Student Number : 9902006N - MSc(Med) Dissertation - School of Medicine - Faculty of Health Sciences / Hepatocellular carcinoma (HCC) is the most common primary malignant tumour of the liver. Aflatoxin B1 (AFB1) is a potent hepatocarcinogen, and dietary iron overload has been shown to contribute to HCC development in black africans. Both are well studied hepatotoxins. The aim of this study was to use a Wistar rat model over a 12 month period to investigate synergy and the extent thereof between AFB1 ingestion and dietary iron overload. 25ug/day of AFB1, reconstituted in DMSO, was administered by gavaging the animals, over a period of 10 days with a 2 day interval in between. The chow diet was supplemented with 0.75% (w/w) ferrocene iron. Experimental subjects were divided into 4 groups. Group 1 was fed the normal chow diet. Group 2 was fed 0.75% (w/w) ferrocene iron alone. Group 3 was gavaged 250μg AFB1 alone. Group 4 was fed the 0.75% (w/w) ferrocene iron and gavaged 250μg AFB1. A number of assays were conducted to investigate synergy. Colorimetric assays were used to measure serum iron, total-iron binding capacity, ALT, AST, GGT, nitrite production, lipid peroxidation and hydroxyproline concentrations. ELISA’s were used to determine ferritin, 8-isoprostane and 8-hydroxyguanosine concentrations. Nontransferrin bound iron was measured using an HPLC method. A chemiluminescent assay was used to measure superoxide anion production. Cytokines were measured using a suspension array system. Mutagenicity was assessed using the Ames mutagenicity assay using salmonella typhimirium strains TA97, TA98, TA100 and TA102. Iron profiling indicated that iron overloading occurred with the ingestion of the ferrocene diet. Biomarkers of oxidative stress, as illustrated by the measurement of 8-hydroxyguanosine and lipid peroxidation, showed additive synergistic effects between the two carcinogens. The anti-inflammatory interleukin-10 was shown to be markedly elevated with the co-administration of the two carcinogens, indicating the elevated inflammatory processes. Additive synergistic effects were noted in terms of the liver disease marker ALT. The salmonella typhimirium strain TA102 used in the Ames mutagenicity test showed increased colony counts with respect to the coadministration of carcinogens (P<0.05), although no synergistic effect was noted. In a few of the presented parameters, the AFB1 group was not significantly different to the control group, although significant differences between the Fe group and the Fe + AFB1 groups were noted. The implication of which is that the presence of AFB1 is increasing the activity of Fe as a carcinogen, thereby acting as a co-carcinogen. Examples of such parameters illustrating this are presented in the results section including serum ALT, serum nitrite, liver and serum lipid peroxidation, liver and serum 8-hydroxyguanosine, some of the mutagenicity assays, and interleukin-10. The conclusion of this study suggests that AFB1 acts as a co-carcinogen in the presence of iron overloading, implying that a synergistic relationship between these two toxins exists.

As aflatoxinas são substâncias naturais que apresentam efeitos tóxicos aos humanos e são reconhecidamente carcinogênicas. Estas substâncias podem estar presentes na dieta humana ou, em casos específicos, no ar respirado. Desta maneira, a exposição humana às aflatoxinas é objeto de muita preocupação. Uma das maneiras mais eficazes de avaliar a exposição humana as aflatoxinas é através da mensuração da presença de biomarcadores da exposição a estas substâncias em fluidos biológicos. Dentre as possibilidades de biomarcadores de exposição às aflatoxinas tem-se que aflatoxina M1 (AFM1), presente na urina e leite humano, é considerada um biomarcador válido. Assim sendo, o objetivo deste trabalho de pesquisa foi avaliar a presença de AFM1 em amostras de urina provenientes de indivíduos residentes na região urbana e rural da cidade de Piracicaba-SP, assim como, de leite de gestantes de Piracicaba e cidades da região. Nos indivíduos doadores de amostras de urina foi levantado também o padrão de ingestão de alimentos com alto risco de conter aflatoxinas, através da aplicação de inquéritos de freqüência alimentar e recordatórios 24 horas. A análise de AFM1 em urina e leite foi realizada por cromatografia liquida de alta eficiência (CLAE) com detecção por fluorescência. A extração e purificação do extrato foram realizadas com auxílio de colunas de imunoafinidade. No total 69 amostras de urina e 18 de leite foram analisadas. Entre as amostras de urina detectou-se a presença de AFM1 em 54 (78%) das amostras, com concentrações variando de 1,8 até 39,9 pg/mL. Não foi observada diferença estatística entre as concentrações médias detectadas entre urinas de indivíduos da zona urbana e rural, bem como no nível de consumo de produtos