HFE gene mutation and oxidative damage biomarkers in patients with myelodysplastic syndromes and its relation to transfusional iron overload: an observational cross-sectional study.

OBJECTIVE: A relation between transfusional IOL (iron overload), HFE status and oxidative damage was evaluated. DESIGN, SETTING AND PARTICIPANTS: An observational cross-sectional study involving 87 healthy individuals and 78 patients with myelodysplastic syndromes (MDS) with and without IOL, seen at University Hospital of the Federal University of Ceará, Brazil, between May 2010 and September 2011. METHODS: IOL was defined using repeated measures of serum ferritin ≥1000 ng/mL. Variations in the HFE gene were investigated using PCR/restriction fragment length polymorphism (RFLP). The biomarkers of oxidative stress (plasmatic malonaldehyde (MDA), glutathione peroxidase (GPx) and superoxide dismutase (SOD)) were determined by spectrophotometry. RESULTS: The HFE gene variations were identified in 24 patients (30.77%) and 5 volunteers (5.74%). The H63D variant was observed in 35% and the C282Y variant as heterozygous in 5% of patients with MDS with IOL. One patient showed double heterozygous variant (C282Y/H63D) and serum ferritin of 11,649 ng/mL. In patients without IOL, the H63D variant was detected in 29.34%. Serum MDA levels were highest in patients with MDS with IOL, with a significant difference when compared with patients without IOL and healthy volunteers, pointing to the relationship between IOL and oxidative stress. The GPx and SOD were also significantly higher in these patients, indicating that lipid peroxidation increase was followed by an increase in antioxidant capacity. Higher ferritin levels were observed in patients with HFE gene variation. 95.7% of patients with MDS with the presence of HFE gene variations had received more of 20 transfusions. CONCLUSIONS: We observed a significant increase in MDA levels in patients with MDS and IOL, suggesting an increased lipid peroxidation in these patients. The accumulation of MDA alters the organisation of membrane phospholipids, contributing to the process of cellular degeneration. Results show that excess iron intensifies the process of cell damage through oxidative stress. TRIAL REGISTRATION NUMBER: Local Ethics Committee (licence 150/2009).

Avaliação da toxicidade aguda do epóxilimoneno em camundongos adultos / Evaluación de la toxicidad aguda en adultos epóxilimoneno ratones / Evaluation of the acute toxicity of limonene epoxide in adult mice

Introdução: a biodiversidade da flora mundial proporciona moléculas importantes no tratamento e na prevenção de várias enfermidades humanas, porém na maioria das vezes não são avaliadas sua toxicidade. Objetivo: avaliar a toxicidade do monoterpenóide epóxilimoneno em camundongos tratados de forma aguda com doses repetidas (25, 50 e 75mg/kg) por via oral em parâmetros bioquímicos e hematológicos. Métodos: quarenta camundongos correspondendo a quatro grupos (n=10/grupo) foram tratados, por via oral de forma aguda com doses repetidas e observados durante 14 dias, com epóxilimoneno nas doses de 25, 50 e 75 mg/kg emulsionado em Tween 80 0,05%dissolvido em solução salina 0,9 % (grupos EL25, EL50 e EL75, respectivamente), e com veículo (Tween 80, 0,05 % dissolvido em solução salina 0,9 %, grupo controle). Resultados: o tratamento não causou nenhuma morte ou sinal de toxicidade nos animais. Discussão: dessa forma, baseado nos resultados obtidos a partir dos estudos hematológicos e bioquímicos em camundongos, pode ser sugerido que a administração do epóxilimoneno não produz efeitos tóxicos sobre a maioria dos parâmetros analisados e que pode ser usado de forma segura em ensaios pré-clínicos. No entanto, mais estudos devem ser realizados para garantir que esse derivado de um monoterpeno natural seja utilizado de forma segura na indústria alimentícia e farmacêutica.

