Alterações morfofisiologicas celulares e nucleares em celulas epiteliais mamarias humanas transformadas por benzo [a] pireno e transfectadas com o oncogene c-Ha-ras : aspectos citoquimicos e de analise de imagem

Orientador: Maria Luiza S. MelloDissertação (mestrado) - Universidade Estadual de Campinas, Instituto de BiologiaResumo: Alterações morfofisiológicas celulares enucleares foram investigadas através de métodos citoquímicos e de análise de imagem em células epiteliais mamárias humanas, MCF-IOF, transformadas com o benzo[a]pireno, e que expressam diferentes estágios da progressão tumoral (clones BPl, BPI-E e BPI-El), e, adicionalmente, num clone transfectado com o oncogene c-Ha-ras (BPl- Tras). Um aumento no índice mitótico foi observado com a progressão neoplásica. Por outro lado, um aumento em células apoptóticas, evidenciadas por uma variante do método de concentração crítica de eletrólitos, foi observado somente nas células BPI-Tras. Relocação de RNA durante a mitose, identificável por resposta metacromática, não diferiu quando se compararam células MCF-lOF transformadas e não transformadas, exceto com relação à presença de corpos semelhantes a nucléolos ("nucleolus-like bodies") encontrados próximos à placa cromossômica na metáfase ou ao fuso nas fases mitóticas subsequentes. Estes corpos não foram abundantes nas células MCF-lOF não transformadas e se tornaram mais escassos ainda nos clones celulares transformados, talvez por menores quantidades de RNA excedentes estarem envolvidas em tais condições. Pleomorfismo nuclear foi predominante nos clones celulares que expressam o fenótipo tumorigênico (BPI-E, BPI-El e BPI-Tras). Anormalidades nucleares tais como, cariorréxis, cariólise, multinucleação e micronúcleos foram mais freqüentes nas células BPl- Tras. Todos os clones exibiram células binucleadas e gigantes, mas nenhuma alteração nas suas frequências puderam ser detectadas quando os vários clones celulares foram comparados entre si. Alguns fenótipos nucleares puderam ser caracterizados nas células transformadas e não transformadas. Entretranto, estes poderiam estar associados com a progressão neoplásica, ou mesmo, com as diferentes fases do ciclo celular. Uma diminuição na área, perímetro e fator forma nucleares, mas um aumento na área e perímetro nucleolares foram encontrados com o avanço do processo tumorigênico nas células transformadas com o benzo[a]pireno. Estas alterações podem ser devidas a uma diminuição nas quantidades Feulgen-DNA (hipodiploidia) e/ou a um aumento nos estados de empacotamento cromatínico, embora um aumento na atividade transcripcional ocorra a nível nucleolar. Em particular, os tamanhos e perímetros nucleares das células BPl- Tras aumentaram quando comparados aos das células BP1, BPI-E e BPI-El, mas não diferiram dos valores para células MCF-IOF não transformadas. O aumento na área e no perímetro nuclear pode estar associado a uma certa aneuploidia promovida pela transfecção com o oncogene c-Ha-ras. Os tamanhos e número de nucléolos nas células BPI-E e BPI-Tras diferiram dos das outras células, e, um aumento da tamanho nucleolar com uma diminuição do número de nucléolos foi observado ocorrer nas células BPI-E e BPI-Tras. Fusão nucleolar e aumento em tamanho certamente refletem atividades metabólicas aumentadas nas células BPI-E e BP1 Tras quando comparadas aos outros clones celulares. Os dados de morfometria nuclear nos clones celulares transformados pelo benzo[a]pireno e transfectados com o oncogene c-Ha-ras indicam a existência de mecanismos morfofuncionais distintos e complexos que envolvem a progressão neoplásica de células MCF-lOF in vitro. O aumento na frequência de células micronucleadas e multinucleadas e aneuploidia nas células BP 1- Tras poderia estar relacionado à instabilidade genética induzida pela transfecção do oncogenerasoAbstract: Cellular and nuclear morphophysiological changes were investigated by cytochemistry and image analysis in clones of human breast epithelial cells, MCF-I0F, transformed by benzo[a]pyrene and expressing different stages of progression to neoplastic transformation (BP1, BPI-E and BPI-El clones), and additionally transfected with the c-Ha-ras oncogene (BP 1- Tras). A mitotic index increase was observed with the neoplastic progression. On the other hand, an increase in frequency of apoptotic celIs, as identified by a critical electrolyte concentration method, was only observed in BP 1- Tras celIs. RNA relocation during mitosis, identified by a metachromatic response, was found to differ when comparing nontransformed and transformed MCF-lOF cells. The exception refers to the presence of nucleolus-like bodies found close to the chromosomal plate at metaphase or to the spindle in subsequent mitotic phases. These bodies were not abundant in nontransformed IvlCF-l OF celIs, and become much more scarce in the transformed celIs, possibly due to decrease in surplus RNA involved. Nuclear pleomorphism was predominant in the cell clones expressmg tumorigenesis phenotypes (BPI-E, BPI-El and BP1- Tras cells). Nuclear abnormalities such as karyorhexis, karyolysis, multinucleation and micronucleation were more frequent in BP 1- Tras celIs. AlI celI clones exhibited binucleate and giant celIs, but no change in their frequencies could be detected when comparing the various cell clones. Some nuclear phenotypes could be characterized in the nontrasformed and transformed cells. However, they may be associated with the neoplastic progression or even with different cell cycle phases. A decrease in nuclear area, perimeter and form fator, but an increase in nucleolar area and perimeter were found with advancing tumorigenesis process in the benzo[ a ]pyrene-transformed cells. These changes may be due to a decrease in Feulgen-DNA amounts (hypodiploidy) and/or increase in chromatin packing states, although an increased transcriptional activity occurs at the nucleolar leveI. In particular, the nuclear sizes and perimeter of BP 1- Tras cells increased as compared to those of BP1, BPI-E and BPI-El cells, but were not found to differ from values ofthe nontransformed MCF-I0F cells. The nuclear area and perimeter increase may be associated to a certain aneuploidy degree promoted by the rastransfection. Nucleolus sizes and numbers in BPI-E and BPI-Tras cells were found to differ from those ofMCF-lOF, BPl and BPI-El cells, and nucleolar sizes increasing with the decrease in nucleolus numbers were observed in BPI-E BP1 Tras cells. Nucleolus fusion and enlargement certainly reflect metabolic activities enhanced in BPI-E and BPI-Tras cells when comparing these to the other cell clones. The nuclear morphometry data on the benzo[ a ]pyrene-transformed cells and on those additionally transfected by the c-Ha-ras oncogene indicated the existence of complex and distint morphofunctional mechanisms involving of the in vitro transformation of the MCF-I0F cells. The increase in frequency of micronuclei and multinucleate cells and in aneuploidy in the BP 1- Tras cell clone could be related to the genomic instability induced by ras-transfectionMestradoBiologia CelularMestre em Ciências Biológica