de risco. Apesar das concentrações de AFM1 detectadas serem inferiores as concentrações médias reportadas em outros países a freqüência de amostras positivas foi bastante elevada mostrando que as populações estudadas estão sendo expostas às aflatoxinas. Assim, melhores avaliações dos níveis de exposição necessitam ser realizados considerando que a amostragem utilizada foi pontual, pode existir variação de contaminação sazonal com aflatoxinas na dieta e a contaminação é heterogênea dentro no alimento. Não foi observada uma correlação entre o nível do consumo de produtos de risco e as concentrações detectadas em amostras de urina. Apenas uma amostra de leite apresentou contaminação detectada; entretanto, o nível de contaminação estava entre o limite de detecção (LD) e o limite de quantificação (LQ). / Aflatoxins are natural substances that present toxic and carcinogenic effects to humans. These substances may be present in human diet or, in specific cases, in the breathing air. Thus, the human exposition to aflatoxins is object of concern. One of the most effective ways to evaluate human exposition to aflatoxins is to measure the presence of biomarkers in biological fluids. Among the possibilities of aflatoxin presence biomarkers, the aflatoxin M1 (AFM1), present in human urine and milk, is considered a valid biomarker. The objective of this work was to evaluate the presence of AFM1 in urine samples from individuals who live in urban and rural areas in the county of Piracicaba, state of São Paulo, Brazil, and in milk of pregnant women from Piracicaba and neighbor cities. Urine-donor individuals were researched in relation to the ingestion of food with high risk of containing aflatoxins through the application of a food frequency questionnaire and 24-hour recall. The analysis of AFM1 in urine and milk was performed through high-performance liquid chromatography (HPLC) with fluorescence detection. The extract purification and extraction were performed with the aid of immunoaffinity columns. Overall, 69 urine and 18 human breast milk samples were analyzed. Among urine samples, the presence of AFM1 was detected in 54 (78%), with concentrations ranging from 1.8 to 39.9 pg/mL. No statistical difference was observed between average concentrations detected in the urine of individuals from urban and rural areas, as well as the consumption of aflatoxin risky food. Although the AFM1 concentrations detected are lower than those reported for other countries, the frequency of positive samples was quite high, showing that the populations studied are exposed to aflatoxins. Thus, further evaluations on the exposition levels should be performed, and considering that the sampling used in this work was punctual, there may be seasonal contamination variations in diet and the contamination level is heterogeneous within a food. No correlation between the consumption of risky food and concentrations detected in urine samples was observed. Only one milk sample presented detected contamination; however, the contamination level was between the limit of detection (LOD) and the limit of quantification (LOQ).

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2016-08-24T13:37:53Z No. of bitstreams: 1 2011- Veronica da Silva Cardoso.pdf: 2067813 bytes, checksum: a52c401412243d162e7030dda12cdf48 (MD5) / Made available in DSpace on 2016-08-24T13:37:53Z (GMT). No. of bitstreams: 1 2011- Veronica da Silva Cardoso.pdf: 2067813 bytes, checksum: a52c401412243d162e7030dda12cdf48 (MD5) Previous issue date: 2011-04-28 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico - CNPq / Piperine interference (amide extracted from black pepper) added to the diet of broiler chickens experimentally intoxicated by aflatoxin B1 (mycotoxin of great importance in the poultry sector) and it?s chemoprotective capacity were the main goal of this work. The experiment was divided into two assays: (i) The first assay was carried out to determine the effects of different concentrations of piperine (0, 60, 120, 180 ppm) and it?s possible toxicity in broiler chickens diets. Ninety six male chicks (Cobb), seven days old were used, being randomly allocated into four experimental groups (n=24) during 35 consecutive days. The following parameters were evaluated: biochemical, hematological, histopathological (proventriculus, gizzard, liver, kidney), histomorphometric (small intestine) and zootecnic. The concentration of 60 ppm of piperine in the diet was safe for broilers, showing better performance of broilers on period from 36 to 42 days old. The concentration of 180 ppm caused leukopenia and concentrations of 120 and 180 ppm was observed decrease in the number of heterophils and monocytes. Hepatotoxicity was observed by elevated AST enzyme activity, histopathological changes and decreased absorption surface in the segments (jejunum and ileum) of