Introducción: la biodiversidad global de la flora proporciona moléculas importantes para el tratamiento y prevención de diversas enfermedades humanas, pero más a menudo no se evalúa su toxicidad. Objetivo: evaluar la toxicidad de lepóxilimoneno monoterpenoide en los ratones tratados de forma aguda con dosis repetidas (25, 50 y 75 mg/kg) por vía oral en los parámetros hematológicos y bioquímicos. Métodos: cuarenta ratones que representan cuatro grupos (n = 10/grupo) fueron tratados con dosis por vía oral de forma aguda repetida y se observaron durante 14 días com elepóxilimonenoa dosis de 25, 50 y 75 mg/kg emulsionados enTween 80 0,05 % disuelto en solución salina 0,9 % (grupos EL25, EL50 y EL75, respectivamente), y vehículo (Tween 80, 0,05 % disuelto en solución salina 0,9 %, grupo de control). Resultados: El tratamientono no causó muertes ni signos de toxicidad en animales. Discusión: de este modo, sobre la base de los resultados obtenidos a partir de los parámetros hematológicos y bioquímicos en ratones, se puede sugerir que la administración de epóxilimoneno no produce efectos tóxicos en la mayoría de los parámetros analizados y se puede utilizar de forma segura en la pre-clínica. Sin embargo, se deben realizar más estudios para asegurar que el derivado de un monoterpeno natural puede ser usado con seguridad en la industria alimentaria y farmacéutica.

Introduction: the biodiversity of global flora provides important molecules for the treatment and prevention of various human diseases, but most often their toxicities are not evaluated. Objective: evaluate the toxicity of monoterpenoid limonene epoxidein mice treated acutely with repeated doses (25, 50 and 75 mg/kg) orally on hematological and biochemical parameters. Methods: forty mice divided infour groups (n = 10/group ) were treated orally andacutely with repeated doses and observed for 14 days. The mice were treatedwith limonene epoxide administered at doses of 25, 50 and 75 mg / kg emulsified with 0.05 % Tween 80 dissolved in 0.9 % saline (groupsEL25, EL50 and EL75, respectively)and withvehicle (0.05 % Tween 80, dissolved in 0.9% saline, control group). Results: the treatment caused no deaths or signs of toxicity in the mice treated. Discussion: thus, based on the results obtained from hematological and biochemical studies in mice, it can be suggested that limonene epoxide does not produce anytoxic effect on most of the parameters analyzed, and can be safely used in pre-clinical-assays However, more studies should be conducted to ensure that this derivative of a natural monoterpenecan be safely used in thefood and pharmaceutical industry.

Impact of iron overload on interleukin-10 levels, biochemical parameters and oxidative stress in patients with sickle cell anemia.

OBJECTIVE: The aim of this study was to evaluate the impact of iron overload on the profile of interleukin-10 levels, biochemical parameters and oxidative stress in sickle cell anemia patients. METHODS: A cross-sectional study was performed of 30 patients with molecular diagnosis of sickle cell anemia. Patients were stratified into two groups, according to the presence of iron overload: Iron overload (n = 15) and Non-iron overload (n = 15). Biochemical analyses were performed utilizing the Wiener CM 200 automatic analyzer. The interleukin-10 level was measured by capture ELISA using the BD OptEIAT commercial kit. Oxidative stress parameters were determined by spectrophotometry. Statistical analysis was performed using GraphPad Prism software (version 5.0) and statistical significance was established for p-values < 0.05 in all analyses. RESULTS: Biochemical analysis revealed significant elevations in the levels of uric acid, triglycerides, very low-density lipoprotein (VLDL), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), urea and creatinine in the Iron overload Group compared to the Non-iron overload Group and significant decreases in the high-density lipoprotein (HDL) and low-density lipoprotein (LDL). Ferritin levels correlated positively with uric acid concentrations (p-value < 0.05). The Iron overload Group showed lower interleukin-10 levels and catalase activity and higher nitrite and malondialdehyde levels compared with the Non-iron overload Group. CONCLUSION: The results of this study are important to develop further consistent studies that evaluate the effect of iron overload on the inflammatory profile and oxidative stress of patients with sickle cell anemia.

Oxidative stress in rat striatum after pilocarpine-induced seizures is diminished by alpha-tocopherol.

