Efeito Cardioprotetor da Suplementação Materna com Resveratrol sobre a Toxicidade Induzida por Doxorrubicina em Cardiomiócitos de Neonatos / Cardioprotective Effect of Maternal Supplementation with Resveratrol on Toxicity Induced by Doxorubicin in Offspring Cardiomyocytes

Resumo Fundamento A doxorrubicina (DOX) é frequentemente usada para tratar muitos tipos de cânceres, apesar da cardiotoxicidade dose-dependente. Como alternativa, o resveratrol é um polifenol que tem demonstrado efeitos cardioprotetores em vários modelos de disfunção cardíaca. Objetivo Este estudo investigou se o tratamento com resveratrol em ratas gestantes protege contra toxicidade induzida por doxorrubicina em cardiomiócitos da ninhada. Métodos Ratas Wistar (n-8) receberam sresveratrol como suplemento alimentar durante a gestação. No nascimento da ninhada, os corações (9-11) foram usados para se obter a cultura primária de cardiomiócitos. A cardiotoxicidade induzida por DOX e os efeitos da suplementação com resveratrol foram avaliados por marcadores de stress oxidativo, tais como oxidação da diclorofluoresceína diacetato, diminuição da atividade de enzimas antioxidantes, e oxidação do teor total de grupos sulfidrila, além da avaliação da viabilidade celular, geração de danos ao DNA, bem como a resposta de reparo aos danos ao DNA. Um valor de p <0,05 foi considerado estatisticamente significativo. Resultados Os cardiomiócitos de neonatos de ratas que receberam suplemento resveratrol apresentaram um aumento (p <0,01) na viabilidade das células, e diminuição (p <0,0001) de células apoptóticas/necróticas após o tratamento com DOX, o que está correlacionado às atividades de enzimas antioxidantes e produção de diclorofluoresceína. Além disso, o resveratrol protegeu os cardiomiócitos de danos ao DNA induzidos por DOX, apresentando uma diminuição (p <0,05) nas quebras de DNA induzidas por stress oxidativo, avaliadas pela atividade de enzimas reparadoras do DNA endonuclease III e formamidopirimidina glicosilase. A suplementação com resveratrol aumentou (p <0,05) a expressão da proteína reparadora Sirt6 nos cardiomiócitos dos filhotes. Conclusão Essa pesquisa indica que a suplementação com resveratrol durante o período gestacional tem um efeito cardioprotetor no coração da ninhada contra a toxicidade induzida por DOX, o que pode se dever a sua função antioxidante, e o aumento na resposta de danos ao DNA.

Abstract Background Doxorubicin (DOX) is frequently used to treat many types of cancers, despite its dose-dependent cardiotoxicity. Alternatively, resveratrol is a polyphenol that has shown useful cardioprotective effects in many heart dysfunction models. Objective This study investigated whether resveratrol treatment in pregnant rats protects against doxorubicin-induced toxicity in offspring cardiomyocytes. Methods Wistar rats (n=8) were supplemented with dietary resveratrol during pregnancy. Upon the offspring's birth, hearts (9-11) were used to obtain the primary culture of cardiomyocytes. DOX-induced cardiotoxicity and the effects of resveratrol supplementation were evaluated by oxidative stress markers, such as dichlorofluorescein diacetate oxidation, decrease in the activity of antioxidant enzymes, and oxidation of total sulfhydryl content, in addition to cell viability evaluation, DNA damage generation, and DNA damage repair response. A value of p<0.05 was considered statistically significant. Results Neonatal cardiomyocytes from resveratrol supplemented rats exhibiting an increase (p<0.01) in cell viability and lower (p<0.0001) apoptotic/necrotic cells after DOX treatment, which correlates with the activities of antioxidant enzymes and dichlorofluorescein production. Moreover, resveratrol protected cardiomyocytes from DOX-induced DNA damage, showing a decrease (p<0.05) in DNA breaks induced by oxidative stress, evaluated by the activity of DNA-repair enzymes endonuclease III and formamidopyrimidine glycosylase. Supplementation with resveratrol increased (p<0.05) the expression of the repair protein Sirt6 in the cardiomyocytes of the pups. Conclusion This research indicates that supplementation with resveratrol during the gestational period has a notable cardioprotective effect on the offspring's heart against DOX-induced toxicity, which may well be due to its antioxidant function, and the increase in the DNA damage repair response.

