Assessment of micronucleus frequency in the peripheral blood of female rats in persistent estrus treated with selective estrogen receptor modulators

The aim of this study was to evaluate micronucleus (MN) frequency in polychromatic erythrocytes (PCE) of female rats in persistent estrus (a model developed to mimic polycystic ovary syndrome) treated with selective estrogen receptor modulators (SERMs, tamoxifen, and raloxifene). Forty female Wistar-Hannover rats were divided into four groups of 10 animals each: Group I (normally cycling rats) and Group II (persistent estrus) both received only vehicle, while Group III (persistent estrus) was treated with tamoxifen (250 mu g/animal/day) and Group IV (persistent estrus) was treated with raloxifene (750 mu g/animal/day). Tamoxifen and raloxifene were given by oral gavage beginning on postnatal day 90 and continuing for 30 consecutive days. Peripheral blood samples were collected from tails 1 day following the last exposure. Blood smears were made on glass slides and stained with 10% Giemsa solution. ANOVA and a Tukey post-hoc test were used for data analysis. Mean percentages of MN were 1.82 +/- 0.13, 5.20 +/- 0.24, 3.32 +/- 0.13, and 3.04 +/- 0.12 in Groups I, II, III, and IV, respectively. The results indicate that tamoxifen and raloxifene similarly reduced the formation of MNPCE of female rats in persistent estrus (P < 0.0001 for Groups III and IV vs. Group II), using the dosages and time periods applied in the present study. The data suggest possibly antimutagenic effects of SERMs under high levels of estrogens. The findings also suggest that this is an interesting animal model for studying the genotoxicity of estrogens. Environ. Mol. Mutagen. 2012. (C) 2011 Wiley Periodicals, Inc.

Trabecular bone loss after administration of the second-generation antipsychotic risperidone is independent of weight gain

Second generation antipsychotics (SGAs) have been linked to metabolic and bone disorders in clinical studies, but the mechanisms of these side effects remain unclear. Additionally, no studies have examined whether SGAs cause bone loss in mice. Using in vivo and in vitro modeling we examined the effects of risperidone, the most commonly prescribed SGA, on bone in C57BL6/J (B6) mice. Mice were treated with risperidone orally by food supplementation at a dose of 1.25 mg/kg daily for 5 and 8 weeks, starting at 3.5 weeks of age. Risperidone reduced trabecular BV/TV, trabecular number and percent cortical area. Trabecular histomorphometry demonstrated increased resorption parameters, with no change in osteoblast number or function. Risperidone also altered adipose tissue distribution such that white adipose tissue mass was reduced and liver had significantly higher lipid infiltration. Next, in order to tightly control risperidone exposure, we administered risperidone by chronic subcutaneous infusion with osmotic minipumps (0.5 mg/kg daily for 4 weeks) in 7 week old female B6 mice. Similar trabecular and cortical bone differences were observed compared to the orally treated groups (reduced trabecular BV/TV, and connectivity density, and reduced percent cortical area) with no change in body mass, percent body fat, glucose tolerance or insulin sensitivity. Unlike in orally treated mice, risperidone infusion reduced bone formation parameters (serum P1NP, MAR and BFR/BV). Resorption parameters were elevated, but this increase did not reach statistical significance. To determine if risperidone could directly affect bone cells, primary bone marrow cells were cultured with osteoclast or osteoblast differentiation media. Risperidone was added to culture medium in clinically relevant doses of 0, 2.5 or 25 ng/ml. The number of osteoclasts was significantly increased by addition in vitro of risperidone while osteoblast differentiation was not altered. These studies indicate that risperidone treatment can have negative skeletal consequences by direct activation of osteoclast activity and by indirect non-cell autonomous mechanisms. Our findings further support the tenet that the negative side effects of SGAs on bone mass should be considered when weighing potential risks and benefits, especially in children and adolescents who have not yet reached peak bone mass. This article is part of a Special Issue entitled: Interactions Between Bone, Adipose Tissue and Metabolism. (C) 2011 Elsevier Inc. All rights reserved.

