Submitted by Mario BC (mario@bc.ufrpe.br) on 2016-06-17T12:26:34Z No. of bitstreams: 1 Victor Hugo de Vasconcelos Calado.pdf: 593050 bytes, checksum: ab20bdddfa55a2c22cb9e115c16dbb93 (MD5) / Made available in DSpace on 2016-06-17T12:26:34Z (GMT). No. of bitstreams: 1 Victor Hugo de Vasconcelos Calado.pdf: 593050 bytes, checksum: ab20bdddfa55a2c22cb9e115c16dbb93 (MD5) Previous issue date: 2016-03-11 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Mycotoxins have been a problem in poultry production to be present in the major ingredients (corn and soybean meal) that make up the poultry feed, leading to problems such as an inadequate use of food and reduction of animal front immunity to various pathogens. Thus it aimed to evaluate the effects of adding mycotoxin adsorbents in broiler diets naturally contaminated with aflatoxin and fumonisin, on the parameters: histopathology, biometric and serological. The experiment was conducted with 60 birds, seven-day-old Cobb commercial line in three treatments: T1 - control (not including mycotoxin adsorbents in diet); T2 - adding 0.5 kg / t. of adsorbent A and T3 inclusion of 1.5 kg / t of adsorbent B. The broilers receive in all treatments, isonutritives diets naturally contaminated by mycotoxins. These broilers were raised up to 60 days old when was made serology tests for NDV and IBDV, and after were sacrificed for histopathologic and biometric analysis. The results were submitted to analysis of variance (ANOVA) with significance level of 5% probability and Tukey test. There were significant differences between treatments (P <0.05) in body weight. The body weight was significantly different between T1 and T2 (P = 0.01400), with a higher weight of the broilers receiving the adsorbent (4.26 ± 0.37). Among the organs analyzed, only the weight of the cecal tonsil significant difference between T1 and T2 (P <0.05; P = 0.03008), and T1 (5,20g ± 1.239) had an average weight slightly higher than average weight of T2 (4,05g ± 1.208). The amounts of immunoglobulins to IBDV showed significantly higher concentrations in T2 (P = 0.041). This contamination of broilers aflatoxin and fumonisin caused histopathological lesions in immune organs, liver and kidneys. However adsorbents used A and B reversed in parts of the lesions, especially in the liver, bursa and thymus. This reversal of lesions led to improved serological tests for IBD (Gumboro bursal-infectious disease) in the group treated with the adsorbent, resulting in broilers with better performance, suggesting that this adsorbent showed better effect on broilers in field conditions. / As micotoxinas têm sido um problema na produção avícola por estarem presentes nos principais ingredientes (milho e farelo de soja) que compõem a ração para aves, levando a problemas como: inadequado aproveitamento dos alimentos e diminuição de imunidade animal frente a patógenos diversos. Assim, objetivou-se avaliar os efeitos da adição de adsorventes de micotoxinas, em dietas de frangos de corte contaminadas naturalmente com aflatoxinas e fumonisinas, sobre os parâmetros: biométricos, sorológicos e histopatológicos. O experimento foi realizado com 60 aves, com sete dias de idade, da linhagem comercial Cobb distribuídas em três tratamentos: T1 – Controle (sem inclusão de adsorventes de micotoxinas na dieta); T2 – inclusão de 0,5 kg/t do adsorvente A (composto por aluminossilicato de sódio e cálcio modificado industrialmente) e T3- inclusão de 1,5 kg/t do adsorvente B(composto por aluminossilicato de sódio e cálcio). As aves receberam, em todos os tratamentos, dietas isonutritivas, feitas com milho sabidamente contaminado, em campo por micotoxinas. Essas aves foram criadas até 60 dias de idade, quando foi realizada sorologia para NDV(vírus da doença de Newcastle) e IBDV(vírus da doença infecciosa bursal) e preparadas para eutanásia para posteriores análises histopatológicas e biométricas. Ao final, o peso vivo foi significativamente diferente entre os tratamentos T1 e T2 (P=0,01400), observando-se maior peso das aves que receberam o adsorvente A (4,26 ± 0,37). Entre os órgãos analisados, apenas o peso da tonsila cecal apresentou diferença significativa entre os tratamentos T1 e T2 (P<0,05; P=0,03008), sendo que T1 (5,20g ± 1,239) apresentou peso médio levemente superior ao peso médio do T2 (4,05g ± 1,208). No teste sorológico, as quantidades de imunoglobulinas para IBDV apresentaram concentrações significativamente maiores no T2 (P=0,041). A intoxicação das aves com aflatoxinas e fumonisinas provocou lesões histopatológicas nos órgãos linfóides (bolsa cloacal, baço, timo e tonsilas cecais), fígado e rins. Contudo os adsorventes usados A e B minimizou as lesões, principalmente no fígado, bursa, e timo. A menor intensidade das lesões proporcionou melhores resultados sorológicos para IBDV no grupo tratado com o adsorvente A, resultando em aves com melhor desempenho, conclui-se sugerindo que este adsorvente produz melhor efeito nas aves em condições de campo.

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).

