Cultured human lymphocytes may be assayed for aryl hydrocarbon hydroxylase (AHH) in whole cell preparations. The optimum assay conditions are pH 8.5, and 1.5 mM Mg++. The reaction is linear with time and cell number, and is inhibited by CO. Estradiol may inhibit induction of AHH by 3-methylcholanthrene, but is a poor competitor for the enzyme. A Caucasian population was assayed for AHH activity. The distribution was lognormal; no difference was found in cultured cells from males and females or smokers and nonsmokers. Cells from relatives of lung cancer patients showed higher activity. An American Indian population showed no difference from the Caucasian population in enzyme level. No linkage was found between AHH and 16a-hydroxylase.

Development of novel ill vitro and ill vivo models for determining primary events in HLRCC tumourigenesis Linda O'Flaherty, Mansfield College Thesis submitted for degree of Doctor of Philosophy Nuffield Department of Clinical Medicine, University of Oxford Hilary Term 2012 Germline mutations of fumarate hydratase (FR), encoding an enzyme of the tricarboxylic acid (TCA) cycle, predispose affected individuals to hereditary leiomyomatosis and renal cell cancer (HLRCC). FH-deficient cells and tissues have been shown to accumulate fumarate, exhibit S-(2-succinyl) cysteine (2SC) protein modifications and to constitutively express hypoxia-inducible factor alpha (HIF -1 a and -20.), under nonnoxic conditions. This thesis presents a phenotypic characterisation of FhI-I- mouse embryonic fibroblasts (MEFs), generated from previously reported conditional Fhl knockout mice, as a new in vitro system for investigating and identifying biochemical and metabolic pathways that are dysregulated as a result of FhI inactivation. These cell lines reproduced the aforementioned phenotypes, in addition to an observed shift from oxidative phosphorylation (OXPHOS) to glycolytic metabolism. Re-expression of either full length, mitochondrial-targeted FH (FhI-I- +FH) or cytoplasmic FH (Fhrl- +FHl'1MTS) in FhI-deficient MEFs was sufficient to reduce intracellular fumarate and to correct for the dysregulation of the Hif pathway. These results were of particular interest as they demonstrated that nonnoxic stabilisation of Hif-Ia occurs independently of the persistent mitochondrial defect observed in Fhrl- +FHl'1MTS MEFs. These findings were corroborated in vivo following the development of transgenic mouse models, ubiquitously expressing either FH or FHl'1MTS in mice with targeted inactivation of FhI in renal tubular cells. Surprisingly, the cytoplasmic-restricted FH (FHl'1MTS) transgene was just as efficient as the transgenic mice expressing mitochondrial- targeted FH at rescuing the cystic phenotype associated with Fh I-deficiency in the kidneys. As the function of cytoplasmic FH has remained poorly understood, these results go some way to extricating a role for this isofonn of FH. The results of this thesis demonstrate that these novel in vitro and in vivo models, used either alone or in combination, are a versatile and robust paradigm for studying altered cell metabolism in not only HLRCC but other diseases associated with metabolic dysregulation.