Cardioprotective Effect of Maternal Supplementation with Resveratrol on Toxicity Induced by Doxorubicin in Offspring Cardiomyocytes. / Efeito Cardioprotetor da Suplementação Materna com Resveratrol sobre a Toxicidade Induzida por Doxorrubicina em Cardiomiócitos de Neonatos.

BACKGROUND: Doxorubicin (DOX) is frequently used to treat many types of cancers, despite its dose-dependent cardiotoxicity. Alternatively, resveratrol is a polyphenol that has shown useful cardioprotective effects in many heart dysfunction models. OBJECTIVE: This study investigated whether resveratrol treatment in pregnant rats protects against doxorubicin-induced toxicity in offspring cardiomyocytes. METHODS: Wistar rats (n=8) were supplemented with dietary resveratrol during pregnancy. Upon the offspring's birth, hearts (9-11) were used to obtain the primary culture of cardiomyocytes. DOX-induced cardiotoxicity and the effects of resveratrol supplementation were evaluated by oxidative stress markers, such as dichlorofluorescein diacetate oxidation, decrease in the activity of antioxidant enzymes, and oxidation of total sulfhydryl content, in addition to cell viability evaluation, DNA damage generation, and DNA damage repair response. A value of p<0.05 was considered statistically significant. RESULTS: Neonatal cardiomyocytes from resveratrol supplemented rats exhibiting an increase (p<0.01) in cell viability and lower (p<0.0001) apoptotic/necrotic cells after DOX treatment, which correlates with the activities of antioxidant enzymes and dichlorofluorescein production. Moreover, resveratrol protected cardiomyocytes from DOX-induced DNA damage, showing a decrease (p<0.05) in DNA breaks induced by oxidative stress, evaluated by the activity of DNA-repair enzymes endonuclease III and formamidopyrimidine glycosylase. Supplementation with resveratrol increased (p<0.05) the expression of the repair protein Sirt6 in the cardiomyocytes of the pups. CONCLUSION: This research indicates that supplementation with resveratrol during the gestational period has a notable cardioprotective effect on the offspring's heart against DOX-induced toxicity, which may well be due to its antioxidant function, and the increase in the DNA damage repair response.

FUNDAMENTO: A doxorrubicina (DOX) é frequentemente usada para tratar muitos tipos de cânceres, apesar da cardiotoxicidade dose-dependente. Como alternativa, o resveratrol é um polifenol que tem demonstrado efeitos cardioprotetores em vários modelos de disfunção cardíaca. OBJETIVO: Este estudo investigou se o tratamento com resveratrol em ratas gestantes protege contra toxicidade induzida por doxorrubicina em cardiomiócitos da ninhada. MÉTODOS: Ratas Wistar (n-8) receberam sresveratrol como suplemento alimentar durante a gestação. No nascimento da ninhada, os corações (9-11) foram usados para se obter a cultura primária de cardiomiócitos. A cardiotoxicidade induzida por DOX e os efeitos da suplementação com resveratrol foram avaliados por marcadores de stress oxidativo, tais como oxidação da diclorofluoresceína diacetato, diminuição da atividade de enzimas antioxidantes, e oxidação do teor total de grupos sulfidrila, além da avaliação da viabilidade celular, geração de danos ao DNA, bem como a resposta de reparo aos danos ao DNA. Um valor de p <0,05 foi considerado estatisticamente significativo. RESULTADOS: Os cardiomiócitos de neonatos de ratas que receberam suplemento resveratrol apresentaram um aumento (p <0,01) na viabilidade das células, e diminuição (p <0,0001) de células apoptóticas/necróticas após o tratamento com DOX, o que está correlacionado às atividades de enzimas antioxidantes e produção de diclorofluoresceína. Além disso, o resveratrol protegeu os cardiomiócitos de danos ao DNA induzidos por DOX, apresentando uma diminuição (p <0,05) nas quebras de DNA induzidas por stress oxidativo, avaliadas pela atividade de enzimas reparadoras do DNA endonuclease III e formamidopirimidina glicosilase. A suplementação com resveratrol aumentou (p <0,05) a expressão da proteína reparadora Sirt6 nos cardiomiócitos dos filhotes. CONCLUSÃO: Essa pesquisa indica que a suplementação com resveratrol durante o período gestacional tem um efeito cardioprotetor no coração da ninhada contra a toxicidade induzida por DOX, o que pode se dever a sua função antioxidante, e o aumento na resposta de danos ao DNA.