Made available in DSpace on 2014-08-20T14:38:49Z (GMT). No. of bitstreams: 1 tese_paulo_Lopes.pdf: 545120 bytes, checksum: 00e0a07af29ecb4fca28d703cd43415b (MD5) Previous issue date: 2008-04-10 / Three experiments were carried out at the Ictyology Laboratory (Departamento de Zootecnia UFPel) to evaluate: a) - aflatoxin effects and b) - adsorptive action of 2 adsorbents (sodium and calcium aluminum-silicate and glucomanane) on growth and hematologic paramaters of jundiá (Rhamdia quelen) fingerlings, fed diets artificially contaminated. The first experiment consisted of 360 fingerlings (initial weight 4 g), raised during 90 days in thermo-regulated recirculation water system. Twelve treatments were compared, with 3 replications. Four levels of aflatoxin inclusion in diets (0, 150, 250 e 350 μgAFkg-1), with and without (0, 0.3 and 0,6%) adsorbent addition (sodium and calcium aluminum-silicate). Negative effect of aflatoxins significantly reduced (P<0.05) growth and weight gain of fingerlings, proportionately to increasing levels of aflatoxis in diet, without mortality occurrence. Adsorbent levels in diet did not diminish the effect of aflatoxins on fish performance. The second experiment evaluated the effect of aflatoxins and 2 adsorbents (sodium and calcium aluminum-silicate) on performance of 189 jundiá juveniles (initial weight 43.13 g),grown during 60 days. Nine treatments with 3 replications, 3 levels of aflatoxin inclusion in diet (0, 50, 100 μgAFkg-1) and 2 adsorbents (0.3%) were tested. Negative action of aflatoxins significantly reduced fish specific growth rate, proportionately to increasing levels of aflatoxins in diet, without mortality occurrence. Only the addition of glumanane to diet significantly (P<0.05) neutralized the negative effects of aflatoxins, in relation to the contaminated group. The third experiment evaluated aflatoxin effects on erythrocytic parameters of jundiá juveniles fed diets with different concentrations of aflatoxins (0, 150, 250 e 350 μgAFkg-1), during 90 days in a thermo-regulated recirculation water system. It was observed that increment of aflatoxin levels in diet significantly reduced hematocrit rate and hemoglobin. Jundiá juveniles fed diets with aflatoxin are susceptible to negative effects, presenting losses in growth, weight gain and reduction of erythrocytic parameters. Addition of 0.3% of glucomanane in diet diminishes the action of aflatoxins on jundiá juveniles. / Três experimentos foram conduzidos no laboratório de Ictiologia do Departamento de Zootecnia (UFPel) para avaliar: a)- efeito das aflatoxinas e b)- ação adsortiva de dois adsorventes (aluminosilicato de sódio e cálcio e glucomanano) no crescimento e sobre parâmetros hematológicos em alevinos e juvenis de jundiá (Rhamdia quelen), alimentados com dieta contaminada artificialmente. O primeiro experimento foi realizado com 360 alevinos (peso inicial 4 g), durante 90 dias, criados em sistema de recirculação da água- termo regulada. Foram testados doze tratamentos com três repetições, e quatro níveis de inclusão de aflatoxinas na dieta (0, 150, 250 e 350 μgAFkg-1), com e sem adição de adsorvente (aluminosilicato de sódio e cálcio) (0, 0,3 e 0,6%). O efeito negativo das aflatoxinas reduziu significativamente (P<0,05) o crescimento e ganho de peso dos alevinos de jundiá, proporcionalmente aos níveis crescentes de aflatoxinas na dieta, sem apresentar mortalidade. Os níveis do adsorvente na dieta, não diminuíram os efeitos das aflatoxinas sobre o desempenho dos peixes. No segundo experimento foi avaliado o efeito das aflatoxinas e de dois adsorventes: aluminosilicato de sódio e cálcio e o glucomanano, sobre o desempenho zootécnico de juvenis de jundiá. Foram utilizados 189 juvenis (peso inicial de 43,13 g), durante 60 dias. Foram testados nove tratamentos com três repetições, três níveis de inclusão de aflatoxinas na dieta (0, 50, 100 μgAFkg-1) e com dois adsorventes (0 e 3%). A ação negativa das aflatoxinas reduziu significativamente o ganho de peso, biomassa final, ganho de peso diário e taxa de crescimento especifico dos animais, proporcionalmente aos níveis crescentes de aflatoxinas na dieta, em relação ao tratamento controle, sem apresentar mortalidade. Apenas a adição de glucomanano na dieta neutralizou significativamente (P<0,05) os efeitos negativos das aflatoxinas em relação ao grupo contaminado. No terceiro experimento foi avaliado o efeito das aflatoxinas sobre os parâmetros eritrocitários de alevinos de jundiá, alimentados com diferentes concentrações de aflatoxinas na dieta (0, 150, 250 e 350 μgAFkg-1) durante 90 dias, num sistema de criação fechado e termo-regulado. Foi observado que o aumento dos níveis de aflatoxinas na dieta, reduziu significativamente a taxa de hematócrito e hemoglobina. Os alevinos de jundiá alimentados com aflatoxinas na dieta são susceptíveis aos efeitos negativos, ocorrendo perdas no crescimento, ganho de peso e redução dos parâmetros eritrocitários. A adição de 0,3% de glucomanano na dieta diminui a ação das aflatoxinas em juvenis de jundiá.