O câncer de mama permanece como o segundo tipo de câncer mais freqüente no mundo e o primeiro entre as mulheres (INCA, 2007). Porém, os mecanismos envolvidos no processo de gênese dos tumores mamários mesmo sendo intensamente estudados nos últimos 30 anos, ainda não são bem definidos. Vários estudos apontam que a susceptibilidade em função da genética é uma causa relevante ao surgimento do tumor, porém não a principal. Outros fatores tais quais o ambiente e dieta tendem a ser mais significantes nesse processo. Para a indução dos tumores em animais, a maioria dos modelos utiliza carcinógenos pertencentes à família dos hidrocarbonetos aromáticos policíclicos, dentre eles o DMBA (7,12-dimetil bezantraceno). O DMBA foi utilizado neste estudo com o objetivo de induzir tumor mamário, estabelecendo-se assim um modelo para estudos futuros, quantificando e classificando as lesões nas diferentes concentrações do carcinógeno, avaliando também a proliferação celular através do método de imunohistoquímica PCNA nos diferentes tumores encontrados. Neste estudo, em todos os grupos houve o desenvolvimento de tumores mamários, sendo estes mais freqüentes nos grupos de 3, 6 e 9 mg. O tipo de tumor mais freqüente foi o Adenocarcinoma A, seguido de Adenoacantoma e Adenocarcinoma Misto em menor freqüência. Sendo assim, concluiu-se através deste trabalho que o DMBA produz um modelo de carcinogênese mamária em camundongos. / The breast cancer remains the second most common cancer type in the world and first among women (INCA, 2008). However, the mechanisms involved in the origin even being intensively studied in the past 30 years, are still not well defined. Several studies suggest that genetic susceptibility is a relevant issue to the tumor development, however others factors can favor tumor growing. Among such factors the environment and diet are considered more significant. For mice tumor induction, most significant carcinogens used belonging to the polycyclic aromatic hydrocarbons family, where DMBA (7,12-dimethyl bezantraceno) take place. The DMBA was used in this study in order to induce mammary tumors, establishing the real conditions for future studies in our mice colony, classifying and quantifying the lesions. Cell proliferation was also evaluated though the immunohistochemistry against PCNA in different tumors classified. In this study, all concentration resulted in breast tumor development, which was more frequently observed in groups of 3, 6 and 9 mg. The most common type tumor regarded was Adenocarcinoma A, followed by Adenoacantoma and Mixed A/B in lower frequency. In conclusion, DMBA was able to produces a model of mammary carcinogenesis in mice.

Tang, Ho Lam. / "December 2010." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 119-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

A aflatoxina B1 (AFB1) é um metabólito de fungos do gênero Aspergillus que crescem naturalmente em alimentos. Devido às condições climáticas e às práticas agrícolas inadequadas, países em desenvolvimento, incluindo o Brasil, possuem alta possibilidade de exposição à AFB1 através de alimentos contaminados. A exposição crônica a essa micotoxina pode acarretar no surgimento de carcinoma hepatocelular e explicar a incidência desse tumor na ausência de fatores como hepatites virais e cirrose. Após a ingestão oral, a AFB1 é biotransformada para a sua forma genotóxica que se liga ao DNA das células hepáticas. Isso gera mutações que podem ser consideradas promotoras da hepatocarcinogênese. Na sequência desse processo, ocorre a formação de novos adutos de aflatoxina que podem se ligar à proteína plasmática ou serem excretados pela urina, respectivamente, AFB1-lisina e AFB1-N7-guanina. Esses compostos podem ser detectados e funcionar como biomarcadores da exposição e da toxicidade da AFB1. A AFB1 foi administrada enteralmente em ratos Wistar, via gavagem, durante 90 dias, sendo essa forma de exposição a mais próxima daquela pela qual os seres humanos estão suceptíveis. Os animais foram divididos em quatro grupos experimentais: Grupo Controle (sem AFB1), AFB50 (50 ppb), AFB100 (100 ppb) e AFB200 (200 ppb), sendo a concentração de AFB1 em parte por bilhão (ppb) por kilograma de dieta consumida. Foram realizadas avaliações de bioquímica plasmática de aspartato aminotransferase (AST) e alanina aminotransferase (ALT); alterações na expressão hepática de genes e proteínas relacionadas ao processo de hepatocarcinogênese (Ciclina D1, p53, ?