Infiltrating CD8(+) T lymphocytes, natural killer cells, and expression of IL-10 and TGF-beta 1 in chemically induced neoplasms in male Wistar rats

The present study aimed to estimate the number of CD8(+) T and natural killer (NK) infiltrating cells and the expression of interleukin-10 (IL-10) and transforming growth factor beta 1 (TGF-beta1) in chemically induced neoplasms in an initiation-promotion bioassay for carcinogenesis. Male Wistar rats were treated with N-nitrosodiethylamine, N-methyl-N-nitrosourea, N-butyl-N-(4-hydroxybutyl) nitrosamine, dihydroxy-di-N-propylnitrosamine, and 1,2-dimethylhydrazine for 4 weeks. Two groups were subsequently exposed through diet to phenobarbital (0.05%) or 2-acetylaminofluorene (0.01%) for 25 weeks. An untreated group was used as a control. Immune cells and cytokines were immunohistochemically evaluated in neoplasms and in surrounding normal tissues at the liver, kidneys, lung, and small and large intestines. When compared to the respective normal tissues, an increased number of NK cells was verified infiltrating the colon, lung, and kidney neoplasms, while the number of CD8+ T cells decreased in the intestine and lung neoplasms. Expression of IL-10 was found mainly in kidney tumors. TGF-beta1 was expressed mainly in the liver and kidneys tumors. The results indicate that the differential occurrence of immune cells between neoplastic and normal tissues could be dependent upon tumor microenvironment

Chemically induced immunotoxicity in a medium-term multiorgan bioassay for carcinogenesis with Wistar rats

A variety of chemicals can adversely affect the immune system and influence tumor development. The modifying potential of chemical carcinogens on the lymphoid organs and cytokine production of rats submitted to a medium-term initiation-promotion bioassay for carcinogenesis was investigated. Male Wistar rats were sequentially initiated with N-nitrosodiethylamine (DEN), N-methyl-N-nitrosourea (MNU), N-butyl-N-(4hydroxybutyl)nitrosamine (BBN), dihydroxy-di-n-propylnitrosamine (DHPN), and 1,2-dimethylhydrazine (DMH) during 4 weeks. Two initiated groups received phenobarbital (PB) or 2-acetyl amino fluorene (2-AAF) for 25 weeks and two noninitiated groups received only PB or 2-AAF. A nontreated group was used as control. Lymphohematopoietic organs, liver, kidneys, lung, intestines, and Zymbal's gland were removed for histological analysis. Interleukin (IL)-2, IL-12, interferon gamma (IFN-gamma), tumor necrosis factor alpha (TNF-alpha), IL-10, and transforming growth factor betal (TGF-beta1) levels were determined by ELISA in spleen cell culture supernatants. At the fourth week, exposure to the initiating carcinogens resulted in cell depletion of the thymus, spleen and bone marrow, and impairment of IL-2, IL-12, and IFN-gamma production. However, at the 30th week, no important alterations were observed both in lymphoid organs and cytokine production in the different groups. The results indicate that the initiating carcinogens used in the present protocol exert toxic effects on the lymphoid organs and affect the production of cytokines at the initiation step of carcinogenesis. This early and reversible depression of the immune surveillance may contribute to the survival of initiated cells facilitating the development of future neoplasia. (C) 2003 Elsevier B.V. All rights reserved

