C. Vasconcelos-Nóbrega

In vivo and in vitro effects of RAD001 on bladder cancer

OBJECTIVE To evaluate the influence of Everolimus (RAD001) on chemically induced urothelial lesions in mice and its influence on in vitro human bladder cancer cell lines. METHODS ICR male mice were given N-butyl-N-(4-hydroxybutyl) nitrosamine in drinking water for a period of 12 weeks. Subsequently, RAD001 was administered via oral gavage, for 6 weeks. At the end of the experiment, all the animals were sacrificed and tumor development was determined by means of histopathologic evaluation; mammalian target of rapamycin (mTOR) expressivity was evaluated by immunohistochemistry. Three human bladder cancer cell lines (T24, HT1376, and 5637) were treated using a range of RAD001 concentrations. MTT assay, terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL), and flow cytometry were used to assess cell proliferation, apoptosis index, and cell cycle analysis, respectively. Immunoblotting analysis of 3 cell line extracts using mTOR and Akt antibodies was performed in order to study the expression of Akt and mTOR proteins and their phosphorylated forms. RESULTS The incidence of urothelial lesions in animals treated with RAD001 was similar to those animals not treated. RAD001 did not block T24 and HT1376 cell proliferation or induce apoptosis. A reduction in cell proliferation rate and therefore G0/G1 phase arrest, as well as a statistically significant induction of apoptosis (P = 0.001), was only observed in the 5637 cell line. CONCLUSION RAD001 seems not to have a significant effect on chemically induced murine bladder tumors. The effect of RAD001 on tumor proliferation and apoptosis was achieved only in superficial derived bladder cancer cell line, no effect was observed in invasive cell lines.

Mitochondrial and liver oxidative stress alterations induced by N-butyl-N-(4-hydroxybutyl)nitrosamine: relevance for hepatotoxicity

The most significant toxicological effect of nitrosamines like N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) is their carcinogenic activity, which may result from exposure to a single large dose or from chronic exposure to relatively small doses. However, its effects on mitochondrial liver bioenergetics were never investigated. Liver is the principal organ responsible for BBN metabolic activation, and mitochondria have a central function in cellular energy production, participating in multiple metabolic pathways. Therefore any negative effect on mitochondrial function may affect cell viability. In the present work, ICR male mice were given 0.05% of BBN in drinking water for a period of 12 weeks and were sacrificed one week later. Mitochondrial physiology was characterized in BBN‐ and control‐treated mice. Transmembrane electric potential developed by mitochondria was significantly affected when pyruvate–malate was used, with an increase in state 4 respiration observed for pyruvate–malate (46%) and succinate (38%). A decrease in the contents of one subunit of mitochondrial complex I and in one subunit of mitochondrial complex IV was also observed. In addition, the activity of both complexes I and II was also decreased by BBN treatment. The treatment with BBN increases the susceptibility of liver mitochondria to the opening of the mitochondrial permeability transition pore. This susceptibility could be related with the increase in the production of H2O2 by mitochondria and increased oxidative stress confirmed by augmented susceptibility to lipid peroxidation. These results lead to the conclusion that hepatic mitochondria are one primary target for BBN toxic action during liver metabolism. Copyright

E-Cadherin and β-Catenin Expression during Urothelial Carcinogenesis Induced by N-butyl-N-(4-hydroxybutyl) nitrosamine in Mice

BACKGROUND The purpose of this study was to determine the efficacy of a combination of gemcitabine and sirolimus in a mouse model of invasive bladder cancer. MATERIALS AND METHODS Gemcitabine (50 mg/kg) and sirolimus (1.5 mg/kg) were administered to animals previously exposed to N-butyl-N-4(hydroxybutyl)nitrosamine in drinking water. Tumour development was determined by histopathological evaluation. RESULTS Both drugs were well tolerated by animals. The incidence of lesions in mice treated with gemcitabine was lower in comparison to those not treated, however this result was not statistically significant. The incidence of invasive bladder cancer in animals treated with sirolimus was statistically lower (20%) than in animals not treated (54%) (p=0.008). The results indicate that this drug combination has no statistical significance on the development of pre-neoplastic urothelial lesions and had only a minor impact on invasive bladder cancer incidence in mice. CONCLUSION The combination of gemcitabine and sirolimus had only a marginal impact on invasive bladder cancer in a mouse model.BACKGROUND E-cadherin and β-catenin are adhesion molecules that promote integrity and stability of the urothelium. A decrease in their expression is associated with more aggressive tumour phenotypes with the ability to invade and metastasize. MATERIAL AND METHODS 45 ICR male mice were used, of which 25 received N-butyl-N-(4-hydroxybutyl)nitrosamine (0.05%) in drinking water for a period of 12 weeks. Immunohistochemical expression was evaluated in all urinary bladder preparations for E-cadherin and for β-catenin. RESULTS Preneoplastic lesions showed staining patterns similar to normal urothelium. In simple and nodular hyperplasia, membrane staining was dominant (66.7-78.6 and 50-100%, respectively). In dysplasia a cytoplasmic pattern was prevalent (86.7-100%). Neoplastic lesions exhibit an abnormal staining pattern (100%) with heterogeneous staining (cytoplasmic, nuclear and membrane staining). A strong correlation was observed between both adhesion molecule staining patterns (r = 0.83; p = 0.039). CONCLUSIONS In mice, as in humans, E-cadherin and β-catenin are valuable tools to investigate cellular adhesion status of urothelium and can be considered as indicators of tumour aggressiveness and evolution.