-catenina, Proibitina, p27Kip1 e Glutationa-S-Transferase-p1-GSTP) por meios das técnicas de imuno-histoquímica e PCR em tempo real. Foram realizadas determinações dos níveis dos adutos da AFB1 no soro, na urina. Os resultados mostraram que houve aumento na expressão de AST e ALT em todos os grupos que receberam AFB1. No grupo AFB200 e, em menor proporção no AFB100, surgiram diversos focos de hepatócitos alterados marcados positivamente com GSTP, que são lesões pré-neoplásicas bem determinadas e consideradas endpoints em ensaios de hepatocarcinogênese experimental. A análise das proteínas hepáticas indicou que as lesões decorrentes da AFB1 nos grupos AFB200 e AFB100 apresentaram superexpressão de ciclina D1, p53, ?-catenina, proibitina, indicando a participação delas em vias que favorecem a hepatocarcinogênese. Adicionalmente, ocorreu uma redução na expressão gênica do gene p27, o que também indica uma condição favorável para a progressão neoplásica para a formação de carcinoma hepatocelular. A quantificação dos níveis de adutos no soro e na urina apontou que a formação desses compostos foi dose-dependente com as diferentes concentrações de AFB1 empregadas. Além disso, houve correlação entre a formação dos adutos com a expressão das proteínas Ciclina D, p53, ?-catenina e Rb. Sendo assim, foi possível, experimentalmente, apontar as principais proteínas envolvidas na hepatocarcinogênese e indicar que os adutos de aflatoxina no soro e na urina podem ser biomarcadores úteis para mensurar a exposição e o dano causado pela ingestão subcrônica de AFB1. / Aflatoxin B1 (AFB1) is metabolite produced by fungi of genus Aspergillus that grows naturally in food. Due to weather conditions and inadequate agricultural practices, developing countries, including Brazil, have high possibility of exposure to AFB1- contamined food. Chronic exposure to this mycotoxin may result in the emergence of hepatocellular carcinoma and explain the incidence of this tumor in the absence of factors such viral hepatitis and cirrhosis. After oral ingestion, AFB1 is biotransformed to its genotoxic form that binds to DNA in liver cells. This leads mutations that may be considered promoters of hepatocarcinogenesis. Following this process, there is the formation of new adducts of aflatoxins that can bind to plasma proteins or are excreted in the urine, respectively, AFB1-lysine and AFB1-N7-guanine. These compounds can be detected and work as biomarkers of exposure and toxicity of AFB1. AFB1 was administered in Wistar rats enterally, via gavage, for 90 days, and this form of exposure is closest which humans are susceptible. The animals were separated into four groups: control group (without AFB1), AFB50 (50 ppb), AFB100 (100 ppb) and AFB200 (200 ppb), in which concentration of AFB1 in part per billion (ppb) per kilogram of diet consumed by animals. It were performed liver biochemistry plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assessments; changes in hepatic expression of genes and proteins related to hepatocarcinogenesis (Cyclin D1, p53, ?-catenin, Prohibitin, p27Kip1 e Glutatione-STransferase-p1-GST-P) by immunohistochemical and real-time PCR techniques. The levels of AFB1 adducts of serum and urine were performed. The results showed increase in AST and ALT levels in all groups receiving AFB1. In group AFB200 and, lesser extent in AFB100, emerged several altered hepatocyte foci positively marked with GST-P, which are well determined preneoplastic lesions and deemed endpoints in experimental hepatocarcinogenesis assays. Analysis of liver proteins indicated that damage from AFB1 in groups AFB200 and AFB100 showed overexpression of cyclin D1, p53, ?-catenin, prohibitin, indicating their participation in ways that favor the hepatocarcinogenesis. Additionally, there was a decrease in gene expression of the p27 gene, which also indicates a favorable condition for neoplastic progression to hepatocellular carcinoma. Quantification of adducts levels in serum and urine showed that the formation of these compounds was dose-dependent with different concentrations of AFB1 employed. In addition, there was a correlation between the formation of adducts with the protein expression of Cyclin D, p53, ?-catenin and Rb. Thus, it was possible experimentally to point out the key proteins involved in hepatocarcinogenesis and indicate that aflatoxin adducts in serum and urine can be useful biomarkers to measure exposure and damage caused by subchronic ingestion of AFB1.

