Neuroprotective Effect of Isobenzofuranones on Hydrogen Peroxide-Mediated Redox Imbalance in Primary Cultures of Hippocampal Neurons

Abstract In live organisms, there is a balance between the production of reactive oxygen species (ROS) and their neutralization. The increased level of these species leads to a condition called redox imbalance. The aim of this study was to evaluate the protective action of isobenzofuranones in primary cultures of hippocampal neurons subjected to redox imbalance. To accomplish this, MTT and LIVE/DEAD assays were initially performed. In the cultures pretreated with isobenzofuranones 1 and 2, there was a higher number of live cells when compared to that in the untreated ones. Regarding redox imbalance, there was a significant increase in the intracellular levels of ROS. The cultures pretreated with isobenzofuranones showed a reduction in ROS levels. Lipid peroxidation caused by oxidative damage was significantly reduced in the cultures pretreated with isobenzofuranones 1 and 2. Taken together, these data show the ability of isobenzofuranones 1 and 2 to significantly minimize cytotoxicity, cell death, intracellular levels of ROS and lipid peroxidation induced by redox imbalance. These results suggest that isobenzofuranones 1 and 2 represent a possible alternative therapy for the neurodegenerative disturbances that are triggered by ROS production increases.

Aqueous extract of Baccharis trimera improves redox status and decreases the severity of alcoholic hepatotoxicity

ABSTRACT The metabolism of ethanol occurs mainly in the liver, promoting increase of reactive oxygen species and nitrogen, leading to redox imbalance. Therefore, antioxidants can be seen as an alternative to reestablish the oxidizing/reducing equilibrium. The aim of this study was to evaluate the antioxidant and hepatoprotective effect of aqueous extract of Baccharis trimera (Less.) DC., Asteraceae, in a model of hepatotoxicity induced by ethanol. The extract was characterized and in vitro tests were conducted in HepG2 cells. It was evaluated the cells viability exposed to aqueous extract for 24 h, ability to scavenging the radical DPPH, besides the production of reactive oxygen species and nitric oxide, and the influence on the transcriptional activity of transcription factor Nrf2 (12 and 24 h) after exposure to 200 mM ethanol. The results showed that aqueous extract was non-cytotoxic in any concentration tested; moreover, it was observed a decrease in ROS and NO production, also promoting the transcriptional activity of Nrf2. In vivo, we pretreatment male rats Fisher with 600 mg/kg of aqueous extract and 1 h later 5 ml/kg of absolute ethanol was administrated. After two days of treatment, the animals were euthanized and lipid profile, hepatic and renal functions, antioxidant status and oxidative damage were evaluated. The treatment with extract improved liver function and lipid profile, reflecting the reduction of lipid microvesicules in the liver. It also promoted an increase of glutathione peroxidase activity, decrease of oxidative damage and MMP-2 activity. These results, analyzed together, suggest the hepatoprotective effect of B. trimera aqueous extract.

Novo papel da proteína XPC na regulação dos complexos da cadeia de transporte de elétrons e desequilíbrio redox / New role of XPC protein in regulating the electron transport chain complexes and redox unbalance