Aflatoxins are metabolites from fungi, that may be injurious to animal health, resulting in reduced production and affecting poultry industry by decrease the growth rate and worsening feed conversion. A study was carried out to evaluate the electrophoresis profile of serum protein in broilers fed with diets containing aflatoxins and natural clinoptilolite clay. A total of 528 male broilers Ross were used, distributed in 6 treatments with 4 replications each: T1 control (without aflatoxins or clinoptilolite), T2 5ppm of aflatoxins, T3 0.25% of clinoptilolite, T4 5ppm of aflatoxins and 0.25% of clinoptilolite, T5 0.5% of clinoptilolite and T6 5ppm of aflatoxins and 0.5% of clinoptilolite. The broilers were placed in 24 boxes and submitted to treatments from 1 to 42 days, when they were slaughtered. Were analyzed the total proteins, albumin fractions, alpha 1, alpha 2, beta and gamma. The aflatoxins decreased (P<0.05) total protein, albumin, and globulins (alpha and beta fractions). However, no effect (P<0.05) of aflatoxins was observed about gamma fraction. The clinoptilolite no modify (P<0.05) the serum proteins. Related to control, broilers fed with diets containing aflatoxins and clinoptilolite presented low (P<0.05) levels of total protein, albumin, and globulins (alpha and beta fractions). In conclusion, aflatoxins change the electrophoresis profile and clinoptilolite is not able to protect against this change / Aflatoxinas são metabólitos de fungos, que podem causar danos à saúde do animal, resultando em redução da produção e afetando à indústria avícola, pela diminuição da taxa de crescimento e péssima conversão alimentar. O objetivo do presente trabalho foi avaliar o perfil eletroforético das proteínas séricas de frangos de corte alimentados com dietas contendo aflatoxinas e/ou argila clinoptilolita natural. Foram utilizados 528 frangos de corte, machos, da linhagem Ross, distribuídos em 6 tratamentos com 4 repetições cada: T1 testemunha (ração sem aflatoxinas ou clinoptilolita), T2 ração com 5ppm de aflatoxinas, T3 ração com 0,25% de clinoptilolita, T4 ração com 5ppm de aflatoxinas e 0,25% de clinoptilolita, T5 ração com 0,5% de clinoptilolita e T6 ração com 5ppm de aflatoxinas e 0,5% de clinoptilolita. Os animais ficaram alojados em 24 boxes, e submetidos aos tratamentos do 1º ao 42º dia, quando foram sacrificados. Foram analisadas as proteínas totais, as frações albumina, alfa 1, alfa 2, beta e gama. Com exceção das médias da fração gama, o teste de Tukey revelou diferenças significativas (P<0,05) nas médias de todas as proteínas totais e frações protéicas nos tratamentos onde as aflatoxinas estavam presentes. A ação das aflatoxinas nas proteínas totais ocorre na síntese de albumina e globulinas (frações alfa e beta). As gamaglobulinas não são afetadas pelas aflatoxinas. Em relação ao controle, as aves alimentadas com dietas com aflatoxinas e clinoptilolita apresentaram baixos (P<0,05) níveis de proteína total, albumina e globulinas (alfa e beta). Conclui-se que as aflatoxinas alteram o perfil eletroforético e a clinoptilolita adicionada na ração, não é capaz de evitar essa alteração

This research had as objective evaluated the biochemistry values and the hepatic and renal enzymatic activity pf broiler chicken submitted to experimental intoxication using aflatoxin with and without addition of sodic montmorilonite on the diet. In this study were used 528 Cobb s broiler chickens that were divided in 6 treatments from 1º to 42º day of life: (T1) control: normal diet; (T2) diet with 5 ppm of aflatoxin; (T3) diet with 0,25% sodic montmorilonite; (T4) diet with 5 ppm of aflatoxin + 0,25% sodic montmorilonite; (T5) diet with 0,50% sodic montmorilonite; (T6) diet with 5 ppm of aflatoxin + 0,50% sodic montmorilonite. The treatment (T2) showed significant (P<0.01) decreased levels of uric acid, albumin, cholesterol, creatinine, triglycerides, globulins and total serum proteins. There was statistically significantly increased on alanine aminotransferase (ALT). Aspartate aminotransferase (AST) did not show up important differences among treatments. In (T3) and (T5) there was not variation on the biochemical values when compared with treatment (T1). The sodic montmorilonite (0,50%) (T6) presented the better results on the reduction of the aflatoxin s adsorption when compared with the sodic montmorilonite from (0,25%) (T4). The outcome of this experiment proposed that high level of diet aflatoxin does cause important biochemistry changes on the enzymatic activity of the broiler chicken and also the alternative treatments using sodic montmorilonite may be used to avoid risks caused by feed contamination by aflatoxins. / Este estudo teve como objetivo avaliar os valores bioquímicos e atividade enzimática hepática e renal em frangos de corte submetidos à intoxicação experimental com aflatoxina com e sem a adição de montmorilonita sódica na dieta. Foram utilizados 528 frangos de corte, da linhagem cobb, divididos em 6 tratamentos do 1º ao 42º dia de vida: (T1) Controle: dieta normal; (T2) Dieta com 5 ppm de aflatoxina; (T3) Dieta com 0,25% montmorilonita sódica; (T4) Dieta com 5 ppm de aflatoxina + 0,25% montmorilonita sódica; (T5) Dieta com 0,50% montmorilonita sódica; (T6) Dieta com 5 ppm de aflatoxina + 0,50% montmorilonita sódica. O tratamento (T2)com aflatoxina demonstrou significante (P< 0.01) diminuição dos níveis de ácido úrico, albumina, colesterol, creatinina, triglicerídeos, globulinas e proteína plasmatica total. Houve significante aumento da alamina amino transferase (ALT) e a enzima aspartato amino transferase (AST) não foi significantemente diferente entre os tratamentos. Nos (T3) e (T5) não houve alteração nas dosagens bioquímicas em relação ao (T1) controle. A montmorilonita sódica (0,50%) (T6) apresentou melhores resultados na redução da adsorção da aflatoxina em relação a montmorilonita sódica (0,25%) (T4). Os resultados obtidos, nas condições em que foi realizado este experimento, sugerem que altos níveis de aflatoxina na dieta causam alterações bioquímicas significativas na atividade enzimática de frangos de corte e que o tratamento paleativo com a montmorilonita sódica pode ser usado para se evitar o risco causado pela contaminação alimentar com aflatoxinas.