L-carnitine protects DNA oxidative damage induced by phenylalanine and its keto acid derivatives in neural cells: a possible pathomechanism and adjuvant therapy for brain injury in phenylketonuria.

Although phenylalanine (Phe) is known to be neurotoxic in phenylketonuria (PKU), its exact pathogenetic mechanisms of brain damage are still poorly known. Furthermore, much less is known about the role of the Phe derivatives phenylacetic (PAA), phenyllactic (PLA) and phenylpyruvic (PPA) acids that also accumulate in this this disorder on PKU neuropathology. Previous in vitro and in vivo studies have shown that Phe elicits oxidative stress in brain of rodents and that this deleterious process also occurs in peripheral tissues of phenylketonuric patients. In the present study, we investigated whether Phe and its derivatives PAA, PLA and PPA separately or in combination could induce reactive oxygen species (ROS) formation and provoke DNA damage in C6 glial cells. We also tested the role of L-carnitine (L-car), which has been recently considered an antioxidant agent and easily cross the blood brain barrier on the alterations of C6 redox status provoked by Phe and its metabolites. We first observed that cell viability was not changed by Phe and its metabolites. Furthermore, Phe, PAA, PLA and PPA, at concentrations found in plasma of PKU patients, provoked marked DNA damage in the glial cells separately and when combined. Of note, these effects were totally prevented (Phe, PAA and PPA) or attenuated (PLA) by L-car pre-treatment. In addition, a potent ROS formation also induced by Phe and PAA, whereas only moderate increases of ROS were caused by PPA and PLA. Pre-treatment with L-car also prevented Phe- and PAA-induced ROS generation, but not that provoked by PLA and PPA. Thus, our data show that Phe and its major metabolites accumulated in PKU provoke extensive DNA damage in glial cells probably by ROS formation and that L-car may potentially represent an adjuvant therapeutic agent in PKU treatment.

Oxidative Stress and DNA Damage of Zebrafish Sperm at Different Stages of the Cryopreservation Process.

Although gamete cryopreservation has facilitated advancement of reproduction research by allowing the storage of cells over prolonged periods of time, during freezing-thawing cycles, cells inevitably suffer from cryoinjuries. Here, we evaluate oxidative stress and DNA damage of zebrafish sperm at different stages of the cryopreservation process. It was generally observed that the freezing and thawing of the samples led to an increase in the generation of reactive oxygen species and the activity of the catalase enzyme and a reduction in the generation of sulfhydryl groups and superoxide dismutase activity. The alkaline comet assay demonstrated that DNA damage increased after equilibration time, with an even greater increase after freezing and thawing. The comet assay modified with the enzyme formamidopyrimidine glycosylase, and Endonuclease III demonstrated greater DNA damage than the standard comet assay, demonstrating a high degree of oxidation of purines and pyrimidines at all stages of cryopreservation. Our results show that the freeze and thaw processes cause greater oxidative stress and DNA damage than cryoprotectant toxicity during exposure at the equilibrium stage.

Lacosamide improves biochemical, genotoxic, and mitochondrial parameters after PTZ-kindling model in mice.

