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Zearalenone (ZEN) is an estrogenic mycotoxin produced by fungi in the genus Fusarium. Fusarium species commonly occur in cereal crops such as corn, wheat, barley, and rice which are used in swine feed. In addition to cereals, co-products of ethanol distillation, brewing, and baking are frequently re-purposed as swine feed. ZEN contamination has been related to severe reproductive issues including pelvic organ prolapse (POP), which describes the displacement of pelvic floor organs from their healthy anatomical positions. A recent rise in the rate of POP in sows highlights a need for more information surrounding the occurrence of ZEN in swine feed and the possible role of ZEN in POP. This research aims to understand the relationship between ZEN contamination in swine feed and swine reproductive health. The primary objectives of this work are to (1) characterize the stress relaxation behavior of vaginal tissue in the swine model, (2) determine how ZEN consumption effects the elasticity of pelvic support tissue. (3) determine where ZEN and a related metabolite accumulate in the swine reproductive tract, (4) evaluate the prevalence of ZEN and related Fusarium mycotoxins in swine feed and ingredients, and (5) track ZEN and related Fusarium mycotoxins throughout production of beer and brewers' spent grains (BSG). Results from this work can help stakeholders to mitigate mycotoxin contamination in swine feed, evaluate swine for ZEN toxicosis, and provide care for animals experiencing POP. / Doctor of Philosophy / Under certain conditions, some fungi produce harmful toxins (mycotoxins) that can compromise the health of humans and animals. Mycotoxin-producing fungi are common in cereal crops such as corn, wheat, and barley which may be fed to humans or animals. Swine are especially sensitive to mycotoxins, making feed composition important for animal welfare and profitability. Zearalenone (ZEN) is a mycotoxin that behaves like estrogen, a hormone frequently associated with female reproductive health. Pelvic organ prolapse (POP) is a condition, common in women, wherein organs in the pelvic floor such as the vagina, rectum, and uterus, descend from their healthy anatomical position. Swine herds are experiencing higher rates of POP, which may be related to ZEN contamination in feed. The overall goal of this research is to understand the relationship between mycotoxin contamination in feed and swine reproductive health. The specific objectives of this research are to (1) characterize the mechanical properties of vaginal tissue in swine, (2) characterize mechanical properties of pelvic support tissue associated with POP, in swine fed ZEN, (3) determine which reproductive tissues which accumulate ZEN in swine, (4) evaluate the prevalence of ZEN and related mycotoxins in swine feed, and (5) track ZEN and related mycotoxins throughout production of beer and brewers' spent grain, a co-product used in animal feed. This research can help animal producers develop strategies to mitigate mycotoxin contamination in feed and improve veterinary care.

De nombreuses espèces de moisissures (Aspergillus, Fusarium, Penicillium…) produisent des molécules toxiques appelées mycotoxines. Malgré des efforts agronomiques croissants, la contamination en mycotoxines dans les produits agricoles est toujours présente. Des stratégies de décontamination sont alors utiles pour limiter l’impact des mycotoxines sur la santé animale ou humaine. La première stratégie que nous avons étudiée est le piégeage par adsorption de mycotoxines sur des spores d’Aspergillus de la section Nigri. Nous avons démontré que les spores vivantes sont aussi efficaces que les spores inactivées à la chaleur, indiquant que les composés responsables de l’adsorption ne sont pas altérés par ce traitement. Le phénomène d’adsorption semble être principalement du à des interactions hydrophobes. Des études restent à faire sur l’efficacité d’un tel procédé in vivo et pour caractériser les composés responsables. La deuxième stratégie étudiée a été la biotransformation d’une mycotoxine particulière, la zéaralénone. Plusieurs microorganismes ont été testés. Parmi eux, il a été démontré que certaines espèces d’Aspergillus avaient la capacité de transformer la zéaralénone en zéaralénone-sulfate. La toxicité de ce composé a été évaluée par test de prolifération de cellules cancéreuses MCF-7. Ces cellules ont la particularité de proliférer en présence de composés oestrogéniques. Il a été montré que la zéaralénone-sulfate provoquait peu de prolifération chez ces cellules, suggérant une toxicité plus faible que la zéaralénone. Des études seront effectuées pour évaluer la stabilité d’un tel conjugué in vivo. / Several species of fungi (Aspergillus, Fusarium, Penicillium…) are