A aflatoxina B1 (AFB1) é um metabólito de fungos do gênero Aspergillus que crescem naturalmente em alimentos. Devido às condições climáticas e às práticas agrícolas inadequadas, países em desenvolvimento, incluindo o Brasil, possuem alta possibilidade de exposição à AFB1 através de alimentos contaminados. A exposição crônica a essa micotoxina pode acarretar no surgimento de carcinoma hepatocelular e explicar a incidência desse tumor na ausência de fatores como hepatites virais e cirrose. Após a ingestão oral, a AFB1 é biotransformada para a sua forma genotóxica que se liga ao DNA das células hepáticas. Isso gera mutações que podem ser consideradas promotoras da hepatocarcinogênese. Na sequência desse processo, ocorre a formação de novos adutos de aflatoxina que podem se ligar à proteína plasmática ou serem excretados pela urina, respectivamente, AFB1-lisina e AFB1-N7-guanina. Esses compostos podem ser detectados e funcionar como biomarcadores da exposição e da toxicidade da AFB1. A AFB1 foi administrada enteralmente em ratos Wistar, via gavagem, durante 90 dias, sendo essa forma de exposição a mais próxima daquela pela qual os seres humanos estão suceptíveis. Os animais foram divididos em quatro grupos experimentais: Grupo Controle (sem AFB1), AFB50 (50 ppb), AFB100 (100 ppb) e AFB200 (200 ppb), sendo a concentração de AFB1 em parte por bilhão (ppb) por kilograma de dieta consumida. Foram realizadas avaliações de bioquímica plasmática de aspartato aminotransferase (AST) e alanina aminotransferase (ALT); alterações na expressão hepática de genes e proteínas relacionadas ao processo de hepatocarcinogênese (Ciclina D1, p53, ?-catenina, Proibitina, p27Kip1 e Glutationa-S-Transferase-p1-GSTP) por meios das técnicas de imuno-histoquímica e PCR em tempo real. Foram realizadas determinações dos níveis dos adutos da AFB1 no soro, na urina. Os resultados mostraram que houve aumento na expressão de AST e ALT em todos os grupos que receberam AFB1. No grupo AFB200 e, em menor proporção no AFB100, surgiram diversos focos de hepatócitos alterados marcados positivamente com GSTP, que são lesões pré-neoplásicas bem determinadas e consideradas endpoints em ensaios de hepatocarcinogênese experimental. A análise das proteínas hepáticas indicou que as lesões decorrentes da AFB1 nos grupos AFB200 e AFB100 apresentaram superexpressão de ciclina D1, p53, ?-catenina, proibitina, indicando a participação delas em vias que favorecem a hepatocarcinogênese. Adicionalmente, ocorreu uma redução na expressão gênica do gene p27, o que também indica uma condição favorável para a progressão neoplásica para a formação de carcinoma hepatocelular. A quantificação dos níveis de adutos no soro e na urina apontou que a formação desses compostos foi dose-dependente com as diferentes concentrações de AFB1 empregadas. Além disso, houve correlação entre a formação dos adutos com a expressão das proteínas Ciclina D, p53, ?-catenina e Rb. Sendo assim, foi possível, experimentalmente, apontar as principais proteínas envolvidas na hepatocarcinogênese e indicar que os adutos de aflatoxina no soro e na urina podem ser biomarcadores úteis para mensurar a exposição e o dano causado pela ingestão subcrônica de AFB1. / Aflatoxin B1 (AFB1) is metabolite produced by fungi of genus Aspergillus that grows naturally in food. Due to weather conditions and inadequate agricultural practices, developing countries, including Brazil, have high possibility of exposure to AFB1- contamined food. Chronic exposure to this mycotoxin may result in the emergence of hepatocellular carcinoma and explain the incidence of this tumor in the absence of factors such viral hepatitis and cirrhosis. After oral ingestion, AFB1 is biotransformed to its genotoxic form that binds to DNA in liver cells. This leads mutations that may be considered promoters of hepatocarcinogenesis. Following this process, there is the formation of new adducts of aflatoxins that can bind to plasma proteins or are excreted in the urine, respectively, AFB1-lysine and AFB1-N7-guanine. These compounds can be detected and work as biomarkers of exposure and toxicity of AFB1. AFB1 was administered in Wistar rats enterally, via gavage, for 90 days, and this form of exposure is closest which humans are susceptible. The animals were separated into four groups: control group (without AFB1), AFB50 (50 ppb), AFB100 (100 ppb) and AFB200 (200 ppb), in which concentration of AFB1 in part per billion (ppb) per kilogram of diet consumed by animals. It were performed liver biochemistry plasma aspartate aminotransferase (AST) and alanine aminotransferase (ALT) assessments; changes in hepatic expression of genes and proteins related to hepatocarcinogenesis (Cyclin D1, p53, ?-catenin, Prohibitin, p27Kip1 e Glutatione-STransferase-p1-GST-P) by immunohistochemical and real-time PCR techniques. The levels of AFB1 adducts of serum and urine were performed. The results showed increase in AST and ALT levels in all groups receiving AFB1. In group AFB200 and, lesser extent in AFB100, emerged several altered hepatocyte foci positively marked with GST-P, which are well determined preneoplastic lesions and deemed endpoints in experimental hepatocarcinogenesis assays. Analysis of liver proteins indicated that damage from AFB1 in groups AFB200 and AFB100 showed overexpression of cyclin D1, p53, ?-catenin, prohibitin, indicating their participation in ways that favor the hepatocarcinogenesis. Additionally, there was a decrease in gene expression of the p27 gene, which also indicates a favorable condition for neoplastic progression to hepatocellular carcinoma. Quantification of adducts levels in serum and urine showed that the formation of these compounds was dose-dependent with different concentrations of AFB1 employed. In addition, there was a correlation between the formation of adducts with the protein expression of Cyclin D, p53, ?-catenin and Rb. Thus, it was possible experimentally to point out the key proteins involved in hepatocarcinogenesis and indicate that aflatoxin adducts in serum and urine can be useful biomarkers to measure exposure and damage caused by subchronic ingestion of AFB1.

