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This thesis aims to assess the human health impact of exposure to estrogenic compounds from the diet. A multi-disciplinary approach is taken to address various aspects of this issue. An introduction to xenoestrogens, including international research priorities, wildlife and human health effects, mechanisms of action, structure activity relationships and additivity of estrogenic effects is provided as background information. An assessment of exposure to a range of naturally occurring and synthetic estrogenic compounds found in food is derived in Chapter 2. The assessment combines new and existing data on food concentration, food consumption and serum levels for each xenoestrogen. Exposure is combined with relative estrogenic potency data from published bioassasy data to estimate risk relative to normal circulating levels of estradiol. Assuming additivity of xenoestrogens, for an average New Zealand male and for post-menopausal women, xenoestrogens in the diet contribute an additional 12-90% of estrogenicity above normal circulating levels. For a pre-menopausal female, the contribution from the diet represents in the order of an additional 2%. The level of exposure determined in this thesis would seem to be of pharmacological relevance, especially for men with low levels of estrogen and for post-menopausal women. Bisphenol A (BPA) is an important monomer used in the manufacture of epoxy resins for internal food can linings. A survey of the BPA content of a range of 80 canned foods available to the New Zealand consumer was undertaken and the results used in the exposure and risk assessments. BPA was detected in all foods analysed except soft drinks, at concentrations ranging from <10-29 µg/kg, except for individual samples of tuna, corned beef and coconut cream that were 109, 98 and 191 µg/kg respectively. None, of over 4000 individual exposure scenarios, exceeded the temporary Tolerable Daily Intake (TDI) of 10 µg/kg body weight per day set by the Scientific Committee on Food in 2002. Intestinal microflora influence the bioavailability of the naturally occurring xenoestrogens genistein and daidzein that contribute significantly to total estrogenicity from the diet. The degradation of genistein and daidzein by the faecal microfloral of 5 human subjects was variable and unpredictable between individuals and within an individual. These findings have important implications for the promotion and prescription of soy foods and supplements for disease prevention and health benefits. The "yeast assay" is one of a number of methods available to measure estrogenicity. This assay was established and validated. In utero exposure to estrogenic compounds at critical periods of sexual differentiation and endocrine development may imprint for health effects observed later in life. Placental transfer of estrogenicity, from 17β-estradiol was studied using the human placental perfusion model and the yeast assay. The placenta provides a protective barrier to the transfer of estrogenicity. Experiments with genistein showed that 5-15% placental transfer occurred, suggesting that in utero exposure might be in the order of 10% of maternal exposure. The thesis concludes with consideration of a genomic approach to substantiate, or refute, the mechanistic link between exposure to xenoestrogens and claimed human health effect. Such an approach offers exciting opportunity to clarify the mode of action of the synthetic versus the naturally occurring xenoestrogens, to confirm or dispute additivity of effect that is an important premise of the exposure assessment, to identify key genes involved in the many possible health effects and thence risk to the individual from dietary exposure to xenoestrogens.

This thesis aims to assess the human health impact of exposure to estrogenic compounds from the diet. A multi-disciplinary approach is taken to address various aspects of this issue. An introduction to xenoestrogens, including international research priorities, wildlife and human health effects, mechanisms of action, structure activity relationships and additivity of estrogenic effects is provided as background information. An assessment of exposure to a range of naturally occurring and synthetic estrogenic compounds found in food is derived in Chapter 2. The assessment combines new and existing data on food concentration, food consumption and serum levels for each xenoestrogen. Exposure is combined with relative estrogenic potency data from published bioassasy data to estimate risk relative to normal circulating levels of estradiol. Assuming additivity of xenoestrogens, for an average New Zealand male and for post-menopausal women, xenoestrogens in the diet contribute an additional 12-90% of estrogenicity above normal circulating levels. For a pre-menopausal female, the contribution from the diet represents in the order of an additional 2%. The level of exposure determined in this thesis would seem to be of pharmacological relevance, especially for men with low levels of estrogen and for post-menopausal women. Bisphenol A (BPA) is an important monomer used in the manufacture of epoxy resins for internal food can linings. A survey of the BPA content of a range of 80 canned foods