Methods for the analysis of transcriptome dynamics.

The transcriptome is the complete set of transcripts in a cell or tissue and includes ribosomal RNA (rRNA), messenger RNA (mRNA), transfer RNA (tRNA), and regulatory noncoding RNA. At steady-state, the transcriptome results from a compensatory variation of the transcription and decay rate to maintain the RNA concentration constant. RNA transcription constitutes the first stage in gene expression, and thus is a major and primary mode of gene expression control. Nevertheless, regulation of RNA decay is also a key factor in gene expression control, involving either selective RNA stabilization or enhanced degradation. Transcriptome analysis allows the identification of gene expression alterations, providing new insights regarding the pathways and mechanisms involved in physiological and pathological processes. Upon perturbation of cell homeostasis, rapid changes in gene expression are required to adapt to new conditions. Thus, to better understand the regulatory mechanisms associated with gene expression alterations, it is vital to acknowledge the relative contribution of RNA synthesis and decay to the transcriptome. To the toxicology field, the study of gene expression regulation mechanisms can help identify the early and mechanistic relevant cellular events associated with a particular response. This review aims to provide a critical comparison of the available methods used to analyze the contribution of RNA transcription and decay to gene expression dynamics. Notwithstanding, an integration of the data obtained is necessary to understand the entire repercussions of gene transcription changes at a system-level. Thus, a brief overview of the methods available for the integration and analysis of the data obtained from transcriptome analysis will also be provided.

The Use of Feathers from Racing Pigeons for Doping Control Purposes.

The development of accurate and reliable analytical methodologies to detect the abuse of doping agents in sport animals is crucial to ensure their welfare, as well as to support continuing social acceptance of these sports. The detection of doping agents in racing pigeons is difficult, especially owing to the disadvantages and limitations of obtaining samples from conventional matrices. The present study aimed to develop and validate an analytical methodology combining a two-step extraction procedure (liquid-liquid extraction and solid-phase extraction) in feathers from racing pigeons with analysis by liquid chromatography tandem mass spectrometry (LC-MS-MS) that enabled the simultaneous detection of a beta-agonist drug (clenbuterol) and three corticosteroids (prednisolone, betamethasone and budesonide). The method was validated concerning linearity (with coefficients of determination always higher than 0.99), accuracy (87.3-112.4%), precision (repeatability and intermediate precision coefficient of variation (CV%) always below 15%), recovery (71.6-98.2%), limits of detection (0.24-0.52 ng/g) and quantification (0.79 and 0. 1.74 ng/g) and specificity. The applicability of the method was performed using feathers from pigeons administered orally with a daily dose of 0.075 mg of betamethasone. The drug was administered during 60 days and successive analyses of feathers were performed, at the end of the administration protocol and also after ceasing the oral administration of the drug, for a three weeks period.

Quantification of doping compounds in faecal samples from racing pigeons, by liquid chromatography-tandem mass spectrometry.

The use of performance enhancing drugs is not only common in humans, but also in animal sports, including racing of horses, greyhounds and pigeons. The development of accurate analytical procedures to detect doping agents in sports is crucial in order to protect the fair-play of the game, avoid financial fraud in the attribution of eventual awards and, even more important, to protect the animals from harmful drugs and/or dangerous dosage regimens. The present study aimed to develop and validate, a method that enabled the screening and confirmation of the presence of a beta-agonist (clenbuterol) and three corticosteroids (betamethasone, prednisolone and budesonide) in faeces from pigeons. The extraction procedure entailed the combination of liquid-liquid extraction with solid-phase extraction and the analysis was performed by liquid- chromatography coupled to tandem mass spectrometry, with a single 15 minute chromatographic run-time. The method was validated concerning selectivity, linearity (with coefficients of determination always >0.99), accuracy (87.5-114.9%), inter-day and intra-day precisions, limits of detection (0.14-1.81 ng/g) and limits of quantification (0.49-6.08 ng/g), stability and extraction recovery (71.0%-99.3%). The method was successfully applied for the analysis of samples from two pigeons that had been orally administered betamethasone, demonstrating its suitability for doping control purposes.

