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The Lusitano horse is an autochthonous Portuguese breed with a growing worldwide expansion. Our objective was to establish reference intervals for haematological parameters using the haematological cell counter LaserCyte (IDEXX). For this purpose, blood samples from 100 healthy adult horses (13 females and 87 males, ranging from 3 to 25 years of age) were analysed. The reference intervals were estimated following the ASVCP guidelines with the Reference Value Advisor software. The obtained reference intervals were 6.4–10.1 × 1012/L for red blood cells, 30.6–45.1% for haematocrit, 11.6–17.1 g/dL for haemoglobin, 42.8–53.2 fL for mean corpuscular volume (MCV), 15.5–20.8 pg for mean corpuscular haemoglobin (MCH), 33.7–39.4 g/dL for mean corpuscular haemoglobin concentration, 17.8–20.3% for red cell distribution width (RDW), 4.5–10.1 × 109/L for white blood cells, 2.2–6.0 × 109/L for neutrophils, 0.9–4.9 × 109/L for lymphocytes, 0.2–0.5 × 109/L for monocytes, 0.1–0.6 × 109/L for eosinophils, 0.0–0.1 × 109/L for basophils, 78.5–172.2 K/mL for platelets, 4.3–9.4 fL for mean platelet volume, 18.8–24.2% for platelet distribution width, and 0.06–0.12% for plateletcrit. LaserCyte equine reference intervals are transferable to the Lusitano horse for 18 of the 22 analytes studied. Regarding age, significant statistical differences were observed for MCV, RDW, neutrophils and lymphocytes between the mean values of young (3–6 years old), middle-aged (7–14 years old) and old (< 15 years old) age groups. MCH means were statistically significantly different between the three age groups. The haematological reference intervals established in this study might represent a valuable and applicable tool for haematological assessment of adult Lusitano horses, providing useful information that helps clinicians to interpret clinical data. [ABSTRACT FROM AUTHOR]

Cancer is the first cause of death in developed countries and the second in developing countries. Concerning the most frequent worldwide-diagnosed cancer, primary liver cancer represents approximately 4% of all new cancer cases diagnosed globally. However, among primary liver cancer, hepatocellular carcinoma is by far the most common histological subtype. Notwithstanding the health promotion and disease prevention campaigns, more than half a million new hepatocellular carcinoma cases are reported yearly, being estimated to growth continuously until 2020. Taking this scenario under consideration and the fact that some aspects concerning hepatocellular carcinoma evolution and metastasize process are still unknown, animal models assume a crucial role to understand this disease. The animal models have also provided the opportunity to screen new therapeutic strategies. The present review was supported on research and review papers aiming the complexity and often neglected chemically induced animal models in hepatocarcinogenesis research. Despite the ongoing debate, chemically induced animal models, namely, mice and rat, can provide unique valuable information on the biotransformation mechanisms against xenobiotics and apprehend the deleterious effects on DNA and cell proteins leading to carcinogenic development. In addition, taking under consideration that no model achieves all hepatocellular carcinoma research purposes, criteria to define the "ideal" animal model, depending on the researchers' approach, are also discussed in this review. [ABSTRACT FROM AUTHOR]

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ABSTRACT 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 © 2011 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

The aim of this paper is to analyse sunitinib malate in vitro ability to enhance cisplatin cytotoxicity in T24, 5637, and HT1376 human urinary bladder-cancer cell lines. Cells were treated with cisplatin (3, 6, 13, and 18 μM) and sunitinib malate (1, 2, 4, 6, and 20 μM), either in isolation or combined, over the course of 72 hours. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, acridine orange, and monodansylcadaverine staining and flow cytometry were performed. The combination index (CI) was calculated based on the Chou and Talalay method. In isolation, cisplatin and sunitinib malate statistically (P < 0.05) decrease cell viability in all cell lines in a dose-dependent manner, with the presence of autophagic vacuoles. A cell cycle arrest in early S-phase and in G0/G1-phase was also found after exposure to cisplatin and sunitinib malate, in isolation, respectively. Treatment of urinary bladder-cancer cells with a combination of cisplatin and sunitinib malate showed a synergistic effect (CI < 1). Autophagy and apoptosis studies showed a greater incidence when the combined treatment was put into use. This hints at the possibility of a new combined therapeutic approach. If confirmed in vivo, this conjugation may provide a means of new perspectives in muscle-invasive urinary bladder cancer treatment. [ABSTRACT FROM AUTHOR]