available to the New Zealand consumer was undertaken and the results used in the exposure and risk assessments. BPA was detected in all foods analysed except soft drinks, at concentrations ranging from <10-29 µg/kg, except for individual samples of tuna, corned beef and coconut cream that were 109, 98 and 191 µg/kg respectively. None, of over 4000 individual exposure scenarios, exceeded the temporary Tolerable Daily Intake (TDI) of 10 µg/kg body weight per day set by the Scientific Committee on Food in 2002. Intestinal microflora influence the bioavailability of the naturally occurring xenoestrogens genistein and daidzein that contribute significantly to total estrogenicity from the diet. The degradation of genistein and daidzein by the faecal microfloral of 5 human subjects was variable and unpredictable between individuals and within an individual. These findings have important implications for the promotion and prescription of soy foods and supplements for disease prevention and health benefits. The "yeast assay" is one of a number of methods available to measure estrogenicity. This assay was established and validated. In utero exposure to estrogenic compounds at critical periods of sexual differentiation and endocrine development may imprint for health effects observed later in life. Placental transfer of estrogenicity, from 17β-estradiol was studied using the human placental perfusion model and the yeast assay. The placenta provides a protective barrier to the transfer of estrogenicity. Experiments with genistein showed that 5-15% placental transfer occurred, suggesting that in utero exposure might be in the order of 10% of maternal exposure. The thesis concludes with consideration of a genomic approach to substantiate, or refute, the mechanistic link between exposure to xenoestrogens and claimed human health effect. Such an approach offers exciting opportunity to clarify the mode of action of the synthetic versus the naturally occurring xenoestrogens, to confirm or dispute additivity of effect that is an important premise of the exposure assessment, to identify key genes involved in the many possible health effects and thence risk to the individual from dietary exposure to xenoestrogens.

The microbial populations in the gastrointestinal tracts of poultry play an important role in normal digestive processes and in maintaining animal health. The purpose of this study was to evaluate the effects of probiotics, prebiotics and synbiotics on the growth parameters, gut ecosystem, histology and immune function. In this study, four experiments one in vitro and three in vivo were conducted using specific pathogen free (SPF) and Hubbard broiler chickens. The first experiment was designed to determine the influence of inulin as an effective prebiotic on lactic acid bacteria (LAB) strains, and to screen LAB for selection as a source of chicken probiotic. Eight strains of LAB were isolated from chicken caeca and three strains from the Plymouth University culture collection were screened for potential probiotic properties for growth in inulin from Jerusalem artichoke (Helianthus tuberosus) and commercial inulin (Frutafit® HD, Netherlands). Lactobacillus animalis JCM 8692 strain isolated from chicken caeca showed the highest auto-aggregation and co-aggregation ability, resistance to acidity and bile salts, strong suppression of pathogens and ability to adhere to epithelial cells compared with other isolated strains. The second experiment was conducted to investigate the influence of commercial inulin and Jerusalem artichoke tubers as prebiotic supplementation on the diversity of the caecal microflora, jejunum histology and immune organ of SPF chickens. This investigation has found that inulin which was extracted from JA had a similar result when compared with commercial inulin and could be a suitable candidate for an inulin source in broiler diets. The third experiment was conducted to investigate the influence of Bactocell® (PRO1) and Lb. animalis (PRO2) as probiotic supplements on broiler chickens. EPEF was significantly increased in probiotic1 and probiotic2 compared with control (311.03, 309.87 and 260.06) respectively. Both types of probiotics supported the growth of chicks healthy and could be a suitable candidate as a source of probiotic in broiler diet. The fourth experiment was conducted to investigate the influence of dietary supplementation of a probiotic (Lb. animalis), a prebiotic JA tuber and a combination of both (Synbiotic) in broiler chickens. Growth performance was improved in all additive supplementation compared with the control group. EPEF was increased in probiotic, prebiotic and synbiotic compared with control (290.8±11.8, 300.9±3.86, 322.1±7.09 and 262.3±5.94) respectively. Beneficial bacteria in the guts of chicks fed probiotic, prebiotic and synbiotic was increased compared with chicks fed control diet. The diversity of microbial population in the gastrointestinal tract of chickens improved due to additives. The intestinal villus lengths and microvilli density was improved in all additives supplementation in comparison with control. Overall, it was concluded that probiotic, prebiotic and synbiotics can positively affect production performance and can improve the gut health.