Aflatoxin M1 (AFM1) is frequently found in milk and dairy products. It is a metabolite formed in cows from aflatoxin B1 (AFB1), contained in animal feeds. In cheese production AFM1 distributes between curds and whey. In this study, cows were fed 64 µg/AFB1/d for the high treatment, and 5 µg/AFB1/d for the low treatment, to obtain milk contaminated with AFM1 over the 0.5 µg/L and under 0.05 µg/L restrictions, respectively. Cheese was manufactured with milk contaminated with AFM1 at 0.8 and 0.03 ìg/kg by the higher and lower treatment, respectively. Two commercial cheeses were elaborated: a hard-aged cheese (cheddar cheese) and soft high moisture cheese (fresco cheese) to evaluate whether the cheese type had any impact on AFM1 analysis. AFM1 was extracted from cheese using immunoaffinity columns. Analyses were carried out by using high pressure liquid chromatography (HPLC) as the reference method and fluorometry as a method of validation. Analysis was by 2-way fixed factor analyses. AFM1 was detected in all samples by both methods of analysis. There were no detectable statistical differences between cheese types (P>0.05). AFM1 content was significantly different between the high and low concentration of AFB1 used to make the cheese type (P<0.01). Our regression model shows a linear relationship between fluorometry and HPLC methods; R2 = 0.9141 from cheddar cheese and R2 = 0.9141 from fresco cheese. There were no statistical differences between methods of analysis (P>0.05). Carryover of AFM1 in cheese detected by fluorometry in cheddar cheese was 163% and 80% for high and low treatments, respectively, and in fresco cheese was 119 and 133 for high and low treatments, respectively. These carryovers are below that reported in the literature. Results suggest that fluorometry is a simple and reliable AFM1 detection method for screening samples of complex matrices such as cheese.

Contamination of peanuts by secondary metabolites of certain fungi, namely aflatoxins present a great health hazard when exposed either at low levels for prolonged times (carcinogenic) or at high levels at once (poisonous). It is important to develop an accurate and rapid measurement technique to trace the aflatoxin and/or source fungi presence in peanuts. Thus, current research focused on development of vibrational spectroscopy based methods for detection and separation of contaminated peanut samples. Aflatoxin incidence, as a chemical contaminant in peanut paste samples, was investigated, in terms of spectral characteristics using FTIR-ATR. The effects of spectral pre-processing steps such as mean-centering, smoothing the 1st derivative and normalizing were studied. Logarithmic method was the best normalization technique describing the exponentially distributed spectral data. Spectral windows giving the best correlation with respect to increasing aflatoxin amount led to selection of fat associated spectral bands. Using the multivariate analysis tools, structural contributions of aflatoxins in peanut matrix were detected. The best region was decided as 3028-2752, 1800-1707, 1584-1424, and 1408-1127 cm-1 giving correlation coefficient for calibration (R2C), root mean square error for calibration (RMSEC) and root mean square error for prediction (RMSEP) of 98.6%, 7.66ppb and 19.5ppb, respectively. Applying the constructed partial least squares model, 95% of the samples were correctly classified while the percentage of false negative and false positive identifications were 16% and 0%, respectively. Aspergillus species of section Flavi and the black fungi, A. niger are the most common colonists of peanuts in nature and the majority of the aflatoxin producing strains are from section Flavi. Seed colonization by selected Aspergillus spp. was investigated by following the chemical alterations as a function of fungal growth by means of spectral readouts. FTIR-ATR was utilized to correlate spectral characteristics to mold density, and to separate Aspergillus at section, species and strain levels, threshold mold density values were established. Even far before the organoleptic quality changes became visually observable (~10,000 mold counts), FTIR distinguished the species of same section. Besides, the analogous secondary metabolites produced increased the similarity within the spectra even their spectral contributions were mostly masked by bulk peanut medium; and led to grouping of species producing the same mycotoxins together. Aflatoxigenic and non-aflatoxigenic strains of A. flavus and A. parasiticus were further studied for measurement capability of FTIR-ATR system in discriminating the toxic streams from just moldy and clean samples. Owing to increased similarity within the collected spectral data due to aflatoxin presence, clean samples (having aflatoxin level lower than 20 ppb, n=44), only moldy samples (having aflatoxin level lower than 300 ppb, n=28) and toxic samples (having aflatoxin level between 300-1200 ppb, n=23) were separated into appropriate classes (with a 100% classification accuracy). Photoacoustic spectroscopy (PAS) is a non-invasive technique and offers many advantages over more traditional ATR system, specifically, for in-field measurements. Even though the sample throughput time is longer compared to ATR measurements, intact seeds can be directly loaded into sample compartment for analysis. Compared to ATR, PAS is more sensitive to high moisture in samples, which in our case was not a problem since peanuts have water content less than 10%. The spectral ranges between: 3600-2750, 1800-1480, 1200-900 cm-1 were assigned as the key bands and full separation between Aspergillus spp. infected and healthy peanuts was obtained. However, PAS was not sensitive as ATR either in species level classification of Aspergillus invasion or toxic-moldy level separation. When run for separation of aflatoxigenic versus non-aflatoxigenic batches of samples, 7 out of 54 contaminated samples were misclassified but all healthy peanuts were correctly identified (15 healthy/ 69 total peanut pods). This study explored the possibility of using vibrational spectroscopy as a tool to understand chemical changes in peanuts and peanut products to Aspergillus invasion or aflatoxin contamination. The overall results of current study proved the potential of FTIR, equipped with either ATR or PAS, in identification, quantification and classification at varying levels of mold density and aflatoxin concentration. These results can be used to develop quality control laboratory methods or in field sorting devices. / Ph. D.