producers of very hazardous mycotoxins. Despite the use of recommended agricultural practices to avoid mold development and mycotoxinogenesis during crop growth, harvesting and storage, contamination by mycotoxins still occurs. Decontamination procedures are useful to reduce the mycotoxin content of contaminated raw materials. The first strategy we studied was to decrease the bioavailability of mycotoxins trapping them with binding agents. For the first time, the adsorption of mycotoxins by conidia of black Aspergilli has been demonstrated. Heat-treated conidia are as efficient as living conidia, which demonstrates that the components involved in adsorption are not affected by heat-treatment. The adsorption phenomenon seems to involved hydrophobic interactions. Further in vivo studies are necessary to develop the application of such a decontamination process to contaminated raw materials and feeds. The second strategy studied was the biodegradation of zearalenone by microorganisms which could offer a pratical and efficient method to alleviate negative effects on animals. For the first time, some Aspergillus niger strains, isolated from grapes, have been shown to transform zearalenone to zearalenone-sulfate. To be sure that zearalenone biotransformation leads to a detoxification, the estrogenic toxicity of zearalenone-sulfate was determined by cell culture studies. An estrogen receptor positive breast cancer cell line (MCF-7) was used to confirm the lost of toxicity by E-screen assay. The test principle is based on the fact that MCF-7 cells proliferate in presence of estrogenic substances. Zearalenone-sulfate was less estrogenic than zearalenone. Further work will focus on the stability of the zearalenone-sulfate in vivo.

著作権情報を一部追加（2023-06-30） / 京都大学 / 新制・論文博士 / 博士(農学) / 乙第13508号 / 論農博第2906号 / 新制||農||1095(附属図書館) / 学位論文||R4||N5408(農学部図書室) / (主査)教授 谷 史人, 教授 入江 一浩, 教授 井上 和生 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Heat stress is detrimental across many, if not most aspects of animal agriculture. Heat stress can have direct effects on the animal, effects on their environment and effects on the feedstuffs they consume. The work presented here will address direct effects on the animal (using dairy cattle) and effects on their feed (using pigs). Dairy cattle were chosen to study direct effects on the animal because they exhibit unique metabolic adaptions during heat stress. One adaptation of interest is a heat-stress induced state of hyperinsulinemia and hypoglycemia while self-limiting feed intake. In regards to effects on feedstuffs, high ambient temperatures are associated with elevated concentrations of mycotoxins in cereal grains. Mycotoxins are problematic because, at sufficient concentrations, they induce specific, toxic effects. Zearalenone is one such mycotoxin that is particularly problematic for swine, as it is a potent phytoestrogen. As such, it can reduce productivity by disrupting reproductive processes. Although these topics are disparate, the overarching goal of this work is to improve agricultural animal productivity during periods of heat stress. Overall, the three objectives of this thesis were to: 1) isolate the production-related effects of hyperinsulinemia with hypoglycemia from heat stress, 2) determine whether glucose supplementation during heat stress could improve or rescue milk production during heat stress, and finally, 3) investigate reproductive tract morphology following different durations of zearalenone consumption in peri-pubertal gilts. The first experiment compared the effects of thermoneutral, thermoneutral + hyperinsulinemic-hypoglycemic clamp (HHC), environmental heat stress, and heat stress + euglycemic clamp (EC) on milk production, milk components, and circulating glucose concentrations. Milk production, as expected, did decrease under heat stress when compared to thermoneutral conditions. Milk production during the HHC was intermediate, which was likely the result of the induced hypoglycemia. Interestingly, the glucose infused during the EC did not improve milk production over heat stress. Baseline blood glucose concentrations declined during heat stress, and those concentrations measured during the thermoneutral period were correlated with the change in blood glucose (from thermoneutral to heat stress). In the second experiment, zearalenone (6 mg) was fed to peri-pubertal gilts for 0, 7 or 21 days. Gilts were monitored daily for observable signs of zearalenone toxicity, and tissues were harvested after 21 days of treatment. No differences in the lengths or weights of any portions of the reproductive tract were observed. Likewise, there were no differences in body weight, carcass weight, dressing percent or liver weight. In summary, heat stress has varied effects on livestock production