small intestine were observed for both 120 and 180 ppm concentrations. (ii) In the second assay, 60 broilers with nine days old divided into four groups: control, piperine (60 ppm added to diet), aflatoxin B1 (0.5 mg of aflatoxin B1.Kg-1 of body weight, orally) and piperine associated aflatoxin B1, were evaluated by effect chemoprotector of piperine against toxics effects of aflatoxin B1 being evaluated for zootecnic, biochemical, histopathological and histomorphometric parameters, toxic heterophils in peripheral blood and genotoxic by comet assay and micronucleus were also determined. No changes in the performance parameters were observed after this experiment. Broiler chickens intoxicated with AFB1 (0.5 mg of aflatoxin B1.kg-1 of body weight ) showed: decreased body weight gain and increased feed conversion; reduced carcass and cuts yields; liver toxicity, with increased relative weight of the liver and heart, macroscopic variations of hepatic parenchyma and increase of liver enzymes activity; kidney enzymes increase without evidence of renal tissue damage macroscopic or microscopic; leukopenia with significant reduction of lymphocytes and heterophils; reduction in absorptive surface due to the reduction of the length and width of the villi of all studied segments of small intestine; presence of toxic heterophils. The cytotoxic and genotoxic effects of aflatoxin B1 described above were significantly reduced or absent in the group of broiler intoxicated with aflatoxin B1 and fed with with piperine. No significant difference between piperine associated aflatoxin B1 in control and piperine groups were observed. The addition of 60 ppm of piperine in the diet of broiler chickens was safe, promoting beneficial effect both in zootecnic parameters and in poultry health, preventing toxic effects of aflatoxin B1in broiler chickens. / A interfer?ncia da piperina (amida extra?da da pimenta do reino) adicionada ? ra??o de frangos de corte intoxicados experimentalmente por aflatoxina B1 (micotoxina de grande relev?ncia no setor av?cola) e sua capacidade quimioprotetora foram o principal objetivo deste trabalho. O experimento foi dividido em dois ensaios: (i) O primeiro ensaio foi realizado para determinar os efeitos de diferentes concentra??es de piperina (0, 60, 120 e 180 ppm) foram avaliados e sua poss?vel toxicidade. Noventa e seis pintos (Cobb), com 7 dias de idade foram divididos aleatoriamente em 4 grupos (n=24), por 35 dias consecutivos. Os par?metros avaliados foram: hematol?gicos, bioqu?micos, histopatol?gicos (proventr?culo, moela, f?gado e rim), histomorfom?trico (intestino delgado) e par?metros zoot?cnicos. A concentra??o de 60 ppm de piperina adicionada ? ra??o foi segura para frangos de corte, tendo ainda resultado em melhor desempenho dos frangos na fase final (36-42 dias de idade). A concentra??o de 180 ppm promoveu leucopenia e nas concentra??es de 120 e 180 ppm foi observada diminui??o do n?mero de heter?filos e mon?citos; hepatotoxicidade, com eleva??o da enzima AST e altera??es histopatol?gicas em ambas as concentra??es; diminui??o da superf?cie de absor??o nos segmentos (jejuno e ?leo) do intestino delgado, por?m, sem altera??o dos par?metros zoot?cnicos. (ii) Para o segundo ensaio com a concentra??o de 60ppm de piperina: 60 frangos com 9 dias de idade, foram divididos em 4 grupos (n=15): grupo controle, grupo aflatoxina B1 (0,5 mg aflatoxina B1.kg-1 de peso vivo por via oral), grupo piperina (60 ppm adicionada ? ra??o) e grupo piperina associada a aflatoxina B1, determinando-se a capacidade quimioprotetora da piperina sendo avaliados os par?metros zoot?cnicos, hematol?gicos, bioqu?micos, histopatol?gicos, histomorfom?tricos, os efeitos genot?xicos da aflatoxina B1 pelo teste do cometa e do micron?cleo, presen?a de heter?filos t?xicos no sangue perif?rico. Os frangos intoxicados com aflatoxina B1 (0,5 mg de aflatoxina B1.Kg-1 de peso vivo) apresentaram: diminui??o do ganho m?dio de peso e piora da convers?o alimentar; diminui??o do rendimento de carca?a e cortes; hepatotoxicidade, com aumento de peso relativo do f?gado e cora??o, varia??es macrosc?picas do par?nquima hep?tico e eleva??o das enzimas hep?ticas; aumento das enzimas renais, sem evid?ncia de les?es macrosc?picas e microsc?picas no tecido renal; leucopenia, com diminui??o significativa de linf?citos e heter?filos; diminui??o da superf?cie de absor??o em fun??o da redu??o do comprimento e largura das vilosidades de todos os segmentos estudados do intestino delgado; presen?a de heter?filos t?xicos. Os efeitos citot?xicos e genot?xicos da aflatoxina B1 foram significativamente reduzidos ou ausentes no grupo piperina associada a aflatoxina B1, sem diferen?a significativa entre o grupo controle e piperina. A ra??o de frangos de corte com 60 ppm de piperina foi segura, promovendo efeito ben?fico tanto nos par?metros zoot?cnicos avaliados, como na sanidade av?cola, por impedir os efeitos t?xicos da aflatoxina B1 em frangos de corte.