Grace S.N. Lau. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1994. / Includes bibliographical references (leaves 335-362). / List of Abbreviations --- p.i / Abstract --- p.1 / Chapter Chapter 1 --- General Introduction and Study Objectives / Chapter 1.1 --- Metabolic activation - role in drug toxicity and carcinogenesis --- p.5 / Chapter 1.2 --- Hepatocellular carcinoma --- p.12 / Chapter 1.2.1 --- Epidemiology --- p.12 / Chapter 1.2.2 --- Aetiological factors --- p.17 / Chapter 1.2.2.1 --- Hepatitis B virus infection --- p.17 / Chapter 1.2.2.2 --- Cirrhosis --- p.24 / Chapter 1.2.2.3 --- Aflatoxins --- p.25 / Chapter 1.2.2.4 --- Other factors --- p.26 / Chapter 1.2.2.5 --- Summary --- p.29 / Chapter 1.3 --- Study objectives --- p.30 / Chapter Chapter 2 --- The Metabolism of Paracetamol in Healthy Subjects andin Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 2.1 --- Introduction --- p.34 / Chapter 2.1.1. --- History of paracetamol --- p.34 / Chapter 2.1.2 --- Pharmacology of paracetamol --- p.37 / Chapter 2.1.3 --- "Absorption, Distribution, Metabolism and Excretion" --- p.38 / Chapter 2.1.3.1 --- Absorption --- p.38 / Chapter 2.1.3.2 --- Distribution --- p.41 / Chapter 2.1.3.3 --- Metabolism --- p.42 / Chapter 2.1.3.4 --- Excretion --- p.57 / Chapter 2.1.4 --- Toxicity and Overdosage --- p.59 / Chapter 2.2 --- Estimation of paracetamol and its metabolites in plasma and urine by high performance liquid chromatography --- p.72 / Chapter 2.2.1 --- Introduction --- p.72 / Chapter 2.2.2 --- Analytical method --- p.76 / Chapter 2.2.2.1 --- Materials --- p.76 / Chapter 2.2.2.2 --- Instrumentation --- p.77 / Chapter 2.2.2.3 --- Collection and storage of samples --- p.79 / Chapter 2.2.2.4 --- Chromatographic conditions --- p.79 / Chapter 2.2.3 --- Urine assay --- p.79 / Chapter 2.2.3.1 --- Preparation of standards and test samples for urine assay --- p.79 / Chapter 2.2.3.2 --- Calculation of results for urine assay --- p.80 / Chapter 2.2.3.3 --- Results of urine assay --- p.81 / Chapter 2.2.3.4 --- Validation of urine assay --- p.81 / Chapter 2.2.4 --- Plasma assay --- p.83 / Chapter 2.2.4.1 --- Preparation of standards and test samples for plasma assay --- p.83 / Chapter 2.2.4.2 --- Calculation of results for plasma assay --- p.91 / Chapter 2.2.4.3 --- Results of plasma assay --- p.91 / Chapter 2.2.4.4 --- Validation of plasma assay --- p.93 / Chapter 2.2.5 --- Summary --- p.99 / Chapter 2.3 --- The pharmacokinetics of paracetamol in healthy subjects --- p.103 / Chapter 2.3.1 --- Introduction --- p.103 / Chapter 2.3.2 --- Study protocol --- p.103 / Chapter 2.3.3 --- Methods --- p.103 / Chapter 2.3.3.1 --- Subjects --- p.103 / Chapter 2.3.3.2 --- Drug administration and sampling --- p.104 / Chapter 2.3.3.3 --- Drug analysis --- p.108 / Chapter 2.3.3.4 --- Calculations --- p.108 / Chapter 2.3.4 --- Pharmacokinetic analysis --- p.109 / Chapter 2.3.5 --- Statistical analysis --- p.113 / Chapter 2.3.6 --- Results --- p.114 / Chapter 2.3.6.1 --- Plasma Results --- p.114 / Chapter 2.3.6.2 --- Urine Results --- p.118 / Chapter 2.3.6.3 --- Pharmacokinetic Results --- p.125 / Chapter 2.3.6.4 --- Statistical Results --- p.134 / Chapter 2.3.7 --- Discussion --- p.145 / Chapter 2.4 --- "The pharmacokinetics of paracetamol in healthy subjects, patients with liver disease and hepatocellular carcinoma" --- p.155 / Chapter 2.4.1 --- Introduction --- p.155 / Chapter 2.4.2 --- Study protocol --- p.156 / Chapter 2.4.3 --- Methods --- p.156 / Chapter 2.4.3.1 --- Subjects --- p.156 / Chapter 2.4.3.2 --- Drug administration and sampling --- p.157 / Chapter 2.4.3.3 --- Drug analysis --- p.160 / Chapter 2.4.3.4 --- Calculations --- p.160 / Chapter 2.4.4 --- Pharmacokinetic analysis --- p.161 / Chapter 2.4.6 --- Results --- p.162 / Chapter 2.4.6.1 --- Plasma Results --- p.162 / Chapter 2.4.6.2 --- Urine Results --- p.162 / Chapter 2.4.6.3 --- Pharmacokinetic Results --- p.179 / Chapter 2.4.7 --- Discussion --- p.194 / Chapter 2.4.8 --- Summary --- p.203 / Chapter Chapter 3 --- Metabolic Activation of Aflatoxin B1 in Healthy Subjects and in Patients with Liver Disease and Hepatocellular Carcinoma / Chapter 3.1 --- General introduction --- p.206 / Chapter 3.1.1 --- Chemical structures and properties --- p.207 / Chapter 3.1.2 --- Contamination of food by aflatoxins --- p.209 / Chapter 3.1.3 --- Metabolism of aflatoxins --- p.210 / Chapter 3.1.4 --- Human