This study evaluated the effect of lacosamide (LCM) on biochemical and mitochondrial parameters after PTZ kindling in mice. Male mice were treated on alternative days for a period of 11 days with LCM (20, 30, or 40 mg/kg), saline, or diazepam (2 mg/kg), before PTZ administration (50 mg/kg). The hippocampi were collected to evaluate free radicals, the activities of superoxide dismutase (SOD), catalase (CAT), and the mitochondrial complexes I-III, II, and II-III, as well as Bcl-2 and cyclo-oxygenase-2 (COX-2) expressions. Hippocampi, blood, and bone marrow were collected for genotoxic and mutagenic evaluations. LCM 40 mg/kg increased latency and decreased percentage of seizures, only on the 3rd day of observation. The dose of 30 mg/kg only showed positive effects on the percentage of seizures on the 2nd day of observation. LCM decreased free radicals and SOD activity and the dose of 40 mg/kg were able to increase CAT activity. LCM 30 and 40 mg/kg improved the enzymatic mitochondrial activity of the complex I-III and LCM 30 mg/kg improved the activity of the complex II. In the comet assay, the damage induced by PTZ administration was reduced by LCM 20 and 30 mg/kg. The dose of 20 mg/kg increased COX-2 expression while the highest dose used, 40 mg/kg, was able to reduce this expression when compared to the group treated with LCM 20 mg/kg. Although LCM did not produce the antiepileptogenic effect in vivo, it showed the neuroprotective effect against oxidative stress, bioenergetic dysfunction, and DNA damage induced by the repeated PTZ administration.

Skim milk powder used as a non-permeable cryoprotectant reduces oxidative and DNA damage in cryopreserved zebrafish sperm.

Cryoprotectants play a vital role in the cryopreservation process, protecting biological samples from freezing damage. Here, we evaluate the effects of the combination and interaction of different extenders with permeable and non-permeable cryoprotectants, on the cryopreservation of Danio rerio sperm, analyzing the effects of cryopreservation through a broad approach to variables. Two extenders were used, Hank's balanced salt solution (HBSS) and Ginsburg's solution. Eight cryoprotective solutions (CS) were used: CS1 (HBSS + Me2SO 8%), CS2 (HBSS + Methanol 8%), CS3 (HBSS + Me2SO 8% + Skim milk powder 15%), CS4 (HBSS + Methanol 8% + Skim milk powder 15%), CS5 (Ginsburg + Me2SO 8%), CS6 (Ginsburg + Methanol 8%), CS7 (Ginsburg + Me2SO 8% + Skim milk powder 15%) and CS8 (Ginsburg + Methanol 8% + Skim milk powder 15%). The samples were cryopreserved in cryovials for 20 min on dry ice, stored in liquid nitrogen, thawed at 38 °C for 10 s, and analyzed. In addition to increasing viability, we show that powdered milk also allows for better preservation of the membrane and normal cell morphology, and protects the sperm cells from DNA damage and oxidative stress caused by cryopreservation.

Intradialytic neuromuscular electrical stimulation reduces DNA damage in chronic kidney failure patients: a randomized controlled trial.

BACKGROUND: Chronic kidney failure (CKF) patients on renal replacement therapies exhibit elevated levels of DNA lesions and this is directly related to high mortality. OBJECTIVE: This study aimed to evaluate the effect of neuromuscular electrical stimulation (NMES) on genomic damage in CKF patients on conventional haemodialysis (HD). METHODS: Twenty-one patients with CKF on HD were randomized into control (CG =10) or neuromuscular electrical stimulation (NMESG = 11) groups. NMES was applied on the quadriceps muscle during the HD session, three times a week, for 8 weeks in NMESG. DNA damage in blood was evaluated by the alkaline comet assay prior to follow-up, after 4 and 8 weeks of intervention. RESULTS: Intradialytic NMES in CKF patients induced a significant decrease in DNA damage after four [49.9 (3.68) vs 101.5 (6.53); p = 0.000] than eight [19.9 (2.07) vs 101.5 (6.53); p = 0.000] weeks compared to baseline. Genomic damage was also significantly less after four [NMESG: 49.9 (3.68) vs CG: 92.9 (12.61); p = 0.001] than after eight [NMESG: 19.9 (2.07) vs CG: 76.4 (11.15); p = 0.000] weeks compared to CG. CONCLUSIONS: This study demonstrates for the first time that intradialytic NMES is able to reduce DNA damage in blood of CKF patients.

Low maternal care is associated with increased oxidative stress in the brain of lactating rats.