Espécies reativas de oxigênio (EROs) são normalmente e continuamente geradas em mitocôndrias, majoritariamente na cadeia de transporte de elétrons (CTE). Harman (1956, 1972 e 1992) teorizou que os radicais livres gerados nas mitocôndrias seriam a principal causa do envelhecimento. De fato, durante o envelhecimento é observado um desequilíbrio entre formação e remoção de EROs, que resulta em estresse redox. Essa condição favorece a formação de lesões oxidadas no DNA, acarretando em mutagênese ou morte celular. Diversos mecanismos moleculares cooperam para o reparo de DNA. Duas vias de reparo de DNA lidam com a maioria das lesões: o reparo por excisão de base (BER) e o reparo por excisão de nucleotídeos (NER). A via BER corrige pequenas modificações de bases que surgem de reações de desaminação, alquilação e oxidação. A via NER é mais versátil, reconhecendo lesões que distorcem a dupla hélice de DNA, como danos induzidos por luz UV e adutos volumos. Pacientes xeroderma pigmentoso (XP-A a XP-G) herdam mutações em um de sete genes que codificam proteínas envolvidas na via NER, ou em um gene que codifica uma polimerase translesão (XP-V). A doença é caracterizada por fotosensibilidade e incidência elevada de neoplasias cutâneas. A proteína XPC atua na etapa de reconhecimento da lesão de DNA na subvia de reparo global do genoma (GG-NER), e sua mutação dá origem aos sintomas clássicos de XP. Novas funções de XPC foram recentemente descritas: i) atuando como cofator na via BER auxiliando as DNA glicosilases OGG1, TDG e SMUG; ii) atuando como cofator transcricional de elementos responsivos a Oct4/Sox2, RXR e PPARα; e iii) na adaptação metabólica na transformação de queratinócitos. Então, propusemo-nos a investigar as relações entre XPC e a manutenção da integridade do DNA mitocondrial, a sensibilidade celular a estresse redox mitocondrial e possíveis alterações bioenergéticas e redox. Para tal, padronizamos um ensaio in vitro de cinética de incisão em DNA plasmidial a fim de investigarmos o possível papel de XPC no reparo de lesões oxidadas em mtDNA. Porém, nossos dados revelaram que XPC não se encontra em mitocôndrias. Apesar disso, células XP-C são mais sensíveis ao tratamento com azul de metileno (AM), antimicina A (AA) e rotenona (ROT), que geram estresse redox mitocondrial. A sensibilidade à AA foi completamente revertida em células corrigidas. Células XP-C apresentaram alterações quanto ao uso dos complexos mitocondriais, com diminuição da taxa de consumo de oxigênio (OCR) via complexo I e um aumento da OCR via complexo II, dependente da presença de XPC. Ademais, a linhagem XP-C apresentou um desequilíbrio redox mitocondrial com maior produção de EROs e menor atividade de GPx. O DNA mitocondrial de células XP-C apresentou níveis elevados de lesão e deleção, que no entanto não retornaram aos níveis encontrados em células selvagens na linhagem XP-C corrigida. Observamos uma acentuada diminuição da expressão de PPARGC1A, um importante regulador de biogênese mitocondrial. Contudo, não foi possível determinar o mecanismo de supressão da expressão de PPARGC1A. Por fim, identificamos que o tipo de mutação em XPC pode estar associado a expressão de PPARGC1A. Esse estudo abre novas possibilidade na investigação do papel de proteína XPC, à parte da instabilidade genômica, na adaptação metabólica e desequilíbrio redox em direção da progressão tumoral

Mitochondria continuously produce reactive oxygen species (ROS), mainly at the electron transport chain. Harman (1956, 1972 e 1992) proposed that normal aging is driven by increased mitochondrially generated free radicals. Indeed, during the course of aging there is an increased imbalance between formation and removal of ROS, leading to redox stress. This condition favours the formation of oxidized DNA lesions, given rise to mutations and cell death. Several molecular mechanisms cooperates to repair the DNA. Two DNA repair pathways deal with the majority of lesions: base excision repair (BER) and nucleotide excision repair (NER). The BER pathway corrects small base modifications that arise from deamination, alkylation and oxidation reactions. The NER pathway is more versitile, recognizing helix-distorting lesions, such as UV-induced damage and bulky adducts. Xeroderma pigmentosum (XP-A to XP-G) patients inherit mutations in one of seven protein-coding genes involved in NER pathway, or in a gene coding a translesion DNA polymerase (XP-V). Photosensitivity and a thousand-fold increased in the risk of developing cutaneous neoplasms are the main clinical features of XP. XPC protein functions in the recognition step of global genome NER (GG-NER) sub-pathway, and mutations in this gene lead to classical XP symptoms. Recently, it has been described that XPC acts: i) as a cofactor in BER pathway through functional interaction with DNA glycosylases OGG1, TDG and SMUG1; ii) as coactivator in transcription at Oct4/Sox2, RXR and PPARα responsive elements; iii) in metabolic shift during keratinocytes transformation. Thus, we sought to investigate a possible role for XPC in the maintenance of mtDNA integrity, cellular sensitivity to mitochondrial redox stress and eventual bioenergetic and redox changes. For this purpose, we established an in vitro plasmid incision assay to investigate the possible role of XPC in the repair of oxidized lesions in mitochondrial DNA. However, our data revealed that XPC did not localized in mitochondria. Nonetheless, XP-C cells are more sensitive to methylene blue, antimycin A (AA) and rotenone treatment, which induce mitochondrial redox stress. The XP-C sensitivity to AA was completely reverted in XPC-corrected cells. XP-C cells presented altered usage of mitochondrial complexes, with decreased oxygen consumption rate (OCR) via complex I and increased OCR through complex II, an XPC-dependent phenomenon. Furthermore, the XP-C cell line showed mitochondrial redox imbalance with increased ROS production and decrease GPx activity. MtDNA from XP-C cells accumulate lesions and deletions, which, however, were found at similar levels in the corrected cell line. We identified a sharp decrease in the expression of PPARGC1A, a master regulator of mitochondrial biogenesis. Nevertheless, it was not possible to determine the mechanism of suppression of PPARGC1A expression. Finally, our results suggest a possible link between the type of XPC mutation and PPARGC1A expression. This study unfolds new possible roles for XPC, aside from its established roles in genomic instability, in metabolic adaptation and redox imbalance towards tumour progression