and the ultimate outcomes are not always predictable. Future work will be aimed at defining the factors that contribute to this variability. / Master of Science / Heat stress can have direct and indirect consequences that affect the efficiency of livestock production. The overall objective of this work is to improve agricultural animal productivity during periods of heat stress. The first study compared milk production, milk characteristics and blood glucose concentrations in the presence or absence of heat stress. Aspects of productivity were also measured when insulin and/or glucose were administered in a manner that mimicked either heat stress or thermoneutral conditions. Heat stress did cause a decline in circulating blood glucose, feed intake, and milk production. Infusion of insulin during thermoneutral conditions caused a decline in milk production but not in feed intake. As a way to compensate for the naturally low circulating glucose during heat stress, glucose was infused during heat stress in the last portion of the experiment. Simply infusing glucose to return the animal to pre-heat stress concentrations did not improve milk production. Further work is needed to determine factors (other than feed intake) that limit milk production during heat stress. The second study investigated whether the mycotoxin, zearalenone, affects the size or weight of the pig's reproductive tract. If differences existed, they would be indicative of potential problems with the function of those tissues. Young female pigs consumed zearalenone for 0, 7 or 21 days and tissues were evaluated 21 days after the experiment began. No changes in tissue weights or lengths were observed. Future experiments should be aimed at determining the concentration and duration of exposure at which zearalenone becomes problematic for young, female pigs.

A number of chemicals may have a negative impact on the environment and wildlife. Endocrine disruptors (EDs), which can mimic estrogen, interfere with natural hormones in organism and can have a negative effect on the reproductive system. Such substances include zearalenone (ZEA) - mycotoxin, produced by the fungi Fusarium. Despite the non-steriodal structure of its molecule, ZEA and its derivates possess potent estrogenic activity. The influence of ZEA on reproductive parameters and changes in expression of selected genes were tested in the outbred line of mice. This study showed significant effects of ZEA on number of reproductive performances. This effect was observed at lower examined dose, to which are humans normally exposed, and at higher doses, both showed changes in the tested parameters.

O presente trabalho visou utilizar o perfil polifásico, envolvendo características fenotípicas e genotípicas, na identificação de Fusarium spp. isolados de grãos de trigo como também investigar a presença das principais toxinas: desoxinivalenol e zearalenona nos grãos de trigo de três regiões produtoras de trigo (São Paulo, Paraná e Rio Grande do Sul). Nossos resultados indicaram que os gêneros mais frequentes no trigo foram Alternaria, Fusarium e Epicoccum. A determinação do genótipo por qPCR demonstrou predomínio de 15-ADON seguido por NIV e 3-ADON. A quantificação de DNA demostrou que o perfil 15-ADON foi responsável por 96% de todo DNA quantificado, seguido por NIV com 3.84% e 3-ADON, com somente 0.06%, indicando que 15-ADON, é o principal genótipo de tricoteceno nos grãos de trigo nacional. A toxina desoxinivalenol foi detectada em todas amostras analisadas, com mediana de 323, 466 e 783 µg/kg em SP, PR e RS, respectivamente. A determinação de zearalenona demonstrou contaminação em 100%, 80% e 42% dos grãos dos Estados do RS, PR e SP e medianas de 843, 100 e 14 µg/kg, respectivamente. / The present work aimed to use a polyphasic approach, by phenotypic and genotypic characteristics for the identification of Fusarium strains from wheat grains as well to investigate the presence of deoxynivalenol and zearalenone on wheat grains from three wheat producers States (Parana, Rio Grande do Sul and Sao Paulo). Our results showed that the main genera found were Alternaria, Fusarium and Epicoccum. Genotype detection from qPCR revealed predominance of 15-ADON, followed by NIV and 3-ADON. The qPCR demonstrated that 15-ADON genotype was responsible for 96% of all DNA quantified, followed by NIV with 3.84% and 3-ADON with 0.06%, indicating that 15-ADON is the main trichothecene genotype from Brazilian wheat grains. The toxin deoxynivalenol was detected in all 150 samples analyzed from wheat grains, with medians of 323, 466 and 783 µg/kg in SP, PR and RS, respectively. The zearalenone determination showed contamination on 100%, 80% and 42% of wheat grains from RS, PR and SP State and medians of 843, 100 and 14 µg/kg, respectively.