A rapid and simple procedure for the establishment of human normal and cancer renal primary cell cultures from surgical specimens.

The kidney is a target organ for the toxicity of several xenobiotics and is also highly susceptible to the development of malignant tumors. In both cases, in vitro studies provide insight to cellular damage, and represent adequate models to study either the mechanisms underlying the toxic effects of several nephrotoxicants or therapeutic approaches in renal cancer. The development of efficient methods for the establishment of human normal and tumor renal cell models is hence crucial. In this study, a technically simple and rapid protocol for the isolation and culture of human proximal tubular epithelial cells and human renal tumor cells from surgical specimens is presented. Tumor and normal tissues were processed by using the same methodology, based on mechanical disaggregation of tissue followed by enzymatic digestion and cell purification by sequential sieving. The overall procedure takes roughly one hour. The resulting cell preparations have excellent viabilities and yield. Establishment of primary cultures from all specimens was achieved successfully. The origin of primary cultured cells was established through morphological evaluation. Normal cells purity was confirmed by immunofluorescent staining and reverse transcription-polymerase chain reaction analysis for expression of specific markers.

Mechanisms underlying the hepatotoxic effects of ecstasy.

3,4-Methylenedioxymethamphetamine (MDMA or ecstasy) is a worldwide illegally used amphetamine-derived designer drug known to be hepatotoxic to humans. Jaundice, hepatomegaly, centrilobular necrosis, hepatitis and fibrosis represent some of the adverse effects caused by MDMA in the liver. Although there is irrefutable evidence of MDMA-induced hepatocellular damage, the mechanisms responsible for that toxicity remain to be thoroughly clarified. One well thought-of mechanism imply MDMA metabolism in the liver into reactive metabolites as responsible for the MDMA-elicited hepatotoxicity. However, other factors, including MDMA-induced hyperthermia, the increase in neurotransmitters efflux, the oxidation of biogenic amines, polydrug abuse pattern, and environmental features accompanying illicit MDMA use, may increase the risk for liver complications. Liver damage patterns of MDMA in animals and humans and current research on the mechanisms underlying the hepatotoxic effects of MDMA will be highlighted in this review.

First report on Cydonia oblonga Miller anticancer potential: differential antiproliferative effect against human kidney and colon cancer cells.

The present study reports the phenolic profile and antiproliferative properties of quince (Cydonia oblonga Miller) leaf and fruit (pulp, peel, and seed) against human kidney and colon cancer cells. The phenolic profiles of quince methanolic extracts were determined by high-performance liquid chromatography (HPLC)/diode array detector (DAD). 5-O-Caffeoylquinic acid was always one of the two major phenolic compounds present in all extracts, except for seed. Our results revealed that quince leaf and fruit extracts exhibited distinctive antiproliferative activities. The extracts from quince leaf showed concentration-dependent growth inhibitory activity toward human colon cancer cells (IC(50) = 239.7 +/- 43.2 microg/mL), while no effect was observed in renal adenocarcinoma cells. Concerning the fruit, seed extracts exhibited no effect on colon cancer cell growth, whereas strong antiproliferative efficiency against renal cancer cells was observed for the highest concentration assayed (500 microg/mL). The antiproliferative activity of pulp and peel extracts was low or absent in the selected range of extract concentrations. This is the first report showing that C. oblonga may be useful as a cancer chemopreventive and/or chemotherapeutic agent.

Comparative effects of 3,4-methylenedioxymethamphetamine and 4-methylthioamphetamine on rat liver mitochondrial function.