A recent study using human fecal samples in conjunction with a culture-independent approach revealed immense diversity of antibiotic resistance (AR) genes in the human gut microflora. We hypothesize that food animal gut microflora also contain diverse and novel AR genes which could contribute to the emergence and transmission of AR in pathogens important in animal and human health. To test this, we examined AR reservoir in chicken gut microflora using a metagenomic, functional cloning method. Total genomic DNA was extracted from individual cecal contents of two free range chickens and two conventionally raised chickens. The DNAs were physically sheered into 1 to 3 kb fragments, cloned into expression vector pZE21-MCS, and transformed into E. coli TOP10 host strain, resulting in four metagenomic libraries of a total size of 108 base pairs per library. The AR transformants from the libraries were selected on plates containing the specific antibiotic of interest; six antibiotics including ampicillin, tetracycline, chloramphenicol, spectinomycin, ciprofloxacin and norfloxacin were used for screening. Plasmids from selected transformants were extracted and subjected to sequence analysis of inserted fragments. Identified AR genes were annotated and aligned with homologs that have been deposited in GenBank. A total of 12 AR genes and 3 AR genes were identified from the microbiome in conventionally raised chickens and free-range chickens, respectively. Of the identified 15 AR genes, 8 genes that confer resistance to ampicillin, spectinomycin or chloramphenicol shared low sequence similarity (58% - 76% at amino acid level) with the corresponding AR genes previously identified using culture-dependent approaches. Notably, among the 8 novel AR genes identified in this study, 4 genes also shared low sequence similarities (59%-76% at amino acid level) with recently identified AR genes in human gut. An E. coli-Campylobacter shuttle vector bearing the flaA sigma 28 promoter was constructed. Two novel genes conferring resistance to ampicillin (FRAmp1.1) and spectinomycin (FRSpe1.1) were cloned into this new expression vector, respectively. The derived vectors have conferred increased AR in C. jejuni, a leading zoonotic bacterial pathogen causing human gastroenteritidis in many industrialized countries. Together, findings from this study showed the effectiveness of the metagenomic approach for examination of AR reservoir in food animals, revealed novel AR resistance genes in chicken gut microflora, and demonstrated the functionality of such AR genes in foodborne human pathogens.

Gastrointestinal microflora has been shown to have a bi-directional relationship with the host immune system. A variety of fermentable carbohydrate polymers largely pass through the small intestine, providing fermentable substrates for gut microflora. Dietary fibre supplementation may provide a strategy for manipulating the intestinal bacterial profile, changing the interaction with the mucosal immune system, thereby modulating the host immune system. We used a BBc rat animal model to evaluate the effects of oat bran and wheat bran dietary fibre on the immune system. Previous collaborative efforts have shown that these dietary fibres can change the intestinal microflora, with wheat bran fibre showing a greater ability to influence colonic microbial community diversity. We have shown that dietary wheat bran fibre led to reduced IL-4 levels in the liver and T lymphocyte numbers in the Mesenteric Lymph Node and may be involved in reduced IgA levels in the cecal contents. In addition, IgA in the cecal contents was decreased while MLN B cell numbers increased in response to dietary wheat bran fibre. It was observed that neither wheat bran or oat bran treatments exerted any pro-inflammatory effects, with oat bran actually improving antioxidant status. These results suggest that both oat and wheat bran fibre treatments induce changes in the intestinal microflora, and that the microflora changes due to wheat fibre are associated with immunomodulatory effects on the host. This type of dietary fibre supplementation could ultimately provide a potential strategy for promoting health through microflora-associated effects on the immune system.