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-05-02T11:42:47Z No. of bitstreams: 1 2012 - Juliana Scofano Barrabin.pdf: 1875258 bytes, checksum: a222dd8183b171aeac5b500db7dbb482 (MD5) / Made available in DSpace on 2017-05-02T11:42:49Z (GMT). No. of bitstreams: 1 2012 - Juliana Scofano Barrabin.pdf: 1875258 bytes, checksum: a222dd8183b171aeac5b500db7dbb482 (MD5) Previous issue date: 2012-06-06 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / Aflatoxins classified by the World Health Organization as carcinogenic to humans and their intake causes many health injuries, from acute toxicities with liver lesions to hepatocellular carcinoma. Many countries already have regulations regarding these contaminants and the European Union is the most critical in relation to food safety. It has a strict regulation not only in relation to maximum permitted levels of aflatoxins in foods, but also concerning the quality of the analytical methods used to measure such contaminants. In Brazil, the National Agency of Sanitary Surveillance (ANVISA) published the resolution RDC N? 7, February 18th, 2011, providing for maximum permitted levels on mycotoxins in food for human consumption and raw materials, setting every two years the limits of these toxins become smaller, tending to be as strict as the European Union. For these goals to be fulfilled high sensitivity, selectivity and accuracy analytical methodologies are needed. This work aimed to develop an analytical method and to calculate validation parameters for determination of aflatoxin in peanuts using ultra performance liquid chromatography and detection by high-resolution mass spectrometry, method equivalent to the analytical quality standards of the European Union, which are worldwide trend. The developed method showed high selectivity and resolution, allowing the unambiguous qualitative analysis of aflatoxins B1, B2, G1 and G2 in peanuts and fulfilled the most rigorous analytical chromatographic criteria established by the EU. The requirements for a quantitative analysis have been partially achieved. Calibration curves were obtained with good linearity, however, was not possible to fix the sensitivity of the method, compromising the accuracy and quantification of the test. / As aflatoxinas s?o atualmente as micotoxinas mais estudadas no mundo. S?o classificadas pela Organiza??o Mundial da Sa?de como carcinog?nicas para humanos e sua ingest?o causa diversos danos ? sa?de, variando de toxicidades agudas com les?es de f?gado ao carcinoma hepatocelular. Muitos pa?ses j? possuem regulamenta??o quanto a estes contaminantes e a Uni?o Europ?ia (UE) ? uma das comunidades mais cr?ticas em rela??o ? legisla??o das micotoxinas com foco na inocuidade alimentar. Possui uma r?gida legisla??o n?o apenas em rela??o aos limites m?ximos permitidos de aflatoxinas em alimentos, mas tamb?m em rela??o ? qualidade dos m?todos anal?ticos utilizados para medir tais contaminantes. No Brasil, a Ag?ncia Nacional de Vigil?ncia Sanit?ria (ANVISA) publicou a resolu??o RDC N? 7 de 18 de fevereiro de 2011, dispondo sobre limites m?ximos tolerados de micotoxinas em alimentos. Essa resolu??o fixa novos limites para aflatoxinas, al?m de ocratoxina A, desoxinivalenol, fumonisinas, patulina e zearalenona em mat?rias primas e alimentos prontos para consumo, estabelecendo metas para que a cada 2 anos os limites dessas toxinas sejam cada vez menores, tendendo a uma maior rigidez, assim como a UE. Para que essas metas possam ser cumpridas s?o necess?rias metodologias anal?ticas de maior sensibilidade, seletividade e exatid?o. Desta forma o presente trabalho visou desenvolver um m?todo anal?tico e avaliar os par?metros de valida??o necess?rios para determina??o de aflatoxinas em amendoim. Utilizou-se cromatografia l?quida de ultra efici?ncia e detec??o por espectrometria de massas (UPLC-MS) de alta resolu??o, metodologia equiparada aos padr?es de qualidade anal?tica da UE. No m?todo desenvolvido obteve-se de 3 a 7 fragmentos para cada aflatoxina permitindo a an?lise qualitativa inequ?voca das AFB1, AFB2, AFG1 e AFG2 em amendoim e cumpriu-se com os mais rigorosos crit?rios anal?ticos cromatogr?ficos estabelecidos pela UE. O m?todo apresentou uma corrida cromatogr?fica de 5 minutos e menor gasto de solventes org?nicos quando comparado ? t?cnica de HPLC. Os limites de detec??o alcan?ados foram de 1,6; 0,396; 1,608 e 1,396 ?g/kg ?g/kg?g/kg?g/kg para as AFB1, AFB2, AFG1 e AFG2, respectivamente, e foram obtidas curvas de calibra??o com boa linearidade entre os valores de 1,6 a 12,0 ?g/kg (AFB1 e AFG1) e 0,396 a 2,97 ?g/kg (AFB2 e AFG2). Foram analisadas 10 amostras de amendoim adquiridas em feira livre do Rio de Janeiro e identificados tra?os das aflatoxinas B1, B2 e G2 em duas delas