Ecstasy, which is used as a recreational drug, is a common street name for 3, 4-methylenedioxymethamphetamine (MDMA). Another drug of abuse chemically related, though less common than MDMA, is the amphetamine derivative 4-methylthioamphetamine (MTA). MDMA and MTA induce different systemic and organ-specific effects, including neurotoxicity, hyperthermia, nephrotoxicity, cardiotoxicity and hepatotoxicity. Therefore, it is clear that MDMA and MTA are responsible for inducing organ toxicity. The mechanisms underlying MDMA and MTA-induced hepatotoxicity are multifactorial, and therefore not completely understood. Recent findings indicate interference with cellular bioenergetics as an important toxicological feature of ecstasy. However, less is known about the involvement of mitochondria in MTA-induced hepatotoxicity. Thus, we compared the direct influence of MDMA and MTA on rat liver mitochondrial function [mitochondrial permeability transition (MPT), mitochondrial oxidative stress, and mitochondrial bioenergetics]. It was shown that MTA (from 0.025 up to 0.1mM) was more potent than MDMA (from 0.2 up to 0.5mM) in decreasing the sensitivity of rat liver mitochondria to MPT. However, higher concentrations of MTA (from 0.5 up to 2mM) were highly toxic to mitochondria. MTA simultaneously increased H(2)O(2) production in a monoamine oxidase (MAO)-dependent way, and uncoupled and inhibited mitochondrial respiration. In contrast, MDMA had only limited or no effects on these mitochondrial parameters. According to these results, it is possible to postulate that, depending on the concentration, MTA can potentially be more efficient in its effects on liver mitochondria than MDMA and, also, that its harmful effects may contribute to its hepatotoxicity.

Acute paraquat poisoning: report of a survival case following intake of a potential lethal dose.

When properly used, paraquat (PQ) is a widely used bipyridil herbicide with a good safety record. Most cases of PQ poisoning result from intentional ingestion, with death resulting from hypoxemia secondary to lung fibrosis in moderate to severe poisonings. With high ingestion volumes (>50 mL of a 20% wt/vol formulation), death results from multiple organ failure and cardiovascular collapse within 1 week after intoxication. The present report describes a successful clinical case regarding the intoxication of a 15-year-old girl by a presumed lethal dose of PQ. The adolescent ingested approximately 50 mL of a commercialized concentrate (20% wt/vol of dichloride salt) formulation of PQ. High serum and urinary levels of PQ confirmed the bad prognosis. However, the therapeutic protocol followed in the present clinical case led to a positive outcome. Besides the measures for decreasing PQ absorption and increasing its elimination, other protective procedures were applied in aiming to reduce the production of reactive oxygen species (ROS), to scavenge ROS, to repair ROS-induced lesions, and to reduce inflammation. The status-of-the-art concerning the biochemical and toxicological aspects of PQ poisoning and the pharmacologic basis of the respective treatment is also presented.

Strenuous exercise aggravates MDMA-induced skeletal muscle damage in mice.

The aim of this study was to investigate the influence of ecstasy (MDMA) administration on body temperature and soleus muscle histology in exercised and non-exercised mice. Charles-River mice were distributed into four groups: Control (C), exercise (EX), MDMA treated (M), and M + EX. The treated animals received an i.p. injection (10 mg/kg) of MDMA (saline for C and EX), and the exercise consisted of a 90 min level run at a velocity of 900 m/h, immediately after the MDMA or saline administration. Body temperature was recorded every 30 min via subcutaneous implanted transponder. Animals were sacrificed 1.5, 25.5, and 49.5 h after i.p. injection and the soleus muscles were removed and processed for light and electron microscopy. The MDMA-treated animals showed a significant increase in body temperature (similar in M and M + EX groups), reaching the peak 90 min after i.p. administration; their temperature remained higher than control for more than 5 h. The EX group evidenced a similar and parallel, yet lower temperature increase during exercise and recovery. Morphological signs of damage were rarely encountered in the EX group; they were more pronounced in M group and even aggravated in M + EX group. In conclusion, MDMA and exercise per se increased body temperature but in conjunction did not have a cumulated effect. However, ecstasy and concomitant physical activity might severely accumulate with regard to skeletal muscle toxicity and may lead to rhabdomyolysis.

Leucoisoprenochrome-o-semiquinone formation in freshly isolated adult rat cardiomyocytes.

