Molecular signatures associated with diuron exposure on rat urothelial mitochondria.

Diuron, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, is a worldwide used herbicide whose biotransformation gives rise to the metabolites, 3-(3,4-dichlorophenyl)-1-methylurea (DCPMU) and 3,4-dichloroaniline (DCA). Previous studies indicate that diuron and/or its metabolites are toxic to the bladder urothelium of the Wistar rats where, under certain conditions of exposure, they may induce successively urothelial cell degeneration, necrosis, hyperplasia and eventually tumors. The hypothesis was raised that the molecular initiating event (MIE) of this Adverse Outcome Pathway is the mitochondrial toxicity of those compounds. Therefore, this study aimed to investigate in vitro the metabolic alterations resulting from urothelial mitochondria isolated from male Wistar rats exposure to diuron, DCPMU and DCA at 10 and 100 µM. A non-targeted metabolomic analysis using mass spectrometry showed discriminative clustering among groups and alterations in the intensity abundance of membrane-associated molecules phosphatidylcholine, phosphatidylinositol and phosphatidylserine, in addition to methylhexanoyl-CoA and, particularly for diuron 100 µM, dehydro-L-gulonate, all of them involved in critical mitochondrial metabolism. Collectively, these data indicate the mitochondrial dysfunction as an MIE that triggers cellular damage and death observed in previous studies.

Chromosomal imbalances in successive moments of human bladder urothelial carcinoma.

OBJECTIVE: To understand developmental characteristics of urinary bladder carcinomas (UBC) by evaluating genomic alterations and p53 protein expression in primary tumors, their recurrences, and in the morphologically normal urothelium of UBC patients. METHODS: Tumors and their respective recurrences, six low-grade and five high-grade cases, provided 19 samples that were submitted to laser microdissection capture followed by high resolution comparative genomic hybridization (HR-CGH). HR-CGH profiles went through two different analyses--all tumors combined or classified according to their respective histologic grades. In a supplementary analysis, 124 primary urothelial tumors, their recurrences, and normal urothelium biopsied during the period between tumor surgical resection and recurrence, were submitted to immunohistochemical analyses of the p53 protein. During the follow-up of at least 21 patients, urinary bladder washes citologically negative for neoplastic cells were submitted to fluorescence in situ hybridization (FISH) to detect copy number alterations in centromeres 7, 17, and 9p21 region. RESULTS AND CONCLUSIONS: HR-CGH indicated high frequencies (80%) of gains in 11p12 and losses in 16p12, in line with suggestions that these chromosome regions contain genes critical for urinary bladder carcinogenesis. Within a same patient, tumors and their respective recurrences showed common genomic losses and gains, which implies that the genomic profile acquired by primary tumors was relatively stable. There were exclusive genomic alterations in low and in high grade tumors. Genes mapped in these regions should be investigated on their involvement in the urinary bladder carcinogenesis. Successive tumors from same patient did not present similar levels of protein p53 expression; however, when cases were grouped according to tumor histologic grades, p53 expression was directly proportional to tumor grades. Biopsies taken during the follow-up of patients with history of previously resected UBC revealed that 5/15 patients with no histologic alterations had more than 25% of urothelial cells expressing the p53 protein, suggesting that the apparently normal urothelium was genomically unstable. No numerical alterations of the chromosomes 7, 17, and 9p21 region were found by FISH during the periods "free-of-neoplasia." Our data are informative for further studies to better understand urinary bladder urothelial carcinogenesis.

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-acetylaminofluorene (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 beta1 (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.

A deteccao de substancias cancerígenas em bioensaios alternativos de média-duracao / The detection of chemical carcinogen in alternative medium-term biossays

Atualmente, o método standard para identificacao de substancias cancerígenas é teste de longa-duracao, con roedores. No Brasil nao há condicoes para a sua execucao, devido á inexistencia de especialistas e de infra-estructura adecuada. En consequencia, o país é dependente dos conhecimentos gerados no exterior sobre o risco que determinados compostos químicos impoem ás populacoes e ao meio ambiente. Além disto, as desvantagens operacionais de teste delonga-duiracao levaram ao desenvolvimento de bioensaios alternativos, mais convenientes. O Instituto Brasileiro de Meio Ambiente(IBAMA) adotou oficialmente o bioensaio de média-duracao para múltiplos orgaos, no rato, como fonte de evidencia do potencial cancerígeno de agentes químicos. Con isto, procura atender a demanda de substancias que precisam ser testadas no país, antes de colocadas no mercado e, ao mesmo tempo, aum,entar o know-how nacional em carcinogénese química e avaliacao de risco cancerígeno

A deteccao de substancias cancerígenas em bioensaios alternativos de média-duracao / The detection of chemical carcinogen in alternative medium-term biossays

Atualmente, o método standard para identificacao de substancias cancerígenas é teste de longa-duracao, con roedores. No Brasil nao há condicoes para a sua execucao, devido á inexistencia de especialistas e de infra-estructura adecuada. En consequencia, o país é dependente dos conhecimentos gerados no exterior sobre o risco que determinados compostos químicos impoem ás populacoes e ao meio ambiente. Além disto, as desvantagens operacionais de teste delonga-duiracao levaram ao desenvolvimento de bioensaios alternativos, mais convenientes. O Instituto Brasileiro de Meio Ambiente(IBAMA) adotou oficialmente o bioensaio de média-duracao para múltiplos orgaos, no rato, como fonte de evidencia do potencial cancerígeno de agentes químicos. Con isto, procura atender a demanda de substancias que precisam ser testadas no país, antes de colocadas no mercado e, ao mesmo tempo, aum,entar o know-how nacional em carcinogénese química e avaliacao de risco cancerígeno(AU)