Maize is susceptible to biotic and abiotic stresses. The most important abiotic stresses in Africa are drought and low soil fertility. Aflatoxin contamination is a potential problem in areas facing drought and low soil fertility. Three studies were conducted to evaluate maize germplasm for tolerance to stress. In the first study, fifteen maize inbred lines crossed in a diallel were evaluated under drought, low N stress, and well-watered conditions at six locations in three countries to estimate general (GCA) and specific combining ability (SCA), investigate genotype x environment interaction, and estimate genetic diversity and its relationship with grain yield and heterosis. GCA effects were not significant for grain yield across environments. Lines with good GCA effect for grain yield were P501 and CML258 across stresses. Lines CML339, CML341, and SPLC7-F had good GCA effects for anthesis silking interval across stresses. Additive genetic effects were more important for grain yield under drought and well-watered conditions. Heterosis estimates were highest in stress environments. Clustering based on genetic distance calculated using marker data from AFLP, RFLP, and SSRs grouped lines according to origin. Genetic distance was positively correlated with grain yield and specific combining ability. In the second study, synthetic hybrids were evaluated at seven locations in three countries to estimate GCA and SCA effects under low N stress and optimal conditions and investigate genotype x environment interaction. GCA effects were significant for all traits across low N stress and optimal conditions. The highest yielding synthetic hybrids involved synthetics developed from stress tolerant lines. Synthetics 99SADVIA-# and SYNA00F2 had good GCA for grain yield across low N stress conditions. Heterosis was highly correlated with grain yield. Optimal environments explained more variation than stress environments. The third study evaluated the agronomic performance and aflatoxin accumulation of single and three-way cross white maize hybrids at five locations in Texas. Inbreds CML343, Tx601W, and Tx110 showed positive GCA effects for grain yield. Significant GCA effects for reduced aflatoxin concentration were observed in lines CML269, CML270, and CML78 across locations. Differences in performance between single and three-way crosses hybrids were dependent mostly on the inbred lines.

The need to assess human exposures to foodborne and environmental carcinogens, particularly in populations at high risk for cancer and disease, has led to the development of chemical-specific biomarkers. Sensitive biomarkers for aflatoxin and polycyclic aromatic hydrocarbons (PAHs) have been useful in providing information on population exposure and reducing associated public health impacts. Aflatoxins are fungal metabolites found in a variety of foods. Among these toxins, aflatoxin B1 (AFB1) is the most predominant and hepatocarcinogenic. Acutely, AFB1 can cause disease and death, necessitating safe and effective intervention strategies. Inclusion of NovaSil (NS) clay in the diet represents a practical, sustainable approach. NS has been shown to prevent aflatoxicosis in multiple animal species by binding aflatoxins in the gastrointestinal tract, reducing toxin bioavailability. Co-exposure to PAHs, hazardous environmental contaminants, has been shown to increase the risk for hepatocellular carcinoma (HCC). Therefore, objectives of this research were to utilize biomarkers to assess aflatoxin and PAH exposures in susceptible populations in Ghana and the U.S. and to evaluate the safety and efficacy of NS intervention in Ghana (a population at risk for aflatoxicosis). After 3-month intervention with 3.0g NS/day, median aflatoxin M1 (an AFB1 metabolite) was significantly reduced (up to 58 percent) compared to the placebo group. Furthermore, no significant differences were found in levels of nutrient minerals between NS and placebo groups at baseline and 3-months suggesting NS can be used to effectively sorb AFB1 without affecting serum concentrations of important minerals. PAH biomarker results showed participants in Ghana were significantly exposed to high levels of PAHs based on the presence of 1-hydroxypyrene (1-OHP) in the majority of urines (98.9 percent). NS treatment had no effect on 1-OHP levels, further confirming the preferential binding of aflatoxins by NS. U.S. population data from a Hispanic community in Texas with an elevated incidence of HCC demonstrated a lower percentage and level of aflatoxin and PAH biomarkers. Aflatoxin M1 excretion, however, was associated with increased consumption of certain foods prone to aflatoxin contamination; thus, some individuals may be more vulnerable to exposure and associated interactions that increase the risk for HCC (e.g., PAHs or hepatitis infection).