O câncer de mama permanece como o segundo tipo de câncer mais freqüente no mundo e o primeiro entre as mulheres (INCA, 2007). Porém, os mecanismos envolvidos no processo de gênese dos tumores mamários mesmo sendo intensamente estudados nos últimos 30 anos, ainda não são bem definidos. Vários estudos apontam que a susceptibilidade em função da genética é uma causa relevante ao surgimento do tumor, porém não a principal. Outros fatores tais quais o ambiente e dieta tendem a ser mais significantes nesse processo. Para a indução dos tumores em animais, a maioria dos modelos utiliza carcinógenos pertencentes à família dos hidrocarbonetos aromáticos policíclicos, dentre eles o DMBA (7,12-dimetil bezantraceno). O DMBA foi utilizado neste estudo com o objetivo de induzir tumor mamário, estabelecendo-se assim um modelo para estudos futuros, quantificando e classificando as lesões nas diferentes concentrações do carcinógeno, avaliando também a proliferação celular através do método de imunohistoquímica PCNA nos diferentes tumores encontrados. Neste estudo, em todos os grupos houve o desenvolvimento de tumores mamários, sendo estes mais freqüentes nos grupos de 3, 6 e 9 mg. O tipo de tumor mais freqüente foi o Adenocarcinoma A, seguido de Adenoacantoma e Adenocarcinoma Misto em menor freqüência. Sendo assim, concluiu-se através deste trabalho que o DMBA produz um modelo de carcinogênese mamária em camundongos. / The breast cancer remains the second most common cancer type in the world and first among women (INCA, 2008). However, the mechanisms involved in the origin even being intensively studied in the past 30 years, are still not well defined. Several studies suggest that genetic susceptibility is a relevant issue to the tumor development, however others factors can favor tumor growing. Among such factors the environment and diet are considered more significant. For mice tumor induction, most significant carcinogens used belonging to the polycyclic aromatic hydrocarbons family, where DMBA (7,12-dimethyl bezantraceno) take place. The DMBA was used in this study in order to induce mammary tumors, establishing the real conditions for future studies in our mice colony, classifying and quantifying the lesions. Cell proliferation was also evaluated though the immunohistochemistry against PCNA in different tumors classified. In this study, all concentration resulted in breast tumor development, which was more frequently observed in groups of 3, 6 and 9 mg. The most common type tumor regarded was Adenocarcinoma A, followed by Adenoacantoma and Mixed A/B in lower frequency. In conclusion, DMBA was able to produces a model of mammary carcinogenesis in mice.