The purpose of this study was to determine whether everolimus, a rapamycin derivative, might significantly enhance the cytotoxicity of gemcitabine, an antitumor drug, in two human bladder-cancer cell lines. Human bladder-cancer T24 and 5637 cells were incubated with gemcitabine and everolimus in a range of concentrations either alone or in combination for 72 h. Flow cytometry, comet assay, MTT method and optical microscopy were used to assess cell proliferation, cell cycle, DNA damage, and morphological alterations. Gemcitabine exerted an inhibitory effect on T24 and 5637 cell proliferation, in a concentration-dependent manner. Everolimus significantly reduced proliferation of 5637 bladder cancer cells (IC30 at 1 μM), whereas T24 demonstrated marked resistance to everolimus treatment. A significant antiproliferative effect was obtained combining gemcitabine (100 nM) with everolimus (0.05–2 μM) with an arrest of cell cycle at S phase. Furthermore, an increase in frequency of DNA damage, apoptotic bodies, and apoptotic cells was observed when T24 and 5637 cancer cells were treated simultaneously with both drugs. Data show that in vitro combination produced a more potent antiproliferative effect when compared with single drugs. [ABSTRACT FROM PUBLISHER]

Some diet profiles are associated with the risk of developing cancer; however, some nutrients show protective effects. Porphyra umbilicalis is widely consumed, having a balanced nutritional profile; however, its potential for cancer chemoprevention still needs comprehensive studies. In this study, we incorporated P. umbilicalis into the diet of mice transgenic for the human papillomavirus type 16 (HPV16), which spontaneously develop pre-malignant and malignant lesions, and determined whether this seaweed was able to block lesion development. Forty-four 20-week-old HPV+/− and HPV−/− mice were fed either a base diet or a diet supplemented with 10% seaweed. At the end of the study, skin samples were examined to classify HPV16-induced lesions. The liver was also screened for potential toxic effects of the seaweed. Blood was used to study toxicological parameters and to perform comet and micronucleus genotoxicity tests. P. umbilicalis significantly reduced the incidence of pre-malignant dysplastic lesions, completely abrogating them in the chest skin. These results suggest that P. umbilicalis dietary supplementation has the potential to block the development of pre-malignant skin lesions and indicate its antigenotoxic activity against HPV-induced DNA damage. Further studies are needed to establish the seaweed as a functional food and clarify the mechanisms whereby this seaweed blocks multistep carcinogenesis induced by HPV. [ABSTRACT FROM AUTHOR]

An abstract of the article "Physiologic parameters variation in ICR mice during a chemical induced liver carcinogenesis experiment," by Nuno M. Paula-Santos, Filipa O. Pereira, and Maria J. Pires is presented.

Abstract: Stereological quantitative methods have revealed the presence of three distinct follicular cell populations (C1–C3) in bovine cumulus–oocyte complexes. Type C3 cells became largely predominant from metestrous to proestrous, with a simultaneously large decrease in the other two cell types. To further characterize these cumulus cell types, cumulus–oocyte complexes from small (1–4mm) and medium (4–8mm) antral follicles (category 1: with a compact multilayered cumulus and a homogeneous ooplasm) were aspirated from ovaries of Holstein–Friesian cows and processed for electron microscopy, ultrastructural cytochemical detection of glycogen and glycoproteins, and immunogold localization of chondroitin sulfate. Each follicular cell type displayed the same ultrastructural characteristics independently of the size of the follicle and the stage of the estrous cycle. Type C1 cells showed morphological characteristics of undifferentiated cells and progressively transformed into type C2 cells. Type C2 cells were characterized by cell extensions, polarized nuclei with evident nucleolar fibrilar centers, steroidogenic characteristics (numerous large lipid droplets, large endoplasmic reticulum vesicles and vacuoles), and synthesis of glycoproteins and chondroitin sulfate by the Golgi apparatus and endoplasmic reticulum. Type C3 cells presented morphologic features of fully differentiated and luteinized cumulus cells. They were characterized by an increase in cytoskeleton filaments, loss of cell extensions and of intercellular junctions, depletion of lipid and glycogen stores, and initiation of glycoprotein and chondroitin sulfate exocytosis. In conclusion, the present study suggests that bovine cumulus cells from small and medium antral follicles follow a complete dynamic functional differentiation process, in which the three cell types seem to correspond to a functional continuum. We identified undifferentiated cells, cells strongly engaged in glycoprotein and proteoglycan synthesis, and cells fully differentiated and secreting. [Copyright &y& Elsevier]