La zéaralénone (ZEN) est une mycotoxine produite par des Fusarium qui contaminent les cultures céréalières. Oestrogéno-mimétique et perturbateur endocrinien, ses effets toxiques concernent l'appareil reproducteur. En amont de l'évaluation du risque lié à une exposition à de faibles doses de ZEN, nous nous sommes intéressés à l'effet de la ZEN sur le niveau d'expression de transporteurs ABC majeurs. Ces pompes d'efflux sont présentes dans les barrières de l'organisme (notamment les barrières testiculaires et placentaires) où elles jouent un rôle dans la protection des tissus contre la toxicité de leurs substrats xénobiotiques. Dans ce travail nous avons d'abord caractérisé le métabolisme et la distribution tissulaire de la ZEN chez le rat, puis avons évalué son effet respectivement à court et long terme sur l'expression des transporteurs ABC après une exposition gestationnelle et néonatale. Nos résultats mettent en évidence des différences de comportement toxicocinétique de la ZEN en fonction du genre et du statut hormonal, et une réelle exposition fœtale et néonatale via la mère. La ZEN module l'expression des transporteurs ABC majeurs in vivo (dans les organes maternels, le foie foetal, le testicule du jeune adulte exposé J1-J5). Les résultats obtenus sur modèles in vitro (lignées sertolienne et placentaire) suggèrent que ces modulations sont liées en partie à l'interaction de la ZEN avec les récepteurs aux oestrogènes. Les conséquences d'une modulation de l'expression des transporteurs ABC induite par la ZEN pourraient être dramatiques pour le développement et la santé des individus à l'âge adulte et doivent être évaluées / Zearalenone (ZEN) is a mycotoxin produced by Fusarium that infect cereal crops. This Estrogenmimetic and endocrine disruptor affects the reproductive system. Upstream of the risk assessment of exposure to low doses of ZEN, we are interested in the effect of ZEN on the expression level of major ABC transporters. These efflux pumps are present in the barriers of organism (including testicular and placental barriers), where they protect tissues against the toxicity of their xenobiotic substrates. In this work, we first characterized the metabolism and tissue distribution of ZEN in rat and then evaluated respectively its short and long-term effect on ABC transporters expression after gestational and neonatal exposure. Our results highlight differences in the toxicokinetic behavior of ZEN by gender and hormonal status, and fetal and neonatal real exposure via the mother. ZEN modulates the expression of major ABC transporters in vivo (in the maternal organs, fetal liver, testis of young adult exposed J1-J5). The results of in vitro models (sertoli and placental cell lines) suggest that these variations are partly due to the interaction of ZEN with the estrogen receptors. The consequences of expression modulations of ABC transporters induced by ZEN could be dramatic for the development and health of individuals in adulthood and should be evaluated

Dentre os desafios do cultivo de trigo, encontram-se as doenças fúngicas e a presença de micotoxinas. Estima-se que as perdas anuais nesse setor sejam bilionárias. Além dos prejuízos econômicos, há preocupações quanto a segurança alimentar humana e animal. Com o objetivo de avaliar a micobiota e a ocorrência de micotoxinas em diferentes estádios de maturação do trigo foram realizados estudos morfológicos, utilizando métodos morfológicos clássicos e moleculares, identificação do perfil genotípico dos fungos produtores de tricotecenos, por PCR em tempo real e determinação de deoxinivalenol (DON) e zearalenona (ZEA), utilizando a Cromatografia Líquida de Alta Eficiência acoplada à Espectrometria de Massas (LC-MS/MS). Os dados obtidos também foram correlacionados com os fatores climatológicos. Os resultados revelaram a predominância do gênero Fusarium nas amostras das duas safras, seguido de