Diuron exposure induces systemic and organ-specific toxicity following acute and sub-chronic exposure in male Wistar rats

Diuron [3-(3,4-dichloropheny1)-1,1-dimethylurea] is a substitute urea herbicide widely used on agricultural crops with potential mutagenic, teratogenic, reproductive and carcinogenic effects. Nonetheless, its toxic potential on the immune system needs a detailed assessment. Thus, in order to evaluate the adverse effect of this herbicide on lymphohematopoietic organs and macrophage activity, male Wistar rats were orally treated with Diuron at 125, 1250 and 2500 ppm for 14, 28 or 90 days. General signs of toxicity were observed in Diuron-treated groups (1250 and 2500 ppm), including reduced food intake and body weight gain, as well as higher relative weights for spleen, kidneys and liver (28 and 90-day toxicity studies) and elevated serum levels of ALT, albumin, total protein, creatinine and urea (28-day toxicity study). Diuron exposure caused a severe depletion of splenic white pulp compartments and cellularity, followed by a decreased number of CD4(+) T lymphocytes, increased extramedullary hematopoiesis and deposition of hemosiderin in red pulp. Despite alteration in macrophage spreading, the macrophagic activity was not significantly affected by the herbicide. Under these experimental conditions, the results suggest that Diuron exerts systemic and target-organ toxicity, mainly at higher concentration. (C) 2011 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Lack of chemoprevention of dietary Agaricus blazei against rat colonic aberrant crypt foci

The mushroom Agaricus blazei (Ab) has been widely used in folk medicine to treat various diseases including cancer. No information is available on its possible protective effects on the development of colon cancer. The potential blocking effect of Ab intake on the initiation stage of colon carcinogenesis was investigated in a short-term (4-week) bioassay using aberrant crypt foci (ACF) as biomarker. Male Wistar rats were given four subcutaneous injections of the carcinogen 1,2-dimethylhydrazine (DMH, 40 mg/kg bw, twice a week), during 2 weeks to induce ACK The diet containing Ab at 5% was given 2 weeks before and during carcinogen treatment to investigate the potential beneficial effects of this edible mushroom on DMH-induced ACF. All groups were killed at the end of the fourth week. The colons were analyzed for ACF formation in 1% methylene blue whole-mount preparations and for cell proliferation in histological sections immunohistochemically stained for the proliferating cell nuclear antigen (PCNA). All DMH-treated rats developed ACF mainly in the middle and distal colon. Agaricus blazei intake at 5% did not alter the number of ACF induced by DMH or the PCNA indices in the colonic mucosa. Thus, the results of the present study did not confirm a chemopreventive activity of Ab on the initiation stage of rat colon carcinogenesis.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Lack of chemopreventive effects of ginger on colon carcinogenesis induced by 1,2-dimethylhydrazine in rats

Ginger (Zingiber officinale Roscoe) has been proposed as a promising candidate for cancer prevention. Its modifying potential on the process of colon carcinogenesis induced by 1,2-dimethylhydrazine (DMH) was investigated in male Wistar rats using the aberrant crypt foci (ACF) assay. Five groups were studied: Groups 1-3 were given four s.c. injections of DMH (40 mg/kg b.w.) twice a week, during two weeks, whereas Groups 4 and 5 received similar injections of EDTA solution (DMH vehicle). After DMH-initiation, the animals were fed a ginger extract mixed in the basal diet at 0.5% (Group 2) and 1.0% (Groups 3 and 4) for 10 weeks. All rats were killed after 12 weeks and the colons were analyzed for ACF formation and crypt multiplicity. The rates of cell proliferation and apoptosis were also evaluated in epithelial colonic crypt cells. Dietary consumption of ginger at both dose levels did not induce any toxicity in the rats, but ginger meal at 1% decreased significantly serum cholesterol levels (p < 0.038). Treatment with ginger did not suppress ACF formation or the number of crypts per ACF in the DMH-treated group. Dietary ginger did not significantly change the proliferative or apoptosis indexes of the colonic crypt cells induced by DMH. Thus, the present results did not confirm a chemopreventive activity of ginger on colon carcinogenesis as analyzed by the ACF bioassay and by the growth kinetics of the colonic mucosa. (c) 2005 Elsevier Ltd. All rights reserved