Phenolic extract of Parkia biglobosa fruit pulp stalls aflatoxin B1 – mediated oxidative rout in the liver of male rats

The effect of phenolic extract of Parkia biglobosa (Jacq.) R. Br. ex G. Don, Fabaceae, pulp on aflatoxin B1 induced oxidative imbalance in rat liver was evaluated. Thirty-five male rats were randomized into seven groups of five animals each. Rats in group A served as control and received vehicle for drug administration (0.5% DMSO) once daily at 24 h intervals for six weeks. Rats in groups B, D, E, F and G, received aflatoxin B1 (167 μg/kg body weight) in 0.5% DMSO for three weeks, starting from the third week of the experimental period. Rats in Group C received 400 mg/kg bodyweight of the extract for six weeks, while groups D, E and F rats were treated with 100, 200 and 400 mg/kg bodyweight of the extract for six weeks respectively. Group G rats received 100 mg/kg body weight of vitamin C. Aflatoxin B1-mediated decrease in the activities of superoxide dismutase, catalase, glutathione peroxidase, glutathione reductase and glucose-6-phosphate dehydrogenase were significantly attenuated. Aflatoxin B1 mediated the elevation in malondialdehyde, conjugated dienes, lipid hydroperoxides, protein carbonyl, and significantly lowered DNA fragmentation percentage. Overall, the phenolic extract of P. biglobosa pulp stalls aflatoxin B1-mediated oxidative rout by enhancing antioxidant enzyme activities leading to decreased lipid peroxidation, protein oxidation and DNA fragmentation.

Association of insulin resistance with redox imbalance in patients with non diabetic hypertension.

Oxidative stress involved in the pathogenesis of several diseases. Hypertension itself acts as source of oxidative stress. Insulin resistance is involved in the pathogenesis of essential hypertension. In the present study an attempt was made to study the association between insulin resistance and oxidative stress in non diabetic hypertensive patients. Two hundred and three, non diabetic hypertensive patients and two hundred and ten, healthy normotensive subjects were enrolled in this study. Malondialdehyde, reduced glutathione, glutathione peroxidase, Fasting insulin and HOMA-IR levels were estimated in both groups. Fasting insulin, reduced glutathione, glutathione peroxidase and MDA shows significant difference between cases and controls. Among the patients HOMA-IR were significantly correlated with lipid peroxides and shows negative correlation between HOMAIR and glutathione peroxidase. Increased HOMA-IR was found in non diabetic hypertensive patients. This study reveals the link between oxidative stress and insulin resistance.

Idiopathic environmental intolerances (IEI): From molecular epidemiology to molecular medicine.

Inherited or acquired impairment of xenobiotics metabolism is a postulated mechanism underlying environment-associated pathologies such as multiple chemical sensitivity, fibromyalgia, chronic fatigue syndrome, dental amalgam disease, and others, also collectively named idiopathic environmental intolerances (IEI). In view of the poor current knowledge of their etiology and pathogenesis, and the absence of recognised genetic and metabolic markers of the diseases. They are often considered “medically unexplained syndromes”,. These disabling conditions share the features of poly-symptomatic multi-organ syndromes, considered by part of the medical community to be aberrant responses triggered by exposure to low-dose organic and inorganic chemicals and metals, in concentrations far below average reference levels admitted for environmental toxicants. A genetic predisposition to altered biotransformation of environmental chemicals, drugs, and metals, and of endogenous low-molecular weight metabolites, caused by polymorphisms of genes coding for xenobiotic metabolizing enzymes, their receptors and transcription factors appears to be involved in the susceptibility to these environment-associated pathologies, along with epigenetic factors. Free radical/antioxidant homeostasis may also be heavily implicated, indirectly by affecting the regulation of xenobiotic metabolizing enzymes, and directly by causing increased levels of oxidative products, implicated in the chronic damage of cells and tissues, which is in part correlated with clinical symptoms. More systematic studies of molecular epidemiology, toxico- and pharmaco-genomics, elucidating the mechanisms of regulation, expression, induction, and activity of antioxidant/detoxifying enzymes, and the possible role of inflammatory mediators, promise a better understanding of this pathologically increased sensitivity to low-level chemical stimuli, and a solid basis for effective individualized antioxidant- and/or chelator-based treatments.