Maternal care is crucial for offspring development and licking/grooming patterns can be induced by sensorial, neuroendocrine, and metabolic variations in the CNS. Important brain functions, such as learning and memory, can be influenced by oxidative stress, which can also modulate pathophysiological processes (e.g., depression, anxiety, and other psychiatric disorders). This study evaluated oxidative stress in the hippocampus (HP), olfactory bulb (OB), and plasma in Low-Licking (LL) and High-Licking (HL) lactating rats through superoxide dismutase (SOD) and catalase (CAT) activities, DNA damage (comet assay), and dihydrodichlorofluorescein (DCF) oxidation assay. Results demonstrate that in the HP of LL, the activities of SOD and CAT were increased compared to HL. In the OB, the activities of SOD and CAT were also increased in LL. The comet assay in the HP showed that LL had higher levels of basal damage and increased levels of DNA breaks than HL. In the OB, LL also had higher levels of DNA damage. In the plasma, no difference was observed in either SOD or CAT activities, but the DCF oxidation assay revealed that LL had higher levels of ROS production than HL. In conclusion, we observed that LL mothers showed evidence of increased oxidative stress when compared to HL, suggesting that variations in maternal behavior might be related to these biochemical parameters.

SUPLEMENTAÇÃO COM ÔMEGA-3 PÓS-RECONSTRUÇÃO DO LIGAMENTO CRUZADO ANTERIOR / SUPPLEMENTATION WITH OMEGA-3 AFTER RECONSTRUCTION OF THE ANTERIOR CRUCIATE LIGAMENT / SUPLEMENTACIÓN CON OMEGA-3 DESPUÉS DE LA RECONSTRUCCIÓN DEL LIGAMENTO CRUZADO ANTERIOR

Introdução: As lesões do ligamento cruzado anterior (LCA) contribuem para a formação de radicais livres de oxigênio que, em excesso, podem desencadear dano oxidativo na articulação do joelho. Objetivo: Avaliar os efeitos da suplementação oral com ômega-3 sobre marcadores de estresse oxidativo em indivíduos submetidos à reconstrução do LCA. Métodos: Este estudo é um ensaio clínico prospectivo, randomizado, controlado e simples cego, com amostra de 25 indivíduos submetidos à reconstrução do LCA, separados aleatoriamente em: grupo ômega-3 (GO), suplementado diariamente com 2 g de ômega-3 durante 15 dias pós-reconstrução do LCA e grupo controle (GC), não suplementado. Foi realizada coleta de sangue e de líquido sinovial imediatamente antes do procedimento cirúrgico e 15 dias pós-reconstrução do LCA. As análises bioquímicas avaliaram os níveis de produtos de lipoperoxidação (MDA); atividade da catalase; grupos sulfidrílicos totais e polifenóis e proteína C reativa (PCR). Resultados: Observou-se diminuição significativa nos níveis de MDA no GO em comparação ao GC (p < 0,05), da mesma forma que a atividade da enzima antioxidante catalase foi significativamente menor no GO quando comparado ao GC (p < 0,001). Também foram observados níveis significativamente elevados de grupos sulfidrílicos totais no plasma dos indivíduos suplementados quando comparados aos do GC (p < 0,001). Além disso, foram observados níveis significativamente maiores de polifenóis (p < 0,05) tanto no plasma quanto no líquido sinovial dos indivíduos que receberam ômega-3 no período pós-cirúrgico comparado ao pré-cirúrgico. Entretanto, não foi observado um efeito protetor da administração do ômega-3 sobre a função anti-inflamatória. Conclusão: Verificamos um efeito protetor do ômega-3 na modulação dos marcadores de estresse oxidativo em indivíduos submetidos à reconstrução do LCA.

Introduction: The injuries of the anterior cruciate ligament (ACL) contribute to the formation of oxygen free radicals, which in excess can trigger oxidative damage in the knee joint. Objective: To evaluate the effects of oral supplementation with omega-3 on markers of oxidative stress in individuals undergoing ACL reconstruction. Methods: This study is a prospective, randomized, controlled, and single blinded clinical trial, with a sample of 25 patients who underwent ACL reconstruction, randomly assigned to: Omega-3 group (OG), supplemented daily with 2 g of omega-3 for 15 days after ACL surgery; and Control group (CG), without supplementation. Blood and synovial fluid collection was performed immediately before the surgical procedure and 15 days after ACL reconstruction. The biochemical analyses assessed the levels of lipid peroxidation products (MDA); catalase activity; total sulfhydryl groups and polyphenols and C-reactive protein (CRP). Results: We verified a significant decrease in the levels of MDA in the OG compared to the CG (p<0.05) and, similarly, that the catalase antioxidant enzyme activity was significantly lower in the OG when compared to the CG (p<0.001). We also observed significantly elevated levels of total sulfhydryl groups in plasma in supplemented individuals when compared to the CG (p<0.001). In addition, significantly higher levels of polyphenols (p<0.05) were observed in both plasma and synovial fluid of individuals who received omega-3 in the post-surgical period compared to pre-surgical. However, no protective effect was observed with the administration of omega-3 on the anti-inflammatory function. Conclusion: The findings suggest that there is a protective effect of omega-3 on the modulation of oxidative stress markers in individuals undergoing ACL reconstruction.