Sustained high levels of circulating catecholamines can lead to cardiotoxicity. There is increasing evidence that this process may result from metal-catalyzed catecholamine oxidation into semiquinones, quinones, and aminochromes. We have previously shown that Cu2+-induced oxidation of isoproterenol into isoprenochrome induces toxic effects in isolated cardiomyocytes. The aim of this study was to characterize the isoproterenol oxidation process and to locate the formation of semiquinone radicals in cardiomyocyte suspensions. Freshly isolated rat cardiomyocytes were incubated with 1 or 10 mM isoproterenol and 20 microM Cu2+ for 4 h. The formation of an isoproterenol oxidation radical was detected in the extracellular medium, cells, membranes, and heavy organelles by electron spin resonance spectroscopy. An electron spin resonance signal assigned to leucoisoprenochrome-o-semiquinone increased in a time-dependent manner in the extracellular medium. A second electron spin resonance signal, characteristic of an immobilized radical, was also found in the cardiomyocytes. The latter was attributed to leucoisoprenochrome-o-semiquinone immobilized on cellular components such as membranes, cytoskeleton, nucleus, and heavy organelles. In addition, the levels of leucoisoprenochrome-o-semiquinone decreased in the presence of glutathione. Computer simulations of the experimental spectra indicate the formation of two distinct isomeric leucoisoprenochrome-o-semiquinone radicals during isoproterenol oxidation. The present study shows that the isoproterenol oxidation in isolated rat cardiomyocytes correlates with the formation of leucoisoprenochrome-o-semiquinone in the cells and in the extracellular medium, suggesting that it might be involved in cardiotoxicity induced by the oxidation of catecholamines.

Synthesis and analysis of aminochromes by HPLC-photodiode array. Adrenochrome evaluation in rat blood.

The catecholamine oxidation process induces cardiotoxicity and neurotoxicity. Catecholamines can oxidize to aminochromes through autoxidation or by enzymatic or non-enzymatic catalysis. Although some toxic effects seem to be related to the formation of aminochromes there is still scarce information concerning the identification and evaluation of these compounds in in vivo models. In this study five catecholamines were oxidized to their respective aminochromes: adrenaline/adrenochrome; noradrenaline/noradrenochrome; dopa/dopachrome; dopamine/dopaminochrome; and isoproterenol/isoprenochrome. The evaluation of the catecholamines oxidation profile was performed by HPLC with photodiode array detection and using either enzymatic (tyrosinase) or non-enzymatic [Ag(2)O, CuSO(4), NaIO(4) and K(3)Fe(CN)(6)] catalytic systems. The NaIO(4) was found to be the most efficient oxidant of catecholamines. An isocratic reverse-phase HPLC method was developed to analyse each pair of catecholamine-aminochrome. The analytical system was then applied to the detection of adrenochrome in rat blood at 490 nm. Thus, adrenochrome was administered i.p. to rats and its concentration in whole blood was monitored after 5, 15 and 25 min. Blood treatment for adrenochrome evaluation consists of an acidification for protein precipitation followed by a rapid neutralization. The results showed a rapid decrease of adrenochrome concentration in blood after its administration. The adrenochrome present in blood was characterized by UV and tandem mass spectrometry.

Cu2+-induced isoproterenol oxidation into isoprenochrome in adult rat calcium-tolerant cardiomyocytes.