Altered expression of CKs 14/20 is an early event in a rat model of multistep bladder carcinogenesis

Cytokeratins (CKs) 14 and 20 are promising markers for diagnosing urothelial lesions and for studying their prognosis and histogenesis. This work aimed to study the immunohistochemical staining patterns of CK14/20 during multistep carcinogenesis leading to papillary bladder cancer in a rat model. Thirty female Fischer 344 rats were divided into three groups: group 1 (control); group 2, which received N‐butyl‐N‐(4‐hydroxybutyl)nitrosamine (BBN) for 20 weeks plus 1 week without treatment; and group 3, which received BBN for 20 weeks plus 8 weeks without treatment. Bladder lesions were classified histologically. CK14 and CK20 immunostaining was assessed according to its distribution and intensity. In control animals, 0–25% of basal cells and umbrella cells stained positive for CK14 and CK20 respectively. On groups 2 and 3, nodular hyperplastic lesions showed normal CK20 and moderately increased CK14 staining (26–50% of cells). Dysplasia, squamous metaplasia, papilloma, papillary tumours of low malignant potential and low‐ and high‐grade papillary carcinomas showed increased CK14 and CK20 immunostaining in all epithelial layers. Altered CK14 and CK20 expression is an early event in urothelial carcinogenesis and is present in a wide spectrum of urothelial superficial neoplastic and preneoplastic lesions.

Epizootic and epidemic dermatophytose outbreaks caused by Trichophyton mentagrophytes from rabbits in Portugal, 2015.

We report an outbreak of dermatophytoses in rabbits, which was the origin of a dermatophytose epidemic in an agricultural school in central Portugal, affecting 15 people. Both the phenotypic characteristics and internal transcribed spacer (ITS) sequence of the dermatophytes isolated from the rabbits and patients were identical, suggesting that a single strain was responsible for both the epizootic and epidemic dermatophytoses and confirming that these two outbreaks were linked. The ITS sequences were also 100% identical to the ITS sequence of five strains isolated from rabbits in Greece and Italy, but different from that of Trichophyton mentagrophytes commonly isolated from dogs and cats. These results suggest that a particular T. mentagrophytes genotype could be prevalent in rabbits in southern Europe.

The N-butyl-N-4-hydroxybutyl Nitrosamine Mouse Urinary Bladder Cancer Model

Urinary bladder cancer (UBC) is a common and complex malignancy, with a multifactorial etiology, like environmental factors, such as cigarette smoking, occupational exposure, and genetic factors.UBC exhibits considerable genotypic and phenotypic heterogeneity. Among all UBC lesions, urothelial carcinoma is the most frequently observed histological type. Despite all the developments made in urologic oncology field, therapeutic options remain inadequate. There is urgency for the identification and development of new antineoplastic drugs to replace or improve current protocols and in vivo models have been proven to be essential for this step. There are different animal models of UBC: Spontaneous and experimentally induced models (genetically engineered, transplantable-xenograft and syngeneic animals- and chemically induced models). N-butyl-N(4-hydroxybutil)nitrosamine (BBN) is the most suitable reagent to generate chemically induced in vivo models of UBC and to study bladder carcinogenesis. BBN has proven, over the years, to be very realistic and reliable. It is bladder specific, and induces high tumor incidence.

Prospective Serosurvey of Coxiella burnetii Antibodies in Selected Sheep of Portugal

Q fever is a zoonotic disease caused by Coxiella burnetii that is highly prevalent across the world. In this study, a prospective serosurvey was performed to study C. burnetii circulation in a population of sheep in the central region of Portugal. Blood from a representative sample of 168 animals was drawn in both 2015 and 2016, and sera were tested for IgG anti-C. burnetii by EIA. In 2015, 7.7% (13/168) animals tested positive for IgG anti-C. burnetii, while in 2016, 17.3% (29/168) tested positive, showing a statistically significant (P = 0.008) increase in anti-C. burnetii seroprevalence. Results support the notion that Q fever is emerging in central Portugal.

Long-term treatment with chaethomellic acid A reduces glomerulosclerosis and arteriolosclerosis in a rat model of chronic kidney disease

The high prevalence of end-stage renal disease emphasizes the failure to provide therapies to effectively prevent and/or reverse renal fibrosis. Therefore, the aim of this study was to evaluate the effect of long-term treatment with chaethomellic acid A (CAA), which selectively blocks Ha-Ras farnesylation, on renal mass reduction-induced renal fibrosis. Male Wistar rats were sham-operated (SO) or subjected to 5/6 renal mass reduction (RMR). One week after surgery, rats were placed in four experimental groups: SO:SO rats without treatment (n=13); SO+CAA: SO rats treated with CAA (n=13); RMR:RMR rats without treatment (n=14); and RMR+CAA:RMR rats treated with CAA (n=13). CAA was intraperitoneally administered in a dose of 0.23μg/kg three times a week for six months. Renal fibrosis was evaluated by two-dimensional ultrasonography and histopathological analysis. The kidneys of the RMR animals treated with CAA showed a significantly decrease in the medullary echogenicity (p<0.05) compared with the RMR rats that received no treatment. Glomerulosclerosis and arteriolosclerosis scores were significantly lower (p<0.001) in the RMR+CAA group when compared with the RMR group. There were no significant differences in interstitial fibrosis, interstitial inflammation and tubular dilatation scores between the RMR+CAA and RMR groups. These data suggest that CAA can be a potential future drug to attenuate the progression of chronic kidney disease.