Transcriptional profile of diuron-induced toxicity on the urinary bladder of male Wistar rats to inform mode of action.

Diuron (3-(3,4-dichlorophenyl)-1,1-dimethylurea) is a substituted urea herbicide that induces rat urinary bladder urothelial tumors at high dietary levels (2500 ppm). The specific mode of action and molecular alterations triggered by diuron, however, have not been clarified. The present study evaluated the dose-dependent effects of mucosal alterations and transcriptional changes in the urinary bladder of rats exposed to diuron. Six-week-old male Wistar rats were treated with 0, 60, 125, 1250, and 2500 ppm of diuron in the diet for 20 weeks. Histologic examination showed urothelial hyperplasia present in rats treated with either 1250 or 2500 ppm of diuron but not 60 or 125 ppm. Comprehensive gene expression analyses of urothelial cell RNA were conducted using Affymetrix microarrays. The numbers of differentially expressed transcripts between each treatment group and control increased with diuron dose. Based on similar histology and gene expression responses, the treatment groups were regrouped into a high-dose (1250 and 2500 ppm) and low-dose group (60 and 125 ppm). These data suggest that persistent exposure to high dietary concentrations of diuron induces oxidative stress, increases cellular metabolism, and enhances cell death that is associated with sustained urothelial hyperplasia.

Cytotoxicity and regenerative proliferation as the mode of action for diuron-induced urothelial carcinogenesis in the rat.

Diuron, a substituted urea herbicide, is carcinogenic to the urinary bladder of rats at high dietary levels. Its proposed carcinogenic mode of action (MOA) includes urothelial cytotoxicity and necrosis followed by regenerative cell proliferation and sustained urothelial hyperplasia. Cytotoxicity could be induced either by urinary solids or by chemical toxicity by diuron and/or metabolites excreted in the urine. Diuron was not genotoxic in a previous single-cell gel (comet) assay, but possible cross-linking activity remained to be evaluated. The present study explored the MOA of diuron and the effect of urinary acidification on the development of urothelial lesions. Male Wistar rats were fed diuron (2500 ppm, about 130 mg/kg of body weight) either with or without NH(4)Cl 10,000 ppm to acidify the urine. Reversibility of urothelial changes was also examined. The animals were euthanized after 15, 25, or 30 weeks. Diuron-fed rats had urinary amorphous precipitate and magnesium ammonium phosphate crystals similar to control animals. Groups treated with diuron + NH(4)Cl showed decreased urinary pH and reduced amounts of urinary crystals and precipitate. Urothelial necrosis and simple hyperplasia were observed by light microscopy and scanning electron microscopy both in diuron- and in diuron + NH(4)Cl-treated groups. Cytotoxicity and proliferative changes were mostly reversible. A modified comet assay developed in vitro with Chinese hamster ovary cells showed that diuron did not induce DNA cross-links. These data suggest that cytotoxicity with consequent regenerative cell proliferation is the predominant MOA for diuron rat urothelial carcinogenesis, the cytotoxicity being chemically induced and not due to urinary solids.

DNA methylation patterns in bladder cancer and washing cell sediments: a perspective for tumor recurrence detection.

BACKGROUND: Epigenetic alterations are a hallmark of human cancer. In this study, we aimed to investigate whether aberrant DNA methylation of cancer-associated genes is related to urinary bladder cancer recurrence. METHODS: A set of 4 genes, including CDH1 (E-cadherin), SFN (stratifin), RARB (retinoic acid receptor, beta) and RASSF1A (Ras association (RalGDS/AF-6) domain family 1), had their methylation patterns evaluated by MSP (Methylation-Specific Polymerase Chain Reaction) analysis in 49 fresh urinary bladder carcinoma tissues (including 14 cases paired with adjacent normal bladder epithelium, 3 squamous cell carcinomas and 2 adenocarcinomas) and 24 cell sediment samples from bladder washings of patients classified as cancer-free by cytological analysis (control group). A third set of samples included 39 archived tumor fragments and 23 matched washouts from 20 urinary bladder cancer patients in post-surgical monitoring. After genomic DNA isolation and sodium bisulfite modification, methylation patterns were determined and correlated with standard clinic-histopathological parameters. RESULTS: CDH1 and SFN genes were methylated at high frequencies in bladder cancer as well as in paired normal adjacent tissue and exfoliated cells from cancer-free patients. Although no statistically significant differences were found between RARB and RASSF1A methylation and the clinical and histopathological parameters in bladder cancer, a sensitivity of 95% and a specificity of 71% were observed for RARB methylation (Fisher's Exact test (p < 0.0001; OR = 48.89) and, 58% and 17% (p < 0.05; OR = 0.29) for RASSF1A gene, respectively, in relation to the control group. CONCLUSION: Indistinct DNA hypermethylation of CDH1 and SFN genes between tumoral and normal urinary bladder samples suggests that these epigenetic features are not suitable biomarkers for urinary bladder cancer. However, RARB and RASSF1A gene methylation appears to be an initial event in urinary bladder carcinogenesis and should be considered as defining a panel of differentially methylated genes in this neoplasia in order to maximize the diagnostic coverage of epigenetic markers, especially in studies aiming at early recurrence detection.

Effect of raloxifene on the vaginal epithelium of postmenopausal women.