Bearded Capuchin or Black-striped Capuchin monkeys (Sapajus lidibinosus) are New World robust capuchin monkeys widely used in medical research. Few data are available concerning hematological reference values for these species, with no studies available from the Northeast region in Brazil. The aim of this study was to determine the hematological reference values for healthy bearded capuchin monkeys and to analyze the influence of sex and age factors. Blood samples were collected from 50 healthy bearded capuchin monkeys housed in captivity. These were analysed for total erythrocyte, hemoglobin, leukocyte and platelet count, packed cell volume (PCV), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC). When considering the age factor, significant differences were reported for total erythrocyte count, PCV, hemoglobin, total leucocytes, band neutrophils, eosinophils and lymphocytes (higher in juveniles). Significant sex-associated differences were noted for total erythrocyte count, PCV, hemoglobin (higher in males) and number of lymphocytes (higher in females). We have reported for the first time the hematological profile of bearded capuchin monkeys in captivity in the state of Paraíba, Brazil. These results can contribute for a better understanding of the normal physiology of capuchin monkeys, while demonstrating that factors such as sex and age influence hematological parameters should be taken into consideration in the hematological evaluation of this species. [ABSTRACT FROM AUTHOR]

Abstract: 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. [Copyright &y& Elsevier]

Cancer is the first cause of death in developed countries and the second in developing countries. Concerning the most frequent worldwide-diagnosed cancer, primary liver cancer represents approximately 4% of all new cancer cases diagnosed globally. However, among primary liver cancer, hepatocellular carcinoma is by far the most common histological subtype. Notwithstanding the health promotion and disease prevention campaigns, more than half a million new hepatocellular carcinoma cases are reported yearly, being estimated to growth continuously until 2020. Taking this scenario under consideration and the fact that some aspects concerning hepatocellular carcinoma evolution and metastasize process are still unknown, animal models assume a crucial role to understand this disease. The animal models have also provided the opportunity to screen new therapeutic strategies. The present review was supported on research and review papers aiming the complexity and often neglected chemically induced animal models in hepatocarcinogenesis research. Despite the ongoing debate, chemically induced animal models, namely, mice and rat, can provide unique valuable information on the biotransformation mechanisms against xenobiotics and apprehend the deleterious effects on DNA and cell proteins leading to carcinogenic development. In addition, taking under consideration that no model achieves all hepatocellular carcinoma research purposes, criteria to define the " ideal" animal model, depending on the researchers' approach, are also discussed in this review.

Background/aim: Hepatocellular carcinoma (HCC) is a frequent and aggressive malignancy associated with multiple environmental risk factors. The chemically-induced mouse model of diethylnitrosamine (DEN) provides useful insight into liver carcinogenesis, namely HCC. This work aimed to study the multistep process of hepato-carcinogenesis, providing a systematic framework for animal studies on this subject., Materials and Methods: Male ICR mice were divided into six control and six DEN-exposed groups. Saline solution and DEN were injected intra-peritoneally, respectively, for eight consecutive weeks. Two groups (DEN vs. control) were euthanized at 8, 15, 22, 29, 36 and 40 weeks after the first administration., Results: Hydropic degeneration, necrosis and apoptosis were acutely induced at eight weeks and onwards. Hyperplastic foci occurred at 29 to 40 weeks along with diffuse dysplastic areas and hepatocellular adenoma. Peliosis hepatis were also identified at 36 and 40 weeks. HCC were only noted at 40 weeks, showing characteristic histological features of malignancy., Conclusion: Results allowed sketching of a timeline of evolution of DEN-induced hepatic lesions in mice, from initial lesions to malignant neoplasms., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Aim: To study the effect of nebivolol on liver fibrosis induced by common bile duct ligation (BDL) in rats., Materials and Methods: After BDL, Wistar rats were divided into three groups (n=24): SO, sham-operated animals; BDL, BDL rats without treatment; BDL+N, BDL rats treated with nebivolol for five weeks. Alanine aminotransferase, aspartate aminotransferase, gamma glutamyltransferase, total bilirubin and albumin levels were assessed. Liver samples collected were stained with hematoxylin-eosin, Masson's trichrom and reticulin., Results: Mortality reached 37.5% in the BDL group, whereas no deaths were observed in the SO and BDL+N groups. The BDL group showed hepatic damage as evidenced by elevation in serum biochemical parameters and fibrosis scores. These pathophysiological changes were attenuated in the BDL+N group. However, there was no significant difference between these two groups., Conclusion: Nebivolol improved the survival rate of animals with BDL, but was unable to significantly improve liver function or reduce liver fibrosis.