Estresse oxidativo em porfiria hepâtica experimental disparadapor succinilacetona - urn inibidor da âcido 5-aminolevulinico desidratase / Oxidative stress in experimental hepatic porphyria triggered bysuccinylacetone — an inhibitor of 5-aminoluvulinic acid dehydratase

Para otimizar urn modelo experimental para o estudo do desbalanço redox em porfirias relacionadas ao acúmulo de ácido 5-aminolevulinico-(ALA), via inibição da ALA desidratase-(ALA-D), ratos foram tratados com o éster metílico de succinilacetona-(SAME), um catabólito da tirosina que inibe fortemente a ALA-D, mimetizando o estado metabólico observado nos portadores de porfirias e tirosinemias. Estabeleceram-se modelos de tratamento agudo por 36 e 18 h. No primeiro, os animais receberam 3 injeções de SAME (10, 40 ou 80 mg/kg, grupos All-IV). No segundo, os animais receberam 3 injeções de 40 mg/kg de SAME, ALA ou éster metílico de ALA (grupos BII-IV), ALA:SAME (30:10 mg/kg, grupo BV), ou 10 mg/kg SAME (grupo BVI). Paralelamente, avaliou-se se os sintomas neurológicos característicos das porfirias decorriam de danos oxidativos mitocondriais. Para isso, aplicou-se uma tecnologia óptica para medidas da difusão da depressão cortical que determinou a oxigenação e o estado redox do cit c em mitocôndrias do córtex cerebral de ratos submetidos ao tratamento crônico com ALA (40 mg/kg), SAME (10 e 40 mg/kg) e ALA:SAME (30:10 mg/kg), a cada 48 h, durante 30 dias. Tratamento agudo/36 h: Os níveis de ALA no plasma, fígado, cérebro e urina e o clearance renal do ALA aumentaram nos grupos tratados. A atividade de ALA-D e a coproporfirina urinaria reduziram. A marcação para proteínas carboniladas, ferro e ferritina aumentou no fígado e cérebro dos grupos tratados, especialmente no All. Os níveis de malondialdeído hepática aumentaram no grupo AIV. A razão GSH/GSH+GSSG e a atividade de GPx cerebrais aumentaram nos grupos AIV e AIII, respectivamente. Consistentemente com estes dados indicando um desbalanço oxidativo induzido pelo SAME, alterações mitocondriais e citosólicas ultraestruturais foram reveladas, especialmente no fígado./Tratamento agudo/18 h: Os níveis de ALA plasmáticos aumentaram nos grupos tratados, exceto em BIV. 0 grupo Bll mostrou aumento dos níveis hepáticos...

To optimize an experimental model for studying redox imbalance in porphyrias related to 5-aminolevulinic acid (ALA) accumulation through the inhibition of ALA dehydratase (ALA-D), rats were treated with methyl ester of succinylacetone (SAME), a tyrosine catabolite that strongly inhibits ALA-D, what mimics the metabolic state observed in patients suffering from porphyrias and tyrosinemias. Models of acute treatment were established during 36 and 18 h. In the first model, animals received 3 injections of SAME (10, 40 or 80 mg/kg, groups All-IV). In the second model, animals received 3 injections of 40 mg/kg SAME, ALA or methyl ester of ALA (groups BII-IV), ALA:SAME (30:10 mg/kg, group BV), or 10 mg/kg SAME (group BVI). Concomitantly, we evaluated if the neurologic symptoms characteristics of porphyrias were a consequence of the oxidative mitochondria! impairment. For this, an optical technology for the measurement of cortical spreading depression was applied. This techonology determined the cerebral oxygenation and the redox state of cit c in mitochondria of the cerebral cortex of rats submitted to a chronic treatment with ALA (40 mg/kg), SAME (10 and 40 mg/kg) and ALA:SAME (30:10 mg/kg), alternate days, during 30 days. Acute treatment/36 h: ALA levels in plasma, liver and urine and clearance of renal ALA increased in treated groups. ALA-D activities and urinary coproporphyrin were found to be decreased. Liver and brain proteins carbonyl, iron and ferritin were higher in the liver of treated groups, especially in All. Liver j malondialdehyde levels were higher in group AIV. Cerebral GSH/GSH+GSSG ratio and GPx activities increased in groups AIV and AIII, respectively. Consistently with these data indicating SAME-induced oxidative imbalance, mitochondrial and cytosolic ultrastructural changes were revealed, especially in the liver. Acute treatment/18 h: Plasma ALA levels increased in all treated groups but BIV. Group BII showed increased hepatic ALA levels…