Maize is susceptible to biotic and abiotic stresses. The most important abiotic stresses in Africa are drought and low soil fertility. Aflatoxin contamination is a potential problem in areas facing drought and low soil fertility. Three studies were conducted to evaluate maize germplasm for tolerance to stress. In the first study, fifteen maize inbred lines crossed in a diallel were evaluated under drought, low N stress, and well-watered conditions at six locations in three countries to estimate general (GCA) and specific combining ability (SCA), investigate genotype x environment interaction, and estimate genetic diversity and its relationship with grain yield and heterosis. GCA effects were not significant for grain yield across environments. Lines with good GCA effect for grain yield were P501 and CML258 across stresses. Lines CML339, CML341, and SPLC7-F had good GCA effects for anthesis silking interval across stresses. Additive genetic effects were more important for grain yield under drought and well-watered conditions. Heterosis estimates were highest in stress environments. Clustering based on genetic distance calculated using marker data from AFLP, RFLP, and SSRs grouped lines according to origin. Genetic distance was positively correlated with grain yield and specific combining ability. In the second study, synthetic hybrids were evaluated at seven locations in three countries to estimate GCA and SCA effects under low N stress and optimal conditions and investigate genotype x environment interaction. GCA effects were significant for all traits across low N stress and optimal conditions. The highest yielding synthetic hybrids involved synthetics developed from stress tolerant lines. Synthetics 99SADVIA-# and SYNA00F2 had good GCA for grain yield across low N stress conditions. Heterosis was highly correlated with grain yield. Optimal environments explained more variation than stress environments. The third study evaluated the agronomic performance and aflatoxin accumulation of single and three-way cross white maize hybrids at five locations in Texas. Inbreds CML343, Tx601W, and Tx110 showed positive GCA effects for grain yield. Significant GCA effects for reduced aflatoxin concentration were observed in lines CML269, CML270, and CML78 across locations. Differences in performance between single and three-way crosses hybrids were dependent mostly on the inbred lines.

Šis mokslinis darbas buvo atliktas Justinas Banelis Lietuvos sveikatos mokslų universitete. Vadovaujant prof. dr . Broniui Bakučiui konsultuojant lekt. dr. Violetai Baliukonienei. Šio tyrimo tema " Mikotoksinus detoksikuojančių profilaktikos priemonių pieniniams galvijams efektyvumo įvertinimas " . Puslapių skaičius 37 , 10 lentelių , 3 nuotraukos . Tyrimai buvo atlikti nuo 01/10/2011 iki 01/10/2012 LSMU VA ir gyvūnų gerovės laboratorijoje. Šio tyrimo tikslas buvo Įvertinti mikotoksinus detoksikuojančio preparato efektyvumą pieninių galvijų bandos sveikatingumui. Biocheminių , kraujo tyrimų parametrų ir pieno sudėties melžiamų karvių ir nustatyti mikotoksinu detoksikacija su komerciniu X adsorbentu. Buvo atrinktos 28 Lietuvos Žalosios veislės kliniškai sveikos vidutinio produktyvumo karvės. Karvės buvo nuo 3,43 ± 0,24 laktacijos trukmės laktacijos buvo 122 ± 15,9 dienų. Pieno išeiga karvės buvo 15,36 kg per dieną ,pieno riebalų vidurkis buvo 4,14 ± 0,25 % , baltymų kiekis 3,06 ± 0,04 % ir karbamido - 23,80 ± 1,35 mg% . Karvių pašarams buvo nustatytas mikotoksinų aflatoksino B1 ( AFL B1) , zearalenono ( ZON ), deoksinivalenolio ( DON ). Zearalenonas buvopagrindinis teršalas ir buvo rasta šiene iki 1000 mikrogramų / kg sausos medžiagos. Deoksinivalenolis buvoantras teršalų ir buvo rasta šieno lygiu iki 600 mikrogramų / kg sausos medžiagos. Didžiausias aflatoksino B1 koncentraciją - 10,0 mikrogramo / kg žemės miežių . Komercinis detoksikuojantis produktas X buvo duotas... [toliau žr. visą tekstą] / This scientific work was done by student Justinas Banelis of Lithuanian University of Health Sciences. He was supervised by prof. dr. Bronius Bakutis and consulted by lect. dr. Violeta Baliukoniene. The topic of this scientific research is “The effects of feeding mycotoxin binding products on the performance of lactating dairy cows“. Page count 37, 10 tables,3 pictures. The experiments was done from 01/10/2011 to 01/10/2012 in LUHS VA in the Animal Welfare Reaserch Laboratory. The aim of this study were to investigate the long-term exposure toxic effects of diet naturally contaminated with a low concentrations mycotoxins (AFL B1, ZON, DON) on biochemical, complete blood count parameters and milk composition of dairy cows and to determined the mycotoxins detoxification by with the commercial X adsorbent. 28 Lithuanian Red clinically healthy medium productive cows were selected. Cows were from 3.43±0.24 lactation and duration of lactation were 122±15.9 days. Milk-yield of cow was 15.36 kg per day, an average of milk fat was 4.14 ±0.25 %, protein content 3.06±0.04 % and urea – 23.80±1.35 mg %. In cow feeds were determined mycotoxins aflatoxin B1 (AFL B1), zearalenone (ZON), deoxynivalenol (DON). Zearalenone was the major contaminant and was found in the hay at level of up to 1000 µg/kg of dry matter. Deoxynivalenol was the second contaminant and was found in the hay at level of up to 600 µg/kg of dry matter. The biggest Aflatoxin B1 content - 10.0 µg/kg in ground barley. The... [to full text]