diseases possibly related to exposure to aflatoxins --- p.226 / Chapter 3.1.4.1 --- Acute aflatoxicosis --- p.226 / Chapter 3.1.4.2 --- Reye's syndrome --- p.227 / Chapter 3.1.4.3 --- Kwashiorkor --- p.228 / Chapter 3.1.4.4 --- Impaired immune function --- p.229 / Chapter 3.1.4.5 --- Hepatocellular carcinoma --- p.230 / Chapter 3.1.5 --- Biochemical and molecular epidemiology of aflatoxins --- p.232 / Chapter 3.2 --- Development of an ELISA method to monitor AFB1 exposure in human serum --- p.237 / Chapter 3.2.1 --- Introduction --- p.237 / Chapter 3.2.2 --- Preparation of all the components necessary for analysing AFB1-albumin adducts by ELISA --- p.243 / Chapter 3.2.2.1 --- Materials --- p.243 / Chapter 3.2.2.2 --- Preparation of rabbit AFB1 antiserum --- p.244 / Chapter 3.2.2.3 --- Preparation of the rat monoclonal antibody --- p.244 / Chapter 3.2.2.4 --- Concentration of cell culture supernatant by ammonium sulphate precipitation --- p.246 / Chapter 3.2.2.5 --- Preparation of the BSA-AFB1 conjugate --- p.248 / Chapter 3.2.2.6 --- Preparation of the immunoaffinity gel --- p.250 / Chapter 3.2.2.7 --- Preparation of the ELISA plates --- p.251 / Chapter 3.2.3 --- General procedures used in the analysis of AFB1- albumin adducts --- p.252 / Chapter 3.2.3.1 --- Competitive ELISA binding assay --- p.253 / Chapter 3.2.3.2 --- Sep-pak C18 cartridge --- p.254 / Chapter 3.2.3.3 --- Immunoaffinity column --- p.255 / Chapter 3.2.3.4 --- Evaporation process --- p.255 / Chapter 3.2.3.5 --- HPLC --- p.256 / Chapter 3.2.3.6 --- Radioactive counting --- p.256 / Chapter 3.2.3.7 --- Albumin isolation --- p.257 / Chapter 3.2.3.8 --- Digestion of albumin --- p.257 / Chapter 3.2.3.9 --- Animal procedures --- p.258 / Chapter 3.2.4 --- Validations --- p.259 / Chapter 3.2.4.1 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.259 / Chapter 3 2.4.2 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.259 / Chapter 3 2.4.3 --- Elution characteristics and capacity of the immunoaffinity column --- p.261 / Chapter 3.2.4.4 --- Comparison of immunoaffinity gels prepared with different affinity gels --- p.261 / Chapter 3.2.4.5 --- Immunoaffinity column experiment of AFB1-lysine --- p.263 / Chapter 3.2.4.6 --- HPLC Analysis of fractions from immunoaffinity column --- p.263 / Chapter 3.2.4.8 --- HPLC analysis of fractions from Sep- Pak C18 cartridge --- p.264 / Chapter 3.2.4.9 --- Digestion of serum albumin by proteinase K --- p.264 / Chapter 3.2.4.10 --- Effect of ethanol in samples to be loaded onto Sep-Pak C18 cartridge --- p.265 / Chapter 3.2.4.11 --- Effect of drying in a vacuum concentrator on recovery of radioactivity of 3H-AFB1 --- p.266 / Chapter 3.2.4.12 --- Evaluation of the overall procedure for the analysis of serum albumin adducts of AFB1 --- p.267 / Chapter 3.2.4.13 --- HPLC analysis of samples obtained after digestion and all clean-up procedures --- p.268 / Chapter 3.2.5 --- Results and discussion --- p.268 / Chapter 3.2.5.1 --- BSA-AFB1 conjugate --- p.268 / Chapter 3.2.5.2 --- Treatment of experimental animals with 3H-AFB1 --- p.270 / Chapter 3.2.5.3 --- Optimisation of antiserum dilution and concentration of coating antigenin ELISA --- p.272 / Chapter 3.2.5.4 --- Analysis of standard AFB1 and AFB1- lysine in ELISA --- p.275 / Chapter 3.2.5.5 --- Sep-Pak C18 cartridge - elution characteristics and capacity --- p.279 / Chapter 3.2.5.6 --- Elution characteristics of immunoaffinity columns --- p.282 / Chapter 3.2.5.7 --- Immunoaffinity column experiment of AFB1-lysine --- p.290 / Chapter 3.2.5.8 --- Digestion of serum albumin by proteinase K --- p.295 / Chapter 3.2.5.9 --- Effect of ethanol in samples to be applied onto Sep-Pak C18 cartridges --- p.297 / Chapter 3.2.5.10 --- Recovery of radioactivity after dryingin a vacuum concentrator --- p.300 / Chapter 3.2.5.11 --- Recovery of the overall clean-up procedure for the analysis of serum albumin adducts of AFB1 --- p.300 / Chapter 3.2.5.12 --- HPLC analysis of samples obtained after all clean-up procedures --- p.305 / Chapter 3.2.5.13 --- The use of rabbit anti-AFB1 anti-serum and rat anti-AFB1 monoclonal antibody --- p.308 / Chapter 3.2.6 --- Summary --- p.309 / Chapter 3.3 --- Monitoring of AFBralbumin adducts in plasma of patients with liver disease and hepatocellular carcinoma --- p.311 / Chapter 3.3.1 --- Introduction --- p.311 / Chapter 3.3.2 --- Material and methods --- p.314 / Chapter 3.3.2.1 --- Subject --- p.314 / Chapter 3.3.2.2 --- Sample collections --- p.315 / Chapter 3.3.2.4 --- Assay for AFB1-albumin adducts --- p.315 / Chapter 3.3.2.5 --- Statistical analysis --- p.318 / Chapter 3.3.3 --- Results and discussion --- p.318 / Chapter Chapter 4 --- Summary and Ideas for Further Studies --- p.330 / Acknowledgements --- p.333 / References --- p.335 / Appendices --- p.364

