to counteract the negative effects of this mycotoxin in livestock. The reaction products of such enzymes need to be thoroughly characterized before technological application as a feed additive can be envisaged. Here, we evaluated the estrogenic activity of the metabolites hydrolyzed zearalenone (HZEN) and decarboxylated hydrolyzed zearalenone (DHZEN) formed by hydrolysis of ZEN by the zearalenone-lactonase Zhd101p. ZEN, HZEN, and DHZEN were tested in two in vitro models, the MCF-7 cell proliferation assay (0.01-500 nM) and an estrogen-sensitive yeast bioassay (1-10,000 nM). In addition, we compared the impact of dietary ZEN (4.58 mg/kg) and equimolar dietary concentrations of HZEN and DHZEN on reproductive tract morphology as well as uterine mRNA and microRNA expression in female piglets (n = 6, four weeks exposure). While ZEN increased cell proliferation and reporter gene transcription, neither HZEN nor DHZEN elicited an estrogenic response, suggesting that these metabolites are at least 50-10,000 times less estrogenic than ZEN in vitro. In piglets, HZEN and DHZEN did not increase vulva size or uterus weight. Moreover, RNA transcripts altered upon ZEN treatment (EBAG9, miR-135a-5p, miR-187-3p and miR-204-5p) were unaffected by HZEN and DHZEN. Our study shows that both metabolites exhibit markedly reduced estrogenicity in vitro and in vivo, and thus provides an important basis for further evaluation of ZEN-degrading enzymes.

MicroRNAs in porcine uterus and serum are affected by zearalenone and represent a new target for mycotoxin biomarker discovery.

The mycotoxin zearalenone (ZEN) poses a risk to animal health because of its estrogenic effects. Diagnosis of ZEN-induced disorders remains challenging due to the lack of appropriate biomarkers. In this regard, circulating microRNAs (small non-coding RNAs) have remarkable potential, as they can serve as indicators for pathological processes in tissue. Thus, we combined untargeted and targeted transcriptomics approaches to investigate the effects of ZEN on the microRNA expression in porcine uterus, jejunum and serum, respectively. To this end, twenty-four piglets received uncontaminated feed (Control) or feed containing 0.17 mg/kg ZEN (ZEN low), 1.46 mg/kg ZEN (ZEN medium) and 4.58 mg/kg ZEN (ZEN high). After 28 days, the microRNA expression in the jejunum remained unaffected, while significant changes in the uterine microRNA profile were observed. Importantly, 14 microRNAs were commonly and dose-dependently affected in both the ZEN medium and ZEN high group, including microRNAs from the miR-503 cluster (i.e. ssc-miR-424-5p, ssc-miR-450a, ssc-miR-450b-5p, ssc-miR-450c-5p, ssc-miR-503 and ssc-miR-542-3p). Predicted target genes for those microRNAs are associated with regulation of gene expression and signal transduction (e.g. cell cycle). Although the effects in serum were less pronounced, receiver operating characteristic analysis revealed that several microRNA ratios were able to discriminate properly between non-exposed and ZEN-exposed pigs (e.g. ssc-miR-135a-5p/ssc-miR-432-5p, ssc-miR-542-3p/ssc-miR-493-3p). This work sheds new light on the molecular mechanisms of ZEN, and fosters biomarker discovery.

Deepoxy-deoxynivalenol retains some immune-modulatory properties of the parent molecule deoxynivalenol in piglets.

Deoxynivalenol (DON) is the most abundant trichothecene in food and feed. It causes both acute and chronic disorders of the human and animal intestine, liver and the immune system. The structural basis for the toxicity of DON has not been fully elucidated. Using the pig as a target and a model species for human, the toxicity of DON and its deepoxy-metabolite (DOM-1) was compared. Animals were exposed by gavage to 1 and 0.5 nmol toxin/kg b.w./day for 2 and 3 weeks respectively. Whatever the dose/duration, DOM-1 was less toxic than DON in terms of weight gain and emesis. In the 3-week experiment, animals were vaccinated with ovalbumin, and their immune response was analyzed in addition to tissue morphology, biochemistry and hematology. DON impaired the morphology of the jejunum and the ileum, reduced villi height, decreased E-cadherin expression and modified the intestinal expression of cytokines. Similarly, DON induced hepatotoxicity as indicated by the lesion score and the blood biochemistry. By contrast, DOM-1 only induced minimal intestinal toxicity and did not trigger hepatotoxicity. As far as the immune response was concerned, the effects of ingesting DOM-1 were similar to those caused by DON, as measured by histopathology of lymphoid organs, PCNA expression and the specific antibody response. Taken together, these data demonstrated that DOM-1, a microbial detoxification product of DON, was not toxic in the sensitive pig model but retained some immune-modulatory properties of DON, especially its ability to stimulate a specific antibody response during a vaccination protocol.