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The aflatoxin B1 (AFB1) is one of the main mycotoxins that can be identified in foods, and has relevance for agricultural economics and public health because of its immunotoxic properties. A functional immune system is a basic requirement for a healthy life in modern animal production. The interaction involving nutrition and immunity is a strategic factor to obtain a high quality performance in the poultry industry. Immunomodulators such as β-glucans have an immunostimulating activity, which enables the host ability to resist opportunistic infections. To contribute to the elucidation of the mechanism of action of β-glucans in broiler chicken lymphocytes, the effects of the concentrations of 0.1, 1 and 10% of β-glucans derived from Saccharomyces cerevisiae were investigated in lymphocytes exposed to increasing concentrations of AFB1 (0, 0.1, 1, 10, 20 μg/ml). Lymphocytes were separated by Ficoll-Histopaque density and cultured in 96 well-plates containing AFB1 and/or β-glucans in a 5% CO2 atmosphere at 39°C. MTT, PicoGreen® and 2'-7'-dichlorofluorescein diacetate cytotoxicity tests were evaluated at 24, 48 and 72 h of incubation. The comet assay to elucidate the DNA damage was also performed. The percentage of viable cells decreased in the presence of 10 μg/ml AFB1 at 48 h (p < 0.05) and 10 and 20 μg/ml AFB1 at 72 h (p < 0.001 and p < 0.01, respectively), when compared to the control group (0 μg/ml). Furthermore, an increase in cell-free DNA in AFB1 concentrations > 1 μg/ml (p < 0.001) and the generation of ROS at 24 h were also observed. DNA damage increased approximately 2.3 fold in lymphocytes exposed to 20 μg/ml of AFB1 when compared to the control group. Conversely, β-glucans showed cytoprotective effects (p < 0.001), and the concentration of 1% reverted the AFB1-induced lymphocyte damage. β-glucans at 10% significantly increased (p < 0.001) the formation of reactive oxygen species (ROS), potentiating the AFB1-induced ROS formation. In conclusion, this study showed that AFB1 and β-glucans exert influence on lymphocyte oxidative metabolism and have dose-dependent potentiating effects. The results also showed genoprotective in vitro effect of β-glucans in poultry lymphocytes exposed to AFB1, being the concentration of 1% β-glucans able to maintain DNA integrity. / A aflatoxina B1 (AFB1) é uma das principais micotoxinas que podem ser identificadas em alimentos, possuindo relevância agroeconômica e para a saúde pública, por ser considerada imunotóxica. Um sistema imune funcional é um requisito básico para uma vida saudável na produção moderna de animais. A interação envolvendo nutrição e imunidade é um fator estratégico para obter um bom desempenho em frangos de corte. Devido as suas atividades imunomodulatórias, substâncias como as β-glucanas proporcionam ao hospedeiro uma maior capacidade de resistir a infecções oportunistas. Para melhor compreender o mecanismo de ação das β-glucanas, sobre os linfócitos de frangos de corte, investigou-se os efeitos das concentrações de 0.1, 1 e 10% de β-glucanas derivadas do fungo Saccharomyces cerevisiae em linfócitos expostos a crescentes concentrações de AFB1 (0, 0.1, 1, 10, 20 μg/ml). Os linfócitos foram separados através do reagente de densidade Ficoll-Histopaque e cultivados em placas de 96 poços, contendo as concentrações de AFB1 e/ou β-glucanas em atmosfera de 5% de CO2 a 39°C durante 24, 48 e 72 h. A citotoxicidade celular foi avaliada através dos testes MTT e PicoGreen®, e a formação de espécies reativas de oxigênio (EROS) através do ensaio 2′-7′- diacetato diclorofluoresceína. Também foi utilizado o teste cometa para elucidar os danos ao DNA. A viabilidade celular reduziu na presença de 10 μg/ml de AFB1 em 48 h (p < 0.05) e em 10 and 20 μg/ml de AFB1 (p < 0.01 e p < 0.001, respectivamente) em 72 h quando comparada ao grupo controle. Além disso, as concentrações de AFB1 > 1 μg/ml aumentaram significativamente (p < 0.001) a liberação de fita dupla de DNA (dsDNA) e a produção de EROS em 24 h. Os danos causados ao DNA foram confirmados através do momento cauda do cometa e aumentaram cerca de 2,3 vezes em linfócitos expostos a 20 μg/ml de AFB1 quando comparados ao grupo controle. Por outro lado, as β-glucanas exerceram efeitos citoprotetores (p < 0.001), sendo que a concentração de 1% foi capaz de reverter os danos genotóxicos causados pela ação da AFB1. Já a concentração de 10% de β-glucanas aumentou significativamente (p < 0.001) a formação de EROS, potencializando a ação da AFB1. Em conclusão, este estudou evidenciou que a AFB1 e as β-glucanas exercem influência sobre o metabolismo oxidativo dos linfócitos e possui efeito potencializador dose-dependente. Os resultados também evidenciaram o efeito genoprotetor in vitro de β-glucanas em linfócitos de frangos expostos a AFB1, sendo a concentração de β-glucanas a 1% capaz de manter a integridade do DNA.