The pituitary hormone prolactin (Prl) is essential for alveolar morphogenesis and plays a role in breast carcinogenesis, however the mechanism that underlies these actions remains to be defined. Alterations in serum Prl provide the primary endocrine signal regulating developmental events in the mammary gland in sexually mature mammals. Prl production and post-translational phosphorylation by the pituitary is regulated by the neuropeptide Galanin (Gal) in response to hypothalamic signals integrating neuronal and endocrine inputs. Prl exerts its effects on the mammary epithelium in two ways, indirectly by modulation of the systemic hormonal environment, for example the release of progesterone from the corpus luteum, and directly by binding to Prl receptors (Prlr) within the mammary epithelium. Prl binding to Prlr initiates signalling predominantly via activation of the Jak2/Stat5 pathway, leading to altered patterns of gene transcription. One of these target genes is the ets transcription factor Elf5, which is required by the epithelium for alveolar morphogenesis. This thesis aims to further our understanding of the mechanisms by which prolactin exerts its influence on the mammary gland during alveolar morphogenesis and carcinogenesis. Transcript profiling revealed a lactation signature of 35 genes in Prlr+/- mice, Gal-/- mice and mice treated with a Prl mutant (S179D) that mimics phosphorylated Prl. We discovered that the majority of changes in gene expression were produced by prolactin rather than by Gal. The action of Gal was predominantly via modulation of Prl phosphorylation and release, as its effects were very similar to that of S179D. Knockout of Elf5 phenocopied knockout of Prlr, resulting in failure of alveolar morphogenesis and reduced expression of milk and lipid synthesis genes. Forced Elf5 expression at puberty resulted in aberrant differentiation of the terminal end buds and milk protein synthesis during ductal morphogenesis. Re-expression of Elf5 in Prlr-/- mammary epithelial cells completely rescued alveolar morphogenesis. These observations indicate that Elf5 is a master regulator of alveolar morphogenesis downstream of the Prlr. Loss of mammary epithelial Prlr resulted in reduced proliferation of low-grade neoplastic lesions resulting in increased tumour latency in the C3(1)/SV40T model of mammary carcinogenesis. There was no change in the growth rate, proliferation nor the morphology of tumours in Prlr-/-/C3(1)/SV40T transplants, thus Prl acts early in carcinogenesis to drive the proliferation of pre-invasive lesions resulting in faster progression to cancer.

Master of Medicine / Non-melanoma skin cancer (NMSC) is the most common human cancer worldwide. Squamous cell carcinoma (SCC) and basal cell carcinoma (BCC) make up almost all NMSC. SCC usually arises from actinic keratosis (AK) as a result of exposure to sunlight. SCC and AK provide a useful clinical model to investigate changes involved in the progression of NMSC. This project examines the expression of Brm, Brg-1, Snail 1 and Snail 2 in the progression of NMSC. Brm and Brg-1 are subunits of the SWI/SNF chromatin-remodelling complex which is involved in regulating the access of cell machinery to DNA by altering the structure of chromatin. It has been suggested that loss of this function is involved in carcinogenesis as the cell is unable to access to DNA normally in order to repair mutations or activate apoptosis. The loss of Brm or Brg-1 has been described in several human cancers. Snail 1 and Snail 2 are zinc-finger transcription factors that are known for their role in epithelial to mesenchymal transition (EMT), a process vital to embryological development. Increased expression of these factors leads to a loss of cell-cell adhesion and a migratory phenotype and has been described in some human cancers. In this project, double-label immunohistochemistry was used to determine the relative expression of these proteins in human SCC, BCC, AK and normal skin. The expression of Snail was unable to be determined due to poor specificity of the antibodies used. The expression of both Brm and Brg-1 proteins was found to be dramatically and consistently decreased in SCC and BCC when compared to normal skin and AK. This loss of Brm and Brg-1 occured as the tumour progressed from benign AK to malignant SCC. This finding suggests that the loss of either Brm or Brg-1 constitutes a key step in carcinogenesis. The results of this study identify Brm and Brg-1 as putative tumour suppressors involved in the progression of non-melanoma skin cancer from benign to malignant.

Bibliography: leaves 159-192. / 11, 192, [58] leaves, [16] leaves of plates : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Discusses the evidence implicating papillomaviruses in the development of cervical intraepithelial neoplasia (CIN) and carcinomas and documents derivation of clones and validation of experimental procedures, epidemiological studies of ano-genital neoplasia, HPV transcription mapping in genital neoplastic lesions and cell lines, and mechanisms of tumor development. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1992