Alternaria e Epicoccum. A atividade de água média para os dois anos foram, 0,608 e 0,886, consecutivamente. Dentro do gênero Fusarium constatou-se predomínio do complexo de espécies Fusarium graminearum (CEFG), identificando F. graminearum sensu stricto, F. meridionale, F. cortaderiae e F. austroamericanum. Os genótipos dos isolados foram, na sua maioria, 15-ADON para F. graminearum s.s, seguido de NIV para as espécies F. meridionale, F. cortaderiae e F. austroamericanum e 3-ADON para as duas últimas. Por PCR em tempo real, a determinação do genótipo nos grãos de trigo nas safras 2014/15 e 2015/16 demonstraram predomínio de 100% e 93% para 15-ADON, respectivamente. A quantificação de DNA dos isolados do CEFG demostrou que o perfil 15-ADON foi responsável por mais de 95% nas duas safras, com valores irrisórios, menores que 3% para 3-ADON e NIV. Os níveis de contaminação pela micotoxina DON nas amostras foi de 100% nas duas safras, com valores variando entre 24,3 a 5530 &#956g/kg. Para ZEA os valores foram 30% e 96%, nas duas coletas consecutivas, variação entre 26,0 a 4360 &#956g/kg. Ambas as micotoxinas foram detectadas em concentrações acima do limite estabelecido pela legislação brasileira, 3000 &#956g/kg para DON e 400 &#956g/kg para ZEA. Os fatores climáticos obtiveram uma correlação positiva tanto na contaminação fúngica quanto no acúmulo de micotoxinas. Este estudo forneceu novas informações sobre a micobiota natural do trigo brasileiro, em diferentes etapas do cultivo, demonstrando a contaminação por DON e alta frequência de ZEA em amostras do provindas do Paraná. / The fungal diseases and the presence of mycotoxins in cereals cause annually, billionaire losses. In addition, there are concerns about human and animal food safety. Due to the wheat culture relevance in feed and foodstuff, the present work aimed to evaluate mycobiota and the occurrence of mycotoxins in different wheat maturation stages. In order to achieve these objectives, morphologic studies using classic and molecular methods, genotype profile and DNA quantification from trichothecene producers, by real-time PCR and determination of deoxynivalenol (DON) and zearalenone (ZEA), using High Performance Liquid Chromatography coupled to Mass Spectrometry (LC-MS/MS), were done. The obtained data were also correlated with the climatic factors. The results showed the predominance of the genus Fusarium followed by Alternaria and Epicoccum. The average water activity over two years were 0.608 and 0.886, consecutively. Within the genus Fusarium, it was identified a predominance of the Fusarium graminearum species complex (FGSC), identifying F. graminearum sensu stricto, F. meridionale, F. cortaderiae and F. austroamericanum. The genotypes of the isolates were 15-ADON for F. graminearum s.s, followed by NIV for the F. meridionale, F. cortaderiae and F. austroamericanum and 3-ADON for F. cortaderiae and F. austroamericanum. In real-time PCR, the determination of genotype in wheat grains in the 2014/15 and 2015/16 harvests showed a predominance of 100% and 93% for 15-ADON, respectively. The DNA quantification of the FGSC isolates showed that the 15-ADON profile was responsible for more than 95% in both harvests. The levels of DON mycotoxin contamination in the samples were 100%, with values ranging from 24.3 to 5530 &#956g/kg. For ZEA the values were 30% and 96%, in the two consecutive collections, ranging from 26,0 to 4360 &#956g/kg. Both mycotoxins were detected in concentrations above the limit established by Brazilian legislation, 3000 &#956g / kg for DON and 400 &#956g/kg for ZEA. The climatic factors obtained a positive correlation both in the fungal contamination and in the accumulation of mycotoxins. This study provided new information on the Brazilian native mycobiota to different stages of cultivation, demonstrating contamination by DON and high frequency of ZEA in samples from Paraná.