OBJECTIVE: The objective was to analyze the effect of raloxifene on the vaginal epithelium of postmenopausal women. STUDY DESIGN: In this non-randomized clinical trial, 80 women (mean age = 60.6 years) were prospectively studied. Forty patients received 60 mg/day of raloxifene (RG), and 40 women constituted a non-treated control group (CG), paired by age and time since menopause. The treated group consisted of patients with osteoporosis of the lumbar spine. Those with a diagnosis of infection in the lower genital tract and using hormone therapy (HT) up to 6 months prior to the study were excluded. Vaginal smears were collected at baseline and after 6 months of intervention. The vaginal maturation value (VMV) was determined, and counts of superficial, intermediate and parabasal cells were performed. Smears were analyzed by only one cytopathologist who was blinded to patient data. The t-test, Wilcoxon test, and Chi-Squared test were used in the statistical analysis. RESULTS: The study groups were homogeneous regarding age, time since menopause, parity, HT use, smoking, and body mass index. No statistically significant differences were observed in VMV median values (RG, 39.7 and 35.7; CG, 50.0 and 50.0, respectively) or in the percentage of superficial, intermediate and parabasal cells between the groups at baseline and after 6 months (p>0.05). There was no significant correlation between VMV and age, time since menopause, previous HT use, or body mass index, in either of the groups. CONCLUSION: Treatment with raloxifene for 6 months has no effect on the maturation of the vaginal epithelium in postmenopausal women with osteoporosis.

Efficacy and safety of a soy isoflavone extract in postmenopausal women: a randomized, double-blind, and placebo-controlled study.

OBJECTIVE: To investigate the efficacy of soy isoflavone on climacteric symptoms in postmenopausal women. DESIGN: In this double-blind, randomized, placebo-controlled study, a total of 80 women (mean age = 55.1 years), who reported 5 or more hot flush episodes per day, were randomized to receive either 250 mg of standardized soy extract (Glycine max AT) a total of 100mg/day of isoflavone (n = 40) or placebo (n = 40). Exclusion criteria included: contra-indication for hormone therapy (HT), chronic gastrointestinal diseases, and users of HT within the preceding 6-months. For 10-months, climacteric symptoms were evaluated using a score card and the menopausal Kupperman index. Compliance and safety were also assessed. At baseline and the end of the study, lipid and hormonal profiles, as well as vaginal, mammographic and ultrasonographic parameters were measured. The t-test, Wilcoxon test and ANOVA were used in the statistical analysis. RESULTS: At baseline, the mean number of hot flushes was 9.6 +/- 3.9 per day in the isoflavone group and 10.1+/-4.9 in the placebo group (p>0.05). After 10 months, there was a significant reduction in frequency of hot flushes among isoflavone users when compared to those on placebo (3.1 +/- 2.3 and 5.9 +/- 4.3, respectively) (p<0.001). Kupperman index mean values showed a significant reduction in both groups. However, soy isoflavone was significantly superior to placebo, in reducing hot flush severity (69.9% and 33.7%, respectively) (p<0.001). Endometrial thickness, mammography, vaginal cytology, lipids and hormonal profile did not change in both groups. No serious adverse event related to isoflavone treatment was reported. CONCLUSIONS: The soy isoflavone extract exerted favorable effects on vasomotor symptoms and good compliance, providing a safe and effective alternative therapeutic for postmenopausal women.

A punçäo aspirativa no diagnóstico citológico de lesöes mamárias / Fine needle aspiration in the cytologic diagnosis of mammary lesions

Foram analisados 68 casos de punçäo aspirativa de mama, dos quais 35,2% foram positivos, 7,3% suspeitos e 57,3% negativos para células neoplásicas. Baseados nos achados citológicos de cada caso, foi feito um diagnóstico presuntivo da doença. Biopsias de 56 casos permitiu fazer a correlaçäo entre os diagnósticos citológicos e histopatológicos na maioria dos casos. Para as lesöes benignas houve uma concordância em 62,5%; a maior porcentagem foi encontrada entre os fibroadenomas (73,0%). Para lesöes malignas da mama, uma correlaçäo cito-hitológica positiva foi observada em 86,3% dos pacientes, sendo 81,8% para carcinomas dutais invasivos e 50,0% para comedocarcinomas. Descrevemos a técnica de aspiraçäo e enfatizamos a importância das características citológicas que resultaram em um bom índice de precisäo diagnóstica

Pesquisa de eosinófilos no muco nasal e eosinofilia sangüínea em pacientes asmáticos e riníticos: valor diagnóstico na alergia de vias respiratórias / Research of eosinophils in the nasal mucous and blood eosinophilia in asmatic and rhinitic patients: diagnostic value in allergy of the respiratory tract

A pesquisa de eosinófilos no muco nasal e a eosinofilia sangüínea säo exames complementares freqüentemente utilizados na suspeita de pacientes alérgicos. Entretanto, segundo vários autores, ainda existem controvérsias sobre a sua real validade no diagnóstico de alergia. Estudamos 147 crianças divididas em 3 grupos: controle - 29 crianças (G1) provenientes do Ambulatório Geral e de Puericultura; asma - 75 crianças (G2) e asma com rinite - 43 crianças (G3) provenientes do Ambulatório de Pneumologia Infantil. A pesquisa de eosinófilos no muco nasal foi feita através de esfregaços fixados com Metanol 99% e corados pelos métodos de Shorr e Hansel e quantificados pelo critério de Shimizu. Na análise dos resultados observou-se: 1) Näo houve diferença no tempo decorrido entre o início dos sintomas até a consulta especializada no grupo G2 e G3. 2) o intervalo entre as crises foi maior em G2 que em G3. 3) G2 tem maior proporçäo de casos graves. 4) Näo foi constatada diferença entre os 3 grupos para glóbulos brancos. 5) A % de eosinófilos sangüíneos foi maior em G2 e G3 que näo diferiram entre si. 6) Em G1 näo se encontram eosinófilos no muco nasal e em G2 e G3 todos apresentaram eosinófilos com a mediana situada em ++ (critério Shimizu). Com estes resultados os autores acreditam que a pesquisa de eosinófilos em pacientes alérgicos é de grande validade, além de consistir em exame simples, econômico e exequível em qualquer serviço médico