Several drugs have shown in vitro and in vivo pharmacological activity against urinary bladder cancer. This review aims at compiling the different drugs evaluated in in vitro and in vivo models of urinary bladder cancer and to review the advantages and limitations of both types of models, as well as the different methodologies applied for evaluating antineoplastic drug activity.

Background: A mouse model in which N-diethylnitrosamine (DEN) induces Hepatocellular carcinoma (HCC) has histological and genetic resemblance to human tumours., Material and Methods: Male ICR mice were divided into control (n=10) and DEN-treated (n=10) groups. DEN was administered via intraperitoneal injection, once a week, for eight consecutive weeks. Animals were euthanized seven weeks after the last administration of DEN and their livers were collected. Plasma albumin, total bilirubin, alanine transaminase and aspartate aminotransferase activity were all measured and liver mitochondrial bioenergetics and oxidative stress were also evaluated., Results: Histologically, pre-neoplastic lesions were identified in the livers of mice from the DEN group. Total plasma bilirubin increased significantly in the group exposed to DEN and mitochondrial complex I and IV were significantly inhibited (p=0.0403 and p=0.0053, respectively)., Conclusion: DEN induced changes in liver bioenergetics and antioxidant capacity towards reactive oxygen species, seven weeks after administration. At this stage, liver tissues in mice exposed to DEN still had the ability to counteract the oxidative effects of DEN by increasing the activity of antioxidant enzymes.

Oral gavage is a widely used method for administering substances to animals in pharmacological and toxicological studies. The authors evaluated whether oral gavage causes behavioral indicators of stress, increased mortality rate, alterations in food and water consumption and body weight or histological lesions in CD-1 mice. Gavage was carried out once per d for 5 d per week over 6 consecutive weeks. The mortality rate of mice in this study was 15%. Mice subjected to gavage did not undergo changes in food or water consumption during the study, and their mean body weights and relative organ weights were similar to those of mice in the control group. Serum cortisol levels at the time of euthanasia in mice in both groups were within the normal range. Histopathology showed acute esophagitis and pleurisy, indicative of perforation of the esophagus, in the two mice that died but no abnormalities in the other mice. The results suggest that animal stress and mortality related to oral gavage can be minimized when the procedure is carried out by an experienced technician.

Background: Sirolimus was originally used as an immunosuppressant drug but recent reports have indicated that it may have other potential biological effects as an anticancer drug. The chemopreventive efficacy of sirolimus was evaluated in an experimental model of invasive urinary bladder cancer., Materials and Methods: ICR mice received N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) in drinking water for a period of twelve weeks. Sirolimus was administered 5 days a week. Animals were sacrificed either one or four weeks after their final treatment. Ki-67 was immunohistochemically analysed in paraffin-embedded tissue., Results: No evidence of host toxicity was found. The incidence of BBN-induced invasive urothelial carcinoma was significantly reduced in mice treated with sirolimus. Preneoplastic and neoplastic lesions exhibited a significant decrease in cellular proliferation., Conclusion: Histopathological and immunohistochemical studies showed that sirolimus reduced tumour incidence and proliferation. Sirolimus should be considered for further in vitro and in vivo studies in order to provide evidence of effectiveness.