Microbial biotransformation of DON: molecular basis for reduced toxicity.

Bacteria are able to de-epoxidize or epimerize deoxynivalenol (DON), a mycotoxin, to deepoxy-deoxynivalenol (deepoxy-DON or DOM-1) or 3-epi-deoxynivalenol (3-epi-DON), respectively. Using different approaches, the intestinal toxicity of 3 molecules was compared and the molecular basis for the reduced toxicity investigated. In human intestinal epithelial cells, deepoxy-DON and 3-epi-DON were not cytotoxic, did not change the oxygen consumption or impair the barrier function. In intestinal explants, exposure for 4 hours to 10 µM DON induced intestinal lesions not seen in explants treated with deepoxy-DON and 3-epi-DON. A pan-genomic transcriptomic analysis was performed on intestinal explants. 747 probes, representing 323 genes, were differentially expressed, between DON-treated and control explants. By contrast, no differentially expressed genes were observed between control, deepoxy-DON and 3-epi-DON treated explants. Both DON and its biotransformation products were able to fit into the pockets of the A-site of the ribosome peptidyl transferase center. DON forms three hydrogen bonds with the A site and activates MAPKinases (mitogen-activated protein kinases). By contrast deepoxy-DON and 3-epi-DON only form two hydrogen bonds and do not activate MAPKinases. Our data demonstrate that bacterial de-epoxidation or epimerization of DON altered their interaction with the ribosome, leading to an absence of MAPKinase activation and a reduced toxicity.

Intestinal toxicity of the masked mycotoxin deoxynivalenol-3-ß-D-glucoside.

Natural food contaminants such as mycotoxins are an important problem for human health. Deoxynivalenol (DON) is one of the most common mycotoxins detected in cereals and grains. Its toxicological effects mainly concern the immune system and the gastrointestinal tract. This toxin is a potent ribotoxic stressor leading to MAP kinase activation and inflammatory response. DON frequently co-occurs with its glucosylated form, the masked mycotoxin deoxynivalenol-3-ß-D-glucoside (D3G). The toxicity of this later compound remains unknown in mammals. This study aimed to assess the ability of D3G to elicit a ribotoxic stress and to induce intestinal toxicity. The toxicity of D3G and DON (0-10 µM) was studied in vitro, on the human intestinal Caco-2 cell line, and ex vivo, on porcine jejunal explants. First, an in silico analysis revealed that D3G, contrary to DON, was unable to bind to the A-site of the ribosome peptidyl transferase center, the main targets for DON toxicity. Accordingly, D3G did not activate JNK and P38 MAPKs in treated Caco-2 cells and did not alter viability and barrier function on cells, as measured by the trans-epithelial electrical resistance. Treatment of intestinal explants for 4 h with 10 µM DON induced morphological lesions and up-regulated the expression of pro-inflammatory cytokines as measured by qPCR and pan-genomic microarray analysis. By contrast, expression profile of D3G-treated explants was similar to that of controls, and these explants did not show histomorphology alteration. In conclusion, our data demonstrated that glucosylation of DON suppresses its ability to bind to the ribosome and decreases its intestinal toxicity.

Rhodococcus erythropolis MTHt3 biotransforms ergopeptines to lysergic acid.

