Erectile dysfunction drugs and oxidative stress in the liver of male rats

AbstractErectile dysfunction (ED) affected the lives of more than 300 million men worldwide. Erectile dysfunction drugs (EDD), known as phosphodiesterase inhibitors (PDEIs), have been used for treatment of ED. It has been shown that oxidative stress plays an important role in the progression of erectile dysfunction. Oxidative stress can be alleviated or decreased by antioxidant enzymes. Therefore, the present study aims at investigating the changes in the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione reductase as well as protein expression of glutathione peroxidase and glutathione S-transferase after treatment of male rats with a daily dose of sildenafil (1.48mg/kg), tadalafil (0.285mg/kg) and vardenafil (0.285mg/kg) for three weeks. In addition, levels of reduced glutathione and malondialdyhyde (MDA) were assayed. The present study showed that sildenafil, vardenafil, and tadalafil treatments significantly decreased the levels of glutathione, MDA and the activity of glutathione reductase. In addition, vardenafil and sildenafil increased the activity of superoxide dismutase and catalase. Interestingly, western immunoblotting data showed that vardenafil induced the activity of glutathione peroxidase (GPX) and its protein expression, whereas tadalafil and sildenafil inhibited such enzyme activity and its protein expression. In addition, the protein expression of GST π isozyme was markedly reduced after treatment of rats with sildenafil. It is concluded that ED drugs induced the activities of both SOD and catalase which consequently decreased MDA level. Therefore, decrement in MDA levels could increase nitric oxide–cGMP level which in turn promotes the erection mechanism

Renal Basolateral Transport of Glucuronides and Other Organic Anions in Rat in Vitro Models

Glucuronidation is a common Phase II biotransformation reaction that increases the hydrophilicity, and thus elimination, of toxins, xenobiotics, and endogenous compounds. Previous studies suggest that the kidney can secrete glucuronide conjugates, but the renal transport mechanisms for glucuronide secretion have not been determined. Based on the chemical nature of glucuronide metabolites, it is hypothesized that organic anion transporter (OAT) proteins along the basolateral membrane of the renal proximal tubule promote renal accumulation of glucuronide conjugates. The purpose of this study was to develop a rat renal proximal tubule model which demonstrates OAT activity and by which the contribution of OAT in the renal accumulation of glucuronide metabolites could be assessed. In the current study two in vitro models were established; freshly isolated renal proximal tubules (IRPTs), and renal cortical slices (RCSs) from the male Fischer 344 rat. These models demonstrate time-, temperature- and probenecid-sensitive uptake of prototypical OAT1 and OAT3 substrates fluorescein (FL) and 14C- p-aminohippuric acid (PAH), and the prototypical OAT 3 selective substrate 3H-estrone sulfate (ES). Accumulation of 14C-4-acetamidophenyl-β-D-glucuronide (AG) was found to be time-dependent, but not temperature- or probenecid-sensitive in IRPTs. In the RCSs, AG uptake was time-dependent, but only minimally temperature- and probenecid-sensitive. Accumulation/inhibition studies with FL, PAH, ES and AG indicate very limited interaction between AG and OAT. PAH uptake was not affected by other glucuronides (i.e. testosterone glucuronide, methylumbelliferyl glucuronide) in IRPTs or RCSs. Studies using RCSs from Sprague-Dawley rats yielded comparable results to those found in Fischer 344 rat. In all models, accumulation of 14C-AG was not inhibited by excess unlabeled AG. These results suggest that in these rat models, AG does not appear to be a substrate for renal basolateral membrane OAT proteins; that the contribution of OAT in the basolateral membrane transport of AG (and potentially other glucuronide metabolites) is minimal; and accumulation of AG does not appear to be a protein carrier-mediated process. Thus, rat renal proximal tubular cells do not appear to facilitate the accumulation of glucuronide conjugates, and thus do not appear to contribute to the renal secretion of glucuronide conjugates

ANÁLISE DO PROTEOMA MITOCONDRIAL DE CÉLULAS DO FÍGADO DE CAMUNDONGOS APOE KNOCKOUT TRATADOS COM SILDENAFIL

Um dos mecanismos propostos sobre o papel dos lipídios plasmáticos na gênese de doenças, é a sua associação com o estresse oxidativo, caracterizado pelo desbalanço redox, e que pode direta ou indiretamente estar relacionado à fisiopatologia das doenças crônicas não transmissíveis. Proteínas são moléculas envolvidas em praticamente todos os fenômenos biológicos, e o entendimento dos fatores que regulam a sua expressão, pode abrir portas para novas estratégias terapêuticas. Assim, o objetivo deste trabalho foi analisar alterações no proteoma mitocondrial de células do fígado (CF) de camundongos apoE knockout (apoE -/-) tratados com sildenafil. Previamente foi evidenciado que os animais apoE -/- apresentam perfil lipídico plasmático característico das dislipidemias, apresentando concentrações plasmáticas médias para triglicerídeos, colesterol total, LDL e VLDL de 5, 14, 6 e 36 vezes maiores que aquelas observadas para o grupo C57, respectivamente. Este aumento não foi revertido após o tratamento. A citometria de fluxo mostrou elevação nos níveis intracelulares nas CF dos animais apoE -/- de ânion superóxido (~82%), peróxido de hidrogênio (~60%) e peroxinitrito (~53%), além do aumento do número de células em apoptose (de ~2% para ~19%). Nossos resultados mostraram que o tratamento com sildenafil previne o aumento das ROS analisadas e inibe a indução de apoptose, sugerindo que o sildenafil possui ação antioxidante. A análise do proteoma mitocondrial por eletroforese bidimensional acoplada à espectrometria de massa (2DE-MS) das CF, mostrou que em ambas as condições experimentais (doença e tratamento), houve mudança no padrão de expressão das proteínas nesse compartimento subcelular. Destaque para o aumento da expressão da urato oxidase no grupo dos animais tratados (~93%) quando comparados com os sem tratamento. Esta enzima é responsável pela degradação do ácido úrico e pode apresentar papel importante na redução da produção de ROS. Os resultados também mostraram que a dislipidemia induz o aumento da transferrina, uma proteína de transporte de Fe, que ao aumentar a oferta desse metal, pode aumentar a formação de H2O2 pela reação de Fenton, contribuindo para o estresse oxidativo. Foi possível observar, entretanto, que o sildenafil reduz (~70%) os níveis dessa proteína quando comparado com o grupo apoE -/- , indicando equilíbrio redox após o tratamento. Após o estudo proteômico mitocondrial, foi possível observar a influência do tratamento com sildenafil na regulação de proteínas-chave na homeostase redox, contribuindo para a compreensão dos mecanismos pelos quais o sildenafil apresenta o efeito antioxidante