Aflatoxins are produced by Aspergillus flavus fungi, mainly A. parasiticus and A. nomius. Aflatoxin B1 (AFB1) is the most common and highly toxic mycotoxin, presents carcinogenic, mutagenic and teratogenic effects. This mycotoxin has been detected in cultures of worldwide importance such as maize, groundnuts, beans, rice, wheat, cotton, sorghum, fruit and also in animal feed. AFB1 exerts its effects after its conversion into liver 8,9-epoxide by the action of cytochrome P-450, which reacts with cellular macromolecules, including proteins, RNA and DNA. Furthermore, there is an increase in levels of reactive oxygen species, altered neurobehavioral performance, damage to motor coordination, and decreased protein levels. Studies show that AFB1 alter the levels of neurotransmitters such as norepinephrine, serotonin and dopamine, and it is known that these changes influence the behavior of animals, also inhibits the activity of the enzyme Na+, K+-ATPase. This enzyme in the brain is essential for the maintenance of the electrochemical gradient, maintenance of resting potential and the release and uptake of neurotransmitters. Thus, a decrease in activity Na+, K+-ATPase could cause increased neuronal excitability, facilitating the occurrence of seizures. Thus, the aim of this study was to investigate the influence of AFB1 in facilitating seizures induced by a subconvulsant dose of pentylenetetrazol (PTZ), and evaluate its toxic effects on the brain, by determining the activity of Na+, K+-ATPase and oxidative stress parameters after acute exposure to AFB1 in rats. EEG recording of the animals was performed after acute oral administration of AFB1 (250 mg/kg) followed by a subconvulsant dose of pentylenetetrazol (30 mg/kg, ip). Prior administration of AFB1 to PTZ reduced the latency of myoclonus, did not alter the total amplitude of the brain waves, and concomitant exposure to PTZ reduced the activity total, α1 and α2/α3 of the enzyme Na+, K+-ATPase in the cerebral cortex. In the hippocampus, the AFB1 and PTZ reduced total and α2/α3 activity of the Na+, K+-ATPase. The AFB1 not alter the activity of catalase (CAT), and glutathione-S-transferase (GST) in the cerebral cortex of animals. We conclude that AFB1 exerts neurotoxic effect, facilitating seizures induced by PTZ possibly by inhibiting Na+, K+-ATPase activity. / As aflatoxinas são produzidas principalmente pelos fungos Aspergillus flavus, A. parasiticus e A. nomius. A aflatoxina B1 (AFB1) é a micotoxina mais frequente e altamente tóxica, apresenta efeitos carcinogênicos, mutagênicos e teratogênicos. Esta micotoxina tem sido detectada em culturas de importância em todo o mundo, como milho, amendoim, feijão, arroz, trigo, algodão, sorgo, frutas e também em rações de animais. A AFB1 exerce seus efeitos após sua conversão hepática em 8,9-epóxido, pela ação de enzimas do citocromo P-450, o qual reage com macromoléculas celulares, incluindo proteínas, RNA e DNA. Além disso, há um aumento nos níveis de espécies reativas de oxigênio, alteração do desempenho neurocomportamental, prejuízos à coordenação motora, e diminuição dos níveis proteicos. Estudos revelam que a AFB1 altera os níveis de neurotransmissores como a norepinefrina, serotonina e dopamina, e sabe-se que estas alterações influenciam no comportamento dos animais, como também inibe a atividade da enzima Na+,K+-ATPase, enzima que no cérebro, é essencial para a manutenção do gradiente eletroquímico, manutenção dos potenciais de repouso e liberação e captação de neurotransmissores. Assim, uma diminuição da atividade Na+,K+-ATPase pode ocasionar aumento da excitabilidade neuronal, facilitando a ocorrência de convulsões. Sendo assim, o objetivo deste trabalho foi investigar a influência da AFB1 em facilitar as convulsões induzidas por uma dose subconvulsivante de pentilenotetrazol (PTZ), e avaliar seus efeitos tóxicos sobre o cérebro, através da determinação da atividade da Na+,K+-ATPase e parâmetros de estresse oxidativo após a exposição aguda à