Alpha-tocopherol has numerous nonenzymatic actions and is a powerful liposoluble antioxidant. The objective of the present study was to evaluate the neuroprotective effects of alpha-tocopherol in rats against oxidative stress caused by pilocarpine-induced seizures. Wistar rats were intraperitoneally treated with 0.9% saline (control group), alpha-tocopherol (200 mg/kg, alpha-tocopherol group), pilocarpine (400 mg/kg, pilocarpine group), or the combination of alpha-tocopherol (200 mg/kg) and pilocarpine (400 mg/kg, i.p.; alpha-tocopherol plus pilocarpine group). After the treatments, all groups were observed for 24 h. The superoxide dismutase (Mn-SOD) and catalase activities, lipid peroxidation and nitrite concentrations were measured using spectrophotometrically methods. To clarify the mechanism of alpha-tocopherol on oxidative stress in pilocarpine model, Western blot analysis of Mn-SOD and catalase in rat striatum were performed. In the pilocarpine group, rats showed a significant increase in lipid peroxidation and nitrite levels. However, there were no alterations on Mn-SOD activity. On the other hand, the catalase activity augmented in pilocarpine group. In the alpha-tocopherol and pilocarpine co-administered rats, antioxidant treatment significantly reduced the lipid peroxidation level and nitrite content and increased the Mn-SOD and catalase activities in rat striatum after seizures. Pilocarpine, alpha-tocopherol plus pilocarpine and alpha-tocopherol groups did not affect of the Mn-SOD and catalase mRNA or protein levels. Our findings strongly support the hypothesis that oxidative stress occurs in striatum during pilocarpine-induced seizures, indicating that brain damage induced by the oxidative process plays a crucial role in seizures pathogenic consequences, which implies that strong protective effect could be achieved using alpha-tocopherol.

Oxidative stress in rat hippocampus caused by pilocarpine-induced seizures is reversed by buspirone.

Temporal lobe epilepsy is the most common form of epilepsy in humans. Oxidative stress is a mechanism of cell death induced by seizures. Buspirone presents anxyolitic and antidepressant effects due to their ability to stimulate 5-HT(1A) receptor. We studied the buspirone effects on oxidative stress in rat hippocampus after seizures and status epilepticus (SE) induced by pilocarpine. In pilocarpine group there was a significant increase in lipid peroxidation and nitrite levels. However, no alteration was observed in superoxide dismutase and catalase activities. Buspirone pretreatment produces significantly reduction of the lipid peroxidation level (60%) and nitrite content (44%) as well as increased the superoxide dismutase (47%) and catalase (40%) activities in rat hippocampus after seizures, when compared with the pilocarpine group. The intraperitoneal injection of buspirone prior to pilocarpine suppressed the behavioral seizure occurrence. According to our results, the oxidative stress is present during seizures. Buspirone exerted anticonvulsant effects associated with the inhibition of the development of oxidative stress. These results suggest a therapeutic use potential of buspirone in epilepsy treatment.

Lipoic acid blocks seizures induced by pilocarpine via increases in delta-aminolevulinic dehydratase and Na+, K+-ATPase activity in rat brain.

In the present study we investigated the effects of lipoic acid (LA) on delta-aminolevulinic dehydratase (delta-ALA-D) and Na(+), K(+)-ATPase activities in rat brain after seizures induction by pilocarpine. Wistar rats were treated with 0.9% saline (i.p., control group), lipoic acid (10mg/kg, i.p., LA group), pilocarpine (400mg/kg, i.p., pilocarpine group), or the combination of LA (10mg/kg, i.p.) with pilocarpine (400mg/kg, i.p.), 30 min before administration of LA (LA plus pilocarpine group). After the treatments all groups were observed for 1h. The enzyme activities (delta-ALA-D and Na(+), K(+)-ATPase) were measured using spectrophotometric methods, and the results were compared with that obtained from saline and pilocarpine-treated animals. Neuroprotective effects of LA against seizures were evaluated based on those enzyme activities. The pilocarpine group showed a reduction in delta-ALA-D and Na(+), K(+)-ATPase activities after seizures. In turn, LA plus pilocarpine abolished the appearance of seizures and reversed the decreased in delta-ALA-D and Na(+), K(+)-ATPase activities produced by seizures, when compared to the pilocarpine seizing group. The results from the present study demonstrate that preadministration of LA abolished seizure episodes induced by pilocarpine in rat, probably by increasing delta-ALA-D and Na(+), K(+)-ATPase activities in rat brain during seizures.