Effects of the antimycobacterial compound 2-phenoxy-1-phenylethanone on rat hepatocytes and formation of metabolites

Context: Neolignans are usually dimers formed by oxidative coupling of allyl and propenyl phenols, and the neolignan analogue, 2-phenoxy-1-phenylethanone (LS-2) is a promising antimycobacterial compound showing very weak cytotoxicity in mammalian cells and lack of acute toxicity in murine models. Objectives: To investigate the mechanism of action of LS-2 in rat hepatocytes by evaluating the activity levels of enzymes related to oxidation status and drug-metabolizing activity. Materials and methods: Hepatocytes were treated with LS-2 from 0.05 up to 1 mM, for 24 and 48 h, and reduced glutathione (GSH), lipid peroxidation and cytochrome P450 enzyme (CYP450) activity were assayed. A homologous series of phenoxazone ethers were used as substrates to measure the enzymatic profile. The biotransformation of LS-2 was studied in hepatocytes by gas chromatography-mass spectrometry (GC-MS) for detection and analysis of possible metabolites. Results: Hepatocytes treated with LS-2 up to 1 mM for 24 or 48 h did not induce the formation of GSH and lipid peroxidation. O-Dealkylation activities of the isoenzymes CYP4501A1, CYP4501A2, CYP4502B1 and CYP4502B2 were also not detected in the hepatocytes treated with LS-2 for 24 or 48 h. Discussion and conclusion: The results indicate that LS-2 or its two detected metabolites, 2-phenoxy-1-phenylethanol and 2,4-(2-hydroxy-2-phenylethoxy) phenol, are not cytotoxic to rat hepatocytes. These compounds maintain a balance between the production of pro-oxidant agents and their respective antioxidant systems. The data show that enzymes related to oxidation status and drug-metabolizing activities are not involved in the mechanism of action of LS-2.

Effect of cations/polycations on the efficiency of formation of a hybrid bilayer membrane that mimics the inner mitochondrial membrane

We aim in this study to characterize the effect of cations and polycations on the formation of hybrid bilayer membranes (HBMs), especially those that mimic the inner mitochondrial membrane (IMM), with a proper composition of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and cardiolipin (CL) adsorbed on an alkanethiol monolayer. HBMs are versatile membrane mimetics that show promising results in sensor technology. Its formation depends on the fusion of vesicles on hydrophobic surfaces, a process that is not well understood at the molecular level. Our results showed to which extend and in which condition the presence of cations and polycations facilitate the formation of HBMs. The required time for lipid layer formation was reduced several times and the lipid layer reaches the expected thickness of 19.5 +/- 1.8 angstrom, in contrast to only 2 +/- 1.5 angstrom usually observed in the absence of cations. In the presence of specific concentrations of spermine and Ca2+ the amount of adsorbed phospholipids on the thiol layer increased nearly 70% compared to that observed when Na+ was used at concentrations 10 times higher. Divalent cations and polycations adsorb specifically on the lipid headgroups destabilizing the hydration forces, facilitating the process of vesicle fusion and formation of lipid monolayers. The concepts and conditions described in the manuscript will certainly help the development of the field of membrane biosensors. (C) 2011 Elsevier B.V. All rights reserved.

Gonadal steroids levels and vitellogenesis in the formation of oocytes in Prochilodus lineatus (Valenciennes) (Teleostei: Characiformes)

The objective of this study was to obtain information about the possible mechanisms related to poor reproductive performance in tropical rheophilic fish. To that effect, cages (Cs) and earthen ponds (EPs) were used as experimental systems to provide unsuitable and suitable conditions, respectively, for curimbata (Prochilodus lineatus) breeders. Fish were maintained under experimental conditions for 18 months, and during this period females were randomly sampled every two months for biometric analysis (n=30), blood (n=5/sampling) and ovary (n=5/sampling). After this period EPs females (EPFs) and Cs females (CFs) were submitted to the induced breeding experiments. The results showed that rearing curimbata for such long time in a cage at this stocking density, reduces its growth, plasma E2 levels and vitellogenesis. During vitellogenesis, the mean plasma estradiol levels of CFs were three times lower than those of EPFs (P<0.01). CFs presented poorer results than EPFs for all the examined parameters of reproductive performance. Taken together these data showed that the reduced estradiol levels during vitellogenesis (and the consequently less intense transition from the previtellogenic to vitellogenic phase) and reduced amounts of yolk are mechanisms associated with the formation of low quality oocytes and shortened and delayed breeding season in this species. Moreover, our data showed that the onset of vitellogenesis (six months before the spawning season) must be considered as a key period related to the formation of oocytes of good quality, and adequate management should be provided throughout the year.