BACKGROUND: Ergopeptines are a predominant class of ergot alkaloids produced by tall fescue grass endophyte Neotyphodium coenophialum or cereal pathogen Claviceps purpurea. The vasoconstrictive activity of ergopeptines makes them toxic for mammals, and they can be a problem in animal husbandry. RESULTS: We isolated an ergopeptine degrading bacterial strain, MTHt3, and classified it, based on its 16S rDNA sequence, as a strain of Rhodococcus erythropolis (Nocardiaceae, Actinobacteria). For strain isolation, mixed microbial cultures were obtained from artificially ergot alkaloid-enriched soil, and provided with the ergopeptine ergotamine in mineral medium for enrichment. Individual colonies derived from such mixed cultures were screened for ergotamine degradation by high performance liquid chromatography and fluorescence detection. R. erythropolis MTHt3 converted ergotamine to ergine (lysergic acid amide) and further to lysergic acid, which accumulated as an end product. No other tested R. erythropolis strain degraded ergotamine. R. erythropolis MTHt3 degraded all ergopeptines found in an ergot extract, namely ergotamine, ergovaline, ergocristine, ergocryptine, ergocornine, and ergosine, but the simpler lysergic acid derivatives agroclavine, chanoclavine, and ergometrine were not degraded. Temperature and pH dependence of ergotamine and ergine bioconversion activity was different for the two reactions. CONCLUSIONS: Degradation of ergopeptines to ergine is a previously unknown microbial reaction. The reaction end product, lysergic acid, has no or much lower vasoconstrictive activity than ergopeptines. If the genes encoding enzymes for ergopeptine catabolism can be cloned and expressed in recombinant hosts, application of ergopeptine and ergine degrading enzymes for reduction of toxicity of ergot alkaloid-contaminated animal feed may be feasible.

Dose-dependent effects on sphingoid bases and cytokines in chickens fed diets prepared with fusarium verticillioides culture material containing fumonisins.

In chickens, the effect of mycotoxins, especially fumonisins (FB), in the gastrointestinal tract (GIT) is not well documented. Thus, this study in broiler chicks determined the effects of consuming diets prepared with Fusarium verticillioides culture material containing FB on intestinal gene expression and on the sphinganine (Sa)/sphingosine (So) ratio (Sa/So; a biomarker of FB effect due to disruption of sphingolipid metabolism). Male broilers were assigned to 6 diets (6 cages/diet; 6 birds/cage) from hatch to 20 days containing 0.4, 5.6, 11.3, 17.5, 47.8, or 104.8 mg FB/kg diet. Exposure to FB altered the Sa/So ratio in all tissues analyzed, albeit to varying extents. Linear dose-responses were observed in the kidney, jejunum and cecum. The liver and the ileum were very sensitive and data fit a cubic and quadratic polynomial model, respectively. Gene expression in the small intestine revealed low but significant upregulations of cytokines involved in the pro-inflammatory, Th1/Th17 and Treg responses, especially at 10 days of age. Interestingly, the cecal tonsils exhibited a biphasic response. Unlike the sphingolipid analysis, the effects seen on gene expression were not dose dependent, even showing more effects when birds were exposed to 11.3 mg FB/kg. In conclusion, this is the first report on the disruption of the sphingolipid metabolism by FB in the GIT of poultry. Further studies are needed to reach conclusions on the biological meaning of the immunomodulation observed in the GIT, but the susceptibility of chickens to intestinal pathogens when exposed to FB, at doses lower than those that would cause overt clinical symptoms, should be addressed.

Effects of orally administered fumonisin B1 (FB1), partially hydrolysed FB1, hydrolysed FB1 and N-(1-deoxy-D-fructos-1-yl) FB1 on the sphingolipid metabolism in rats.

Fumonisin B1 (FB1) is a Fusarium mycotoxin frequently occurring in maize-based food and feed. Alkaline processing like nixtamalisation of maize generates partially and fully hydrolysed FB1 (pHFB1 and HFB1) and thermal treatment in the presence of reducing sugars leads to formation of N-(1-deoxy-D-fructos-1-yl) fumonisin B1 (NDF). The toxicity of these metabolites, in particular their effect on the sphingolipid metabolism, is either unknown or discussed controversially. We produced high purity FB1, pHFB1a+b, HFB1 and NDF and fed them to male Sprague Dawley rats for three weeks. Once a week, urine and faeces samples were collected over 24 h and analysed for fumonisin metabolites as well as for the sphinganine (Sa) to sphingosine (So) ratio by validated LC-MS/MS based methods. While the latter was significantly increased in the FB1 positive control group, the Sa/So ratios of the partially and fully hydrolysed fumonisins were indifferent from the negative control group. Although NDF was partly cleaved during digestion, the liberated amounts of FB1 did not raise the Sa/So ratio. These results show that the investigated alkaline and thermal processing products of FB1 were, at the tested concentrations, non-toxic for rats, and suggest that according food processing can reduce fumonisin toxicity for humans.

Deoxynivalenol (DON) sulfonates as major DON metabolites in rats: from identification to biomarker method development, validation and application.