Resumo: Os efeitos da aflatoxina e fumonisina e sua associação, sobre a produção e a qualidade dos ovos em poedeiras comerciais, foram avaliados bem como a eficácia de um adsorvente glucano em promover a redução e/ou eliminação total destes efeitos. Foram utilizadas 168 poedeiras com 37 semanas de idade, por um período experimental de 56 dias. O delineamento experimental foi inteiramente casualizado em esquema fatorial 3x2 + 1 (3 tratamentos com micotoxinas: aflatoxina (AF), fumonisina (FU) e aflatoxina + fumonisina (AF + FU); 2 tratamentos com e sem adsorvente; e grupo controle sem micotoxinas e adsorvente), totalizando 7 tratamentos e 6 repetições com 4 aves/gaiola. As rações foram contaminadas, isoladamente ou em combinação, com 1ppm de AF e 25ppm de FU e o adsorvente foi incluído na concentração de 2kg/ton ração. Os tratamentos com presença de AF apresentaram as menores porcentagens (p = 0,0594) de postura (76,72% para AF e 77,38% para AF+FU). A massa de ovos obteve a menor média (p<0,05) no tratamento com AF+FU (49,49 g) que foi diferente do grupo controle (64,06 g). O tratamento com AF apresentou maior espessura e resistência de casca comparado ao grupo controle e ao tratamento com FU. O uso do adsorvente no tratamento com AF reduziu a resistência da casca voltando aos valores próximos aos do controle. As alterações observadas neste estudo são indicativas da toxicidade da aflatoxina na concentração de 1 ppm. Os efeitos da fumonisina foram menos evidentes em função da baixa dose utilizada neste estudo (25mg/kg). O uso de glucano na concentração de 2 kg/ton foi efetivo em reverter alguns dos efeitos tóxicos da aflatoxina e, em menor extensão da fumonisina, quando estas micotoxinas estavam separadamente na ração de poedeiras comerciais / Abstract: The present study was conducted to evaluate the effects of aflatoxin and fumonisin and their association on production and egg quality in laying hens and was also evaluated the effectiveness of an adsorbent glucan to promote the reduction and/or total elimination of these effects. One hundred and sixty-eight 37-wk-old laying hens were used for a trial period of 56 days. The experimental design was a completely randomized with 3x2+ 1 factorial arrangement (three treatments with mycotoxins: aflatoxin (AF), fumonisin (FU) + fumonisin and aflatoxin (AF + FU), two treatments with and without adsorbent, and the control group without mycotoxin and adsorbent), totaling seven treatments, six replicates and four birds per cage. The mycotoxins were added to rations, singly and in combination, at levels of 1 ppm AF and 25 ppm FU and the adsorbent was included in the dosage of 2kg/ton .The treatments with the presence of AF had the lowest percentages (p = 0,0594) of stance (76,72% to AF and 77,38% to AF + FU). The egg mass received the lowest average (p<0,05) in the treatment with AF + FU (49,49 g) which was different from the control group (64,06 g). Treatment with AF showed higher values of thickness and peel strength compared to the control group and treatment with FU. The use of the adsorbent in the treatment with AF reduced the strength of the shell back to values near baseline. The changes observed in this study are indicative of the toxicity of aflatoxin at a concentration of 1mg/kg. The effects of fumonisin were less evident because of the low levels used in this study (25mg/kg). The level of glucan added to the diets was effective to prevent some of the toxic effects of aflatoxins and to a lesser extent of fumonisin, when the mycotoxins were separately in the diet of laying hens / Orientador: Denise Rangel da Silva Sartori / Coorientador: José Roberto Sartori / Banca: Alice Eiko Murakami / Banca: Daniela Felipe Pinheiro / Mestre