AFB1 em ratos. Foi realizado o registro eletroencefalográfico dos animais após a administração oral aguda de AFB1 (250 μg/kg) seguida por uma dose subconvulsivante de pentilenotetrazol (30 mg/kg, i.p.). A administração prévia da AFB1 ao PTZ reduziu a latência das mioclonias, não alterou a amplitude global das ondas cerebrais, e a exposição concomitante ao PTZ reduziu a atividade total, α1 e α2/α3 da enzima Na+,K+-ATPase no córtex cerebral. No hipocampo, a AFB1 e o PTZ reduziram a atividade total e α2/α3 da Na+,K+-ATPase. A AFB1 não alterou a atividade da catalase (CAT) e da glutationa-S-transferase (GST) no córtex cerebral dos animais. Concluímos que a AFB1 exerce efeito neurotóxico, facilitando as convulsões induzidas por PTZ, possivelmente devido à redução da atividade da enzima Na+,K+-ATPase.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Aflatoxins are toxic metabolites produced by Aspergillus flavus, A. parasiticus and A. nominus genus fungi and can occur as natural contaminants of foods. Aflatoxins cause a variery of effects in poultry, including poor performance, liver pathology, immunosupression and changes in relative organ weights. Determination of biochemical toxic effects of aflatoxins is important for diagnosis of aflatoxicosis in broiler chickens. The aim of this research was to evaluate the serum mineral and the hepatic function of broiler chicken experimentally intoxicated using 3 ppm of aflatoxin and submitted to different concentrations of clay sodic montmorilonite on the diet. In this study were used 720 Cobb s broiler chickens that were divided in 6 treatments from 1º to 42º day of life: (T1) control: normal diet; (T2) diet with 3 ppm of aflatoxin; (T3) diet with 0,25% sodic montmorilonite; (T4) diet with 3 ppm of aflatoxin + 0,25% sodic montmorilonite; (T5) diet with 0,50% sodic montmorilonite; (T6) diet with 3 ppm of aflatoxin + 0,50% sodic montmorilonite. The treatment T2 showed significantly (P<0.05) decreased in serum concentrations of total protein, albumin, globulins and aspartate aminotransferase (AST); there was significant decreased on uric acid in treatment T4; in treatment T6 there was significant decrease in serum phosphorus levels; the serum calcium and magnesium were not affected by treatments. The aflatoxin in the diet (3ppm) causes changes in the hepatic function of broiler chickens. The use of sodic montmorilonite is effective in preventing the toxic effects of aflatoxins. However, addition of 0,50% sodic montmorilonite in the diet causes decrease in phosphorus levels. / Aflatoxinas são metabólitos tóxicos produzidos pelos fungos Aspergillus flavus, A. parasiticus e A. nominus, podendo ocorrer como contaminantes naturais dos alimentos. Esta toxina causa uma variedade de efeitos nas aves, incluindo diminuição da performance, doenças hepáticas, imunossupressão e mudanças no peso relativo dos órgãos. A determinação de seus efeitos bioquímicos tóxicos é importante para o diagnóstico de aflatoxicose em frangos de corte. Este estudo teve como objetivo avaliar os minerais séricos e a função hepática em frangos de corte submetidos à intoxicação experimental com 3 ppm de aflatoxina, e submetidos a diferentes concentrações da argila montmorilonita sódica na dieta. Foram utilizados 720 frangos de corte, da linhagem Cobb, divididos em 6 tratamentos do 1º ao 42º dia de vida: (T1) Controle: dieta normal; (T2) dieta com 3 ppm de aflatoxina; (T3) dieta com 0,25% de montmorilonita sódica; (T4) dieta com 3 ppm de aflatoxina + 0,25% de montmorilonita sódica; (T5) dieta com 0,50% de montmorilonita sódica; (T6) dieta com 3 ppm de aflatoxina + 0,50% de montmorilonita sódica. A dieta contendo somente aflatoxina (T2) resultou em uma significante (P<0.05) diminuição na concentração sérica de proteínas totais, albumina, globulinas e aspartato amino transferase (AST); a concentração sérica de ácido úrico diminuiu significativamente no tratamento T4; foi encontrada uma diminuição significante nos níveis séricos de fósforo no tratamento T6; os níveis séricos de cálcio e magnésio não foram afetados pelos tratamentos. A aflatoxina na concentração de 3 ppm na dieta causa alteração na função hepática de frangos de corte. O uso da montmorilonita sódica é eficaz na prevenção dos efeitos tóxicos da aflatoxina. No entanto, a adição de 0,5% de montmorilonita sódica na dieta com aflatoxina diminui os níveis séricos de fósforo.