Sustained high levels of circulating catecholamines may induce cardiotoxicity. There is increasing evidence that this could result from catecholamine oxidation into aminochromes, which is catalyzed by transition metals. In fact, it has already been shown that copper-induced oxidation of the beta-agonist isoproterenol decreases the viability of isolated cardiomyocytes. Thus, the aim of this work was to contribute for the clarification of the mechanisms underlying the toxic effects of isoproterenol, Cu2+ and their concomitant effect in isolated rat cardiomyocytes. Freshly isolated calcium-tolerant cardiomyocytes from adult rat were incubated with 1 mM isoproterenol, 20 microM Cu2+ or with both during 4 h. Isoproterenol and its aminochrome (isoprenochrome), and reduced and oxidized glutathione were measured at each hour in the incubation medium and in the cells. The intracellular activities of the selenium-dependent glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were determined after 4 h of incubation. Isoprenochrome was found in both cells and incubation medium in samples incubated with isoproterenol alone. However, in the isoproterenol plus Cu2+ samples, a greater depletion of isoproterenol accompanied by a proportional increase of isoprenochrome was observed. This higher ISO oxidation resulted in the depletion of intracellular glutathione and in the release of oxidized glutathione to the incubation medium. The content of total glutathione (intra- and extracellular) and the intracellular activity of the selenium-dependent glutathione peroxidase, glutathione reductase, and glutathione-S-transferase were also decreased in the isoproterenol plus Cu2+ samples. These results seem to indicate that the oxidative stress resulting from catecholamine/transition metal association may contribute to catecholamine cardiotoxicity.

O ato de doar sangue sob a ótica de técnicos e doadores / Blood donation act in the point of view of technicians and donors

Taking as a premise the fact that the majority of blood donations are linked to a receiver, that is, the donation act generally occurs in situations where someone needs the blood, this investigation's objective was: to comprehend the signification of the donation blood act in respect to the people involved in this process. The interest in effecting a study with this point of view came from the need to know the signification of the donation blood act, and to try to discover ways that can give contributions to strategies to attract qualified blood donors. This is a prospective qualitative study where a semi-structured interview was used for data collection and thematic analysis for data handling. In this view, the article shows the significance of the donation act for technicians with intermediate school level and the donors, that it is linked to private motivation, while with graduated technicians, the signification is based on public motivation

Estudo de pacientes com asma brônquica moderada tratados com flunisolida / Study of patients with moderate bronchial asthma treated with flunisolide

O uso dos costicosteróides inalatórios é o tratamento de escolha na asma brônquica quando há sintomas persistentes. A flunisolida é um dos esteróides tópicos utilizados há mais tempo e é atualmente comercializada no Brasil. Foi objetivo desse estudo observar a evoluçÒo de pacientes com asma moderada em tratamento adequado com esta droga. Trinta e quatro asmáticos moderados foram seguidos por 2 semanas ("run-in") + 12 semanas em tratamento com flunisolida 1mg/dia, através de aerocâmera 300, e fenoterol spray quando necessário. Todos foram instruídos sobre a doença, controle ambiental, uso de medicaçöes inalatórias e recebiam plano de açÒo escrito para crise. As medicaçöes foram fornecidas gratuitamente e a adesÒo ao tratamento assegurada através de controle do peso do spray. Os pacientes eram reavaliados a cada duas semanas, registrando seus sintomas em escalas analógicas e respondendo a questionário para o estabelecimento de índice clínico de hiperreatividade brônquica (ICHB). Espirometria, diários de PFE, consumo de broncodilatador e "escore" de ausculta torácica (EAT) também foram obtidos a cada visita. Reatividade à metacolina foi avaliada no início e no final do estudo. Dezessete de trinta e um pacientes que concluíram o estudo tiveram sua asma reclassificada como leve (p< 0,001). Houve tendência à melhora do VEF(1) (67,8 para 71,6 por cento), com elevaçÒo significativa (p<0,05) da média do PFE (302,3 para 338,5 - à noite) e do seu valor mínimo (47,2 para 60,5 por cento). Reduçäo significante (p<0,05) do ICHB, tosse, chiado, dispnéia e opressäo também foram observados. Näo houve mudança significativa do EAT, entretanto. Rouquidäo, náusea e cefaléia ocorreram em menos de 10 por cento dos voluntários. Nenhum paciente abandonou o estudo por efeitos adversos da droga. Em conclusäo, encontramos resposta clínica favorável na maioria dos adultos asmáticos moderados em uso de flunisolida, que foi bem tolerada. Surpreendentemente, nem a ausculta torácica, nem o VEF(1) foram instrumentos suficientemente sensíveis para detectar a evoluçäo favorável dos pacientes, claramente demonstrada por outros parâmetros.