Objectives: D/L-Nebivolol is a lypophilic beta1-adrenergic antagonist which is devoid of intrinsic sympathomimetic activity and can increase nitric oxide (NO) bioavailability with its subsequent vasodilating properties. The purpose of the present work was to assess the effect of long-term nebivolol administration on both renal damage and endothelial dysfunction induced by renal mass reduction (RMR) in rats. Atenolol, which does not increase NO bioavailability, was included in the study as a comparative beta-adrenoceptor antagonist., Methods: Rats were subjected to both right nephrectomy and surgical removal of two-thirds of the left kidney in order to retain approximately one-sixth of the total renal mass. One week after ablation, rats were distributed randomly according to the following experimental groups: control group containing RMR rats without treatment; RMR rats treated daily with nebivolol for 6 months (drinking water, 8 mg/kg per day); and RMR rats treated daily with atenolol for 6 months (drinking water, 80 mg/kg per day). A group of sham-operated animals was also included., Results: Administration of either nebivolol or atenolol similarly reduced arterial pressure in comparison with RMR untreated animals; however, animals receiving nebivolol presented lower levels of collagen type I expression as well as lower glomerular and interstitial fibrosis than those receiving atenolol. Urinary excretion of oxidative stress markers were also lower in animals receiving nebivolol than in rats treated with atenolol. Furthermore, nebivolol prevented RMR-induced endothelial dysfunction more efficiently than atenolol., Conclusions: Nebivolol protects against renal fibrosis, oxidative stress and endothelial dysfunction better than equivalent doses, in terms of arterial pressure reduction, of atenolol in a hypertensive model of renal damage induced by RMR.

The use of chemical compounds benefits society in a number of ways. Pesticides, for instance, enable foodstuffs to be produced in sufficient quantities to satisfy the needs of millions of people, a condition that has led to an increase in levels of life expectancy. Yet, at times, these benefits are offset by certain disadvantages, notably the toxic side effects of the chemical compounds used. Exposure to these compounds can have varying effects, ranging from instant death to a gradual process of chemical carcinogenesis. There are three stages involved in chemical carcinogenesis. These are defined as initiation, promotion and progression. Each of these stages is characterised by morphological and biochemical modifications and result from genetic and/or epigenetic alterations. These genetic modifications include: mutations in genes that control cell proliferation, cell death and DNA repair--i.e. mutations in proto-oncogenes and tumour suppressing genes. The epigenetic factors, also considered as being non-genetic in character, can also contribute to carcinogenesis via epigenetic mechanisms which silence gene expression. The control of responses to carcinogenesis through the application of several chemical, biochemical and biological techniques facilitates the identification of those basic mechanisms involved in neoplasic development. Experimental assays with laboratory animals, epidemiological studies and quick tests enable the identification of carcinogenic compounds, the dissection of many aspects of carcinogenesis, and the establishment of effective strategies to prevent the cancer which results from exposure to chemicals.

N-butyl-N-(4-hydroxybutyl)-nitrosamine (BBN)-induced urothelial carcinogenesis is a useful model for studying urothelial carcinogenesis. Here, the DNA content and the expression of Ki-67 and p53 in urothelial lesions induced by BBN and treated with mitomycin C (MMC) and Bacillus Calmette-Guérin (BCG) were investigated. Female Fisher 344 rats were distributed into five groups treated with 0.05% BBN in their drinking water for 20 weeks. Ten animals were used as negative control. Intravesical instillations were performed with MMC, BCG and physiological saline solution (PSS), once per week, for 6 weeks. The animals were sacrificed 1 week after the last intravesical instillation. DNA ploidy analysis was carried out by static cytometry. Ki-67 and p53 were analysed immunohistochemically in paraffin-embedded tissue. The incidence of lesions developed in rats with PSS was greater than in rats instilled with MMC and BCG. The incidence of aneuploidy was lower in tumours treated with MMC and BCG. Low- and high-grade papillary carcinoma treated with MMC and BCG showed a decrease in labelling index and an increase of apoptotic index. The proliferative index was correlated with the apoptotic index (r=0.438, p<0.01). Significant correlations were also found between the proliferative index and lesion, and the apoptotic index and lesion (r=0.425, p<0.01 and r=0.275, p<0.01), respectively. A significant correlation was found between ploidy and the apoptotic index (r=0.245, p<0.05). Our results provide information on the biological behaviour of chemically-induced bladder tumours treated with MMC and BCG.