Made available in DSpace on 2017-07-10T18:13:29Z (GMT). No. of bitstreams: 1 Milton Cezar de Moura.pdf: 472197 bytes, checksum: ab2bbd7e5f98658de30016d4f0740ac7 (MD5) Previous issue date: 2011-08-11 / This study aimed to evaluated the use of probiotics Bacillus cereus var. Toyo and Bacillus subtilis C-3102 added to the diet for juvenile Nile tilapia strain of GIFT (Genetically Improved Farmed Tilapia) in order to verify the colonization of the gut epithelium and water, the influence on the bacterial microflora, the production performance, the indexes of body composition, the chemical composition and parameters of water quality. Two experiments were conducted: culture in cages with young individuals by 127 days (E1) and culture in ponds with adults by 201 days (E2). There was used the Center for Environmental Research in Aquaculture (CPAA/Toledo-PR) twenty ponds with 8.4 m3 of water and cages with useful volumes of 0.175 m3. The fishes from each experiment were randomly assigned to four treatments with five replicates. The treatments consisted of diets for each phase of commercial cultivation, added 0.5% B. cereus var. Toyoi (BC), 0.5% B. subtilis C-3102 (BS), 0.5% of the combination of two probiotics (BC+BS) and without addition probiotics (SP). In experiment E1, it was found that B. cereus var. Toyo and B. subtilis C-3102 colonized the gut epithelium of fishes and water culture, but did not influence the other parameters (P > 0.05). Survival ranged from 80% to 90% and feed conversion of 1.69 ± 0.29 to 1.96 ± 0.94 for treatments BS and SP. The CP levels ranged from 13.08% to 13.78% for BC and BC+BS and vicerosomatic index ranged from 9.71% to 10.9% for BS and BS+BC, respectively. In experiment E2 was also observed that the probiotics colonize the intestines and water cultivation, but did not influence the other parameters (p > 0.05). In this experiment, the indexes of production performance were influenced by the average temperature of the water that stood at 20.7ºC and is below the optimal range for the species. Weight and specific growth of the fishes ranged from 324.81 g day-1 and 398.51 g day-1, and 0.62% day-1 to 0.73% day-1, respectively, for BC and BS. Feed conversion ranged from 2.40 ± 0.24 to 2.92 ± 0.41 for BS and BC and survival ranged from 84.5% to 86% for BC and SP. The probiotics B. cereus var. Toyo and B. subtilis C-3102 used individually and in combination colonized the gut epithelium of fishes and cultivation water, however, did not influence the gut microflora, the production performance, the indexes of body composition, the chemical composition and parameters of water quality. / O presente objetivou avaliar a utilização dos probióticos Bacillus cereus var. Toyoi e Bacillus subtilis C-3102 adicionados à dieta para juvenis de tilápia do Nilo da linhagem GIFT (Genetically Improved Farmed Tilapia), a fim de verificar a colonização do epitélio intestinal e água do cultivo, a influência sobre a microflora bacteriana, o desempenho zootécnico, os índices de composição corporal, composição centesimal e os parâmetros da qualidade da água. Realizaram-se dois experimentos: cultivo em tanques rede com indivíduos jovens por 127 dias (E1) e cultivo em viveiros com indivíduos adultos por 201 dias (E2). Utilizou-se no Centro de Pesquisa em Aquicultura Ambiental (CPAA/Toledo-PR), vinte viveiros escavados com 8,4 m3 de água e tanques rede com volumes úteis de 0,175 m3. Os peixes de cada experimento foram distribuídos aleatoriamente em quatro tratamentos com cinco repetições. Os tratamentos foram constituídos de rações comerciais para cada fase de cultivo, adicionadas de 0,5% de B. cereus var. Toyoi (BC), 0,5% de B. subtilis C-3102 (BS), 0,5% da combinação dos dois probióticos (BC+BS) e sem adição de probióticos (SP). No experimento E1, verificou-se que os probióticos contendo B. cereus var. Toyoi e B. subtilis C-3102 colonizaram o epitélio intestinal dos peixes e a água do cultivo, mas não influenciaram os demais parâmetros analisados (p > 0,05). A sobrevivência variou de 80% a 90% e a conversão alimentar de 1,69 ± 0,29 a 1,96 ± 0,94 para os tratamentos BS e SP. Os níveis de PB variaram de 13,08% a 13,78% para BC+BS e BC e o índice vicerosomático variou de 9,71% a 10,9% para BC+BS e BS, respectivamente. No experimento E2, observou-se também, que os probióticos colonizaram o intestino e a água do cultivo, mas não influenciaram os demais parâmetros (p > 0,05). A temperatura média da água ficou em 20,7ºC, estando abaixo da faixa ótima para a espécie. O peso e o crescimento específico dos peixes apresentaram variação de 324,81 g dia-1 e 398,51 g dia-1; e 0,62 % dia-1 a 0,73 % dia-1, respectivamente para BC e BS. A conversão alimentar variou de 2,40 ± 0,24 a 2,92 ± 0,41 para BS e BC e a sobrevivência variou de 84,5% a 86% para BC e SP. Os probióticos B. cereus var. Toyoi e B. subtilis C-3102 utilizados individualmente e combinados realizaram a colonização do epitélio intestinal dos peixes e da água do cultivo, contudo, não influenciaram a microflora bacteriana intestinal, o desempenho zootécnico, os índices de composição corporal e centesimal e os parâmetros de qualidade da água.