Introducción: Las lesiones del ligamento cruzado anterior (LCA) contribuyen a la formación de radicales libres de oxígeno, que en exceso pueden desencadenar daño oxidativo en la articulación de la rodilla. Objetivo: Evaluar los efectos de la suplementación oral con ácidos grasos omega-3 en los marcadores de estrés oxidativo en pacientes sometidos a reconstrucción del LCA. Métodos: Este estudio es un ensayo clínico prospectivo, aleatorizado, controlado, simple ciego, con una muestra de 25 pacientes que se sometieron a la reconstrucción del LCA, divididos aleatoriamente en: grupo omega-3 (GO), suplementado diariamente con 2 g de omega-3 por 15 días después de la reconstrucción del LCA y grupo control (GC), sin suplementos. Se hizo la recogida de sangre y de líquido sinovial inmediatamente antes de la cirugía y 15 días después de la reconstrucción del LCA. El análisis bioquímico evaluó los niveles de productos de la peroxidación lipídica (MDA); la actividad catalasa; polifenoles y el total de grupos sulfhidrilo y la proteína C reactiva (PCR). Resultados: Se observó una disminución significativa en los niveles de MDA en GO en comparación con el GC (p < 0,05) de la misma manera que la actividad de la enzima antioxidante catalasa fue significativamente menor en GO en comparación con el CG (p < 0,001). También se observaron niveles significativamente elevados de grupos sulfhidrilo totales en plasma de individuos suplementados en comparación con el GC (p < 0,001). Además, se observaron niveles significativamente más altos de polifenoles (p < 0,05) en el plasma y en el líquido sinovial de pacientes que recibieron ácidos grasos omega-3 en el postoperatorio en comparación con el preoperatorio. Sin embargo, no se observó un efecto protector con la administración de omega-3 en la función anti-inflamatoria. Conclusión: Se encontró un efecto protector del omega-3 en la modulación de marcadores de estrés oxidativo en pacientes sometidos a la reconstrucción del LCA.

Quercitrin, a glycoside form of quercetin, prevents lipid peroxidation in vitro.

Reactive oxygen species have been demonstrated to be associated with a variety of diseases including neurodegenerative disorders. Flavonoid compounds have been investigated for their protective action against oxidative mechanisms in different in vivo and in vitro models, which seems to be linked to their antioxidant properties. In the present study, we examine the protective mechanism of quercitrin, a glycoside form of quercetin, against the production of TBARS induced by different agents. TBARS production was stimulated by the incubation of rat brain homogenate with Fe2+, Fe2+ plus EDTA, quinolinic acid (QA), sodium nitroprusside (SNP) and potassium ferricyanide ([Fe(CN)6]3-). Quercitrin was able to prevent the formation of TBARS induced by pro-oxidant agents tested; however, it was more effective against potassium ferricyanide ([Fe(CN)6]3-, IC50=2.5), than quinolinic acid (QA, IC50=6 microg/ml) and sodium nitroprusside (SNP, IC50=5.88 microg/ml) than Fe2+ (Fe2+, IC50=14.81 microg/ml), Fe2+ plus EDTA (Fe2+ plus EDTA, IC50=48.15 microg/ml). The effect of quercitrin on the Fenton reaction was also investigated (deoxyribose degradation). Quercitrin caused a significant decrease in deoxyribose degradation that was not dependent on the concentration. Taken together, the data presented here indicate that quercitrin exhibits a scavenger and antioxidant role, and these effects probably are mediated via different mechanisms, which may involve the negative modulation of the Fenton reaction and NMDA receptor.