Deoxynivalenol (DON) is a trichothecene mycotoxin regularly occurring in cereals. Rats are often used to study toxicokinetics of DON and related compounds, yet only about 30 % of the administered dose is typically recovered. Recently, it was reported that DON is partly metabolised to previously undetected DON- and deepoxy-DON (DOM) sulfonate in rats and tentative structures were proposed. The present work describes the production and characterisation of DON-, DOM- and DON-3-glucoside (D3G) sulfonates of three different series; the development and validation of liquid chromatography tandem mass spectrometry (LC-MS/MS)-based methods for determination of DON, DOM, D3G and their sulfonates in rat faeces and urine; and application of the methods to samples from a DON and D3G feeding trial with rats. In addition to previously produced DON sulfonates (DONS) 1, 2 and 3, D3G sulfonates 1, 2 and 3; and DOM sulfonates (DOMS) 2 and 3 were synthesised, purified and characterised. The developed methods showed apparent recoveries of all investigated compounds between 68 and 151 % in faeces and between 48 and 113 % in urine. The recovery of DON, D3G and their metabolites from faeces and urine of rats (n = 6) administered in a single dose of 2.0 mg/kg b.w. DON or the equimolar amount of D3G was 75 ± 9 % for the DON group and 68 ± 8 % for the D3G group. DON-, DOM- and D3G sulfonates excreted in faeces accounted for 48 and 47 % of the total amount of administered DON and D3G. Urinary excretion of sulfonates was <1 %. In both treatment groups, DONS 2 was the major metabolite 0-24 h after treatment, whereas DOMS 2 was predominant thereafter. The developed methods can also be used for investigation of DON (conjugate) sulfonate formation in other animal species.

Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in pigs.

Plants can metabolize the Fusarium mycotoxin deoxynivalenol (DON) by forming the masked mycotoxin deoxynivalenol-3-ß-D-glucoside (D3G). D3G might be cleaved during digestion, thus increasing the total DON burden of an individual. Due to a lack of in vivo data, D3G has not been included in the various regulatory limits established for DON so far. The aim of our study was to contribute to the risk assessment of D3G by determination of its metabolism in pigs. Four piglets received water, D3G (116 µg/kg b.w.) and the equimolar amount of DON (75 µg/kg b.w.) by gavage on day 1, 5 and 9 of the experiment, respectively. Additionally, 15.5 µg D3G/kg b.w. were administered intravenously on day 13. Urine and feces were collected for 24 h and analyzed for DON, D3G, deoxynivalenol-3-glucuronide (DON-3-GlcA), deoxynivalenol-15-GlcA (DON-15-GlcA) and deepoxy-deoxynivalenol (DOM-1) by UHPLC-MS/MS. After oral application of DON and D3G, in total 84.8±9.7% and 40.3±8.5% of the given dose were detected in urine, respectively. The majority of orally administered D3G was excreted in form of DON, DON-15-GlcA, DOM-1 and DON-3-GlcA, while urinary D3G accounted for only 2.6±1.4%. In feces, just trace amounts of metabolites were found. Intravenously administered D3G was almost exclusively excreted in unmetabolized form via urine. Data indicate that D3G is nearly completely hydrolyzed in the intestinal tract of pigs, while the toxin seems to be rather stable after systemic absorption. Compared to DON, the oral bioavailability of D3G and its metabolites seems to be reduced by a factor of up to 2, approximately.

Biotransformation approaches to alleviate the effects induced by fusarium mycotoxins in swine.

Mycotoxin mitigation is of major interest as ingestion of mycotoxins results in poor animal health, decreased productivity, as well as substantial economic losses. A feed additive (FA) consisting of a combination of bacteria (Eubacterium BBSH797) and enzyme (fumonisin esterase FumD) was tested in pigs for its ability to neutralize the effects of mono- and co-contaminated diets with deoxynivalenol (DON) and fumonisins (FB) on hematology, biochemistry, tissue morphology, and immune response. Forty-eight animals, allocated into eight groups, received one of eight diets for 35 days: a control diet, a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg), or both toxins, and the same four diets with FA. Inclusion of FA restored the circulating number of neutrophils of piglets fed the FB and DON + FB diets. Similarly, FA counteracted the minor changes observed on plasma concentrations of albumin and creatinine. In lung, the lesions induced by the ingestion of FB in mono- and co-contaminated diets were no longer observed after addition of FA in these diets. Lesions recorded in the liver of pigs fed either of the contaminated diets with FA were partly reduced, and the increased hepatocyte proliferation was totally neutralized when FA was present in the co-contaminated diet. After 35 days of exposure, the development of the vaccinal response was significantly improved in animals fed diets supplemented with FA, as shown by results of lymphocyte proliferation, cytokine expression in spleen, and the production of specific Ig. Similarly, in jejunum of animals fed diets with FA, occurrence of lesions and upregulation of pro-inflammatory cytokines were much less obvious. The ameliorative effects provided by FA suggest that this approach would be suitable in the control of DON and FB that commonly co-occur in feed.

Metabolism of the masked mycotoxin deoxynivalenol-3-glucoside in rats.

Deoxynivalenol-3-ß-D-glucoside (D3G), a plant metabolite of the Fusarium mycotoxin deoxynivalenol (DON), might be hydrolyzed in the digestive tract of mammals, thus contributing to the total dietary DON exposure of individuals. Yet, D3G has not been considered in regulatory limits set for DON for foodstuffs due to the lack of in vivo data. The aim of our study was to evaluate whether D3G is reactivated in vivo by investigation of its metabolism in rats. Six Sprague-Dawley rats received water, DON (2.0 mg/kg body weight (b.w.)) and the equimolar amount of D3G (3.1 mg/kg b.w.) by gavage on day 1, 8 and 15, respectively. Urine and feces were collected for 48 h and analyzed for D3G, DON, deoxynivalenol-glucuronide (DON-GlcA) and de-epoxy deoxynivalenol (DOM-1) by a validated LC-tandem mass spectrometry (MS/MS) based biomarker method. After administration of D3G, only 3.7±0.7% of the given dose were found in urine in the form of analyzed analytes, compared to 14.9±5.0% after administration of DON, and only 0.3±0.1% were detected in the form of urinary D3G. The majority of administered D3G was recovered as DON and DOM-1 in feces. These results suggest that D3G is little bioavailable, hydrolyzed to DON during digestion, and partially converted to DOM-1 and DON-GlcA prior to excretion. Our data indicate that D3G is of considerably lower toxicological relevance than DON, at least in rats.

The low intestinal and hepatic toxicity of hydrolyzed fumonisin B1 correlates with its inability to alter the metabolism of sphingolipids.

Fumonisins are mycotoxins frequently found as natural contaminants in maize, where they are produced by the plant pathogen Fusarium verticillioides. They are toxic to animals and exert their effects through mechanisms involving disruption of sphingolipid metabolism. Fumonisin B1 (FB1) is the predominant fumonisin in this family. FB1 is converted to its hydrolyzed analogs HFB1, by alkaline cooking (nixtamalization) or through enzymatic degradation. The toxicity of HFB1 is poorly documented especially at the intestinal level. The objectives of this study were to compare the toxicity of HFB1 and FB1 and to assess the ability of these toxins to disrupt sphingolipids biosynthesis. HFB1 was obtained by a deesterification of FB1 with a carboxylesterase. Piglets, animals highly sensitive to FB1, were exposed by gavage for 2 weeks to 2.8 µmol FB1 or HFB1/kg body weight/day. FB1 induced hepatotoxicity as indicated by the lesion score, the level of several biochemical analytes and the expression of inflammatory cytokines. Similarly, FB1 impaired the morphology of the different segments of the small intestine, reduced villi height and modified intestinal cytokine expression. By contrast, HFB1 did not trigger hepatotoxicity, did not impair intestinal morphology and slightly modified the intestinal immune response. This low toxicity of HFB1 correlates with a weak alteration of the sphinganine/sphingosine ratio in the liver and in the plasma. Taken together, these data demonstrate that HFB1 does not cause intestinal or hepatic toxicity in the sensitive pig model and only slightly disrupts sphingolipids metabolism. This finding suggests that conversion to HFB1 could be a good strategy to reduce FB1 exposure.

Chronic ingestion of deoxynivalenol and fumonisin, alone or in interaction, induces morphological and immunological changes in the intestine of piglets.

Deoxynivalenol (DON) and fumonisins (FB) are mycotoxins produced by Fusarium species, which naturally co-occur in animal diets. The gastrointestinal tract represents the first barrier met by exogenous food/feed compounds. The purpose of the present study was to investigate the effects of DON and FB, alone and in combination, on some intestinal parameters, including morphology, histology, expression of cytokines and junction proteins. A total of twenty-four 5-week-old piglets were randomly assigned to four different groups, receiving separate diets for 5 weeks: a control diet; a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg); or both toxins. Chronic ingestion of these contaminated diets induced morphological and histological changes, as shown by the atrophy and fusion of villi, the decreased villi height and cell proliferation in the jejunum, and by the reduced number of goblet cells and lymphocytes. At the end of the experiment, the expression levels of several cytokines were measured by RT-PCR and some of them (TNF-α, IL-1ß, IFN-γ, IL-6 and IL-10) were significantly up-regulated in the ileum or the jejunum. In addition, the ingestion of contaminated diets reduced the expression of the adherent junction protein E-cadherin and the tight junction protein occludin in the intestine. When animals were fed with a co-contaminated diet (DON+FB), several types of interactions were observed depending on the parameters and segments assessed: synergistic (immune cells); additive (cytokines and junction protein expression); less than additive (histological lesions and cytokine expression); antagonistic (immune cells and cytokine expression). Taken together, the present data provide strong evidence that chronic ingestion of low doses of mycotoxins alters the intestine, and thus may predispose animals to infections by enteric pathogens.

Enzyme characteristics of aminotransferase FumI of Sphingopyxis sp. MTA144 for deamination of hydrolyzed fumonisin B1.

Fumonisins are carcinogenic mycotoxins that are frequently found as natural contaminants in maize from warm climate regions around the world. The aminotransferase FumI is encoded as part of a gene cluster of Sphingopyxis sp. MTA144, which enables this bacterial strain to degrade fumonisin B(1) and related fumonisins. FumI catalyzes the deamination of the first intermediate of the catabolic pathway, hydrolyzed fumonisin B(1). We used a preparation of purified, His-tagged FumI, produced recombinantly in Escherichia coli in soluble form, for enzyme characterization. The structure of the reaction product was studied by NMR and identified as 2-keto hydrolyzed fumonisin B(1). Pyruvate was found to be the preferred co-substrate and amino group receptor (K (M) = 490 µM at 10 µM hydrolyzed fumonisin B(1)) of FumI, but other α-keto acids were also accepted as co-substrates. Addition of the co-enzyme pyridoxal phosphate to the enzyme preparation enhanced activity, and saturation was already reached at the lowest tested concentration of 10 µM. The enzyme showed activity in the range of pH 6 to 10 with an optimum at pH 8.5, and in the range of 6°C to 50°C with an optimum at 35°C. The aminotransferase worked best at low salt concentration. FumI activity could be recovered after preincubation at pH 4.0 or higher, but not lower. The aminotransferase was denatured after preincubation at 60°C for 1 h, and the residual activity was also reduced after preincubation at lower temperatures. At optimum conditions, the kinetic parameters K (M) = 1.1 µM and k (cat) = 104/min were determined with 5 mM pyruvate as co-substrate. Based on the enzyme characteristics, a technological application of FumI, in combination with the fumonisin carboxylesterase FumD for hydrolysis of fumonisins, for deamination and detoxification of hydrolyzed fumonisins seems possible, if the enzyme properties are considered.

Individual and combined effects of subclinical doses of deoxynivalenol and fumonisins in piglets.

SCOPE: Deoxynivalenol (DON) and fumonisins (FB) are the most frequently encountered mycotoxins produced by Fusarium species and most commonly co-occur in animal diets. These mycotoxins were studied for their toxicity in piglets on several parameters including plasma biochemistry, organ histopathology and immune response. METHODS AND RESULTS: Twenty-four 5-wk-old animals were randomly assigned to four different groups, receiving separate diets for 5 wk, a control diet, a diet contaminated with either DON (3 mg/kg) or FB (6 mg/kg) or both toxins. At days 4 and 16 of the trial, the animals were subcutaneously immunized with ovalbumin to assess their specific immune response. The different diets did not affect animal performance and had minimal effect on hematological and biochemical blood parameters. By contrast, DON and FB induced histopathological lesions in the liver, the lungs and the kidneys of exposed animals. The liver was significantly more affected when the two mycotoxins were present simultaneously. The contaminated diets also altered the specific immune response upon vaccination as measured by reduced anti-ovalbumin IgG level in the plasma and reduced lymphocyte proliferation upon antigenic stimulation. Because cytokines play a key role in immunity, the expression levels of IL-8, IL-1ß, IL-6 and macrophage inflammatory protein-1ß were measured by RT-PCR at the end of the experiment. The expression of these four cytokines was significantly decreased in the spleen of piglets exposed to multi-contaminated diet. CONCLUSION: Taken together, our data indicate that ingestion of multi-contaminated diet induces greater histopathological lesions and higher immune suppression than ingestion of mono-contaminated diets.