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Desenvolvimento vegetativo e parâmetros fisiológicos em genótipos de amendoim dob déficit hídrico e inoculados com rizóbiosBarbosa, Daniela Duarte 10 March 2016 (has links)
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Previous issue date: 2016-03-10 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / Peanut (Arachis hypogaea L.) is a herbaceous oilseed original in Latin America and cultivated in many regions of Brazil due to great capacity for environmental adaptation. But some biotic and abiotic environmental factors such as water stress, affect the peanut production capacity, however negative economic impacts that vary depending on the duration of the drought and the phenological phase of the plants. In this context aimed to evaluate the interaction of nitrogen-fixing microorganisms, as promoters of tolerance to drought in three peanut genotypes, assessing physiological and vegetative growth parameters. We used three genotypes of peanut and two cultivars (BRS Havana and CNPA 76 AM) and an advanced line (2012-4), the treatments were formed from the inoculation of three strains of rhizobia (SEMIA 6144, 123-10A and SEMIA 4077) , a nitrogenous chemical source (ammonium sulfate) and without nitrogen witness and two water regimes. The experimental design was completely randomized with factorial 3 x 5 x 2 with 6 repetitions. Data were analyzed using the statistical program SISVAR version 5.6, which were submitted to variance analysis by T test and comparison of means by Tukey test at 5% significance. The variables of gas exchange were affected by drought and decreased the plant height, shoot dry weight, dry weight of nodules and number of nodes. The drought also reduced gas exchange in peanut genotypes, however, the rhizobia mitigated the effects of drought. There was an increase in proline content in plants under water stress and antioxidant enzymes acted in accordance with the needs of the plant defense mechanisms. The genotypes studied were tolerant to drought in the presence of strains 123-10A and SEMIA 6144, and 123-10A was more responsive in most of the variables analyzed. / O amendoim (Arachis hypogaea L.) é uma oleaginosa herbácea originária da América Latina e cultivada em várias regiões do Brasil, por sua grande capacidade de adaptação ambiental. Porém, alguns fatores ambientais bióticos e abióticos, como o estresse hídrico, afetam a capacidade de produção do amendoim, desencadeando impactos econômicos negativos, que variam dependendo da duração da estiagem e da fase fenológica das plantas. Nesse contexto, objetivou-se avaliar a interação de microrganismos fixadores de nitrogênio, como promotores de tolerância ao déficit hídrico em três genótipos de amendoim, avaliando-se parâmetros fisiológicos e de crescimento vegetativo. Foram usados três genótipos de amendoim, sendo duas cultivares (BRS Havana e CNPA 76 AM) e uma linhagem avançada (2012-4), os tratamentos foram formados a partir da inoculação de três isolados de rizóbio (SEMIA 6144, 123-10A e SEMIA 4077), uma fonte química nitrogenada (sulfato de amônia) e a testemunha sem nitrogênio e dois regimes hídricos. O delineamento experimental foi inteiramente casualizado com fatorial de 3 x 5 x 2 com 6 repetições. Os dados obtidos foram analisados por meio do Programa estatístico SISVAR versão 5.6, os quais foram submetidos à análise de variância pelo teste T e a comparação de médias pelo de Teste de Tukey a 5% de significância. As variáveis de trocas gasosas foram afetadas pelo déficit hídrico e houve redução da altura de planta, massa seca da parte aérea, peso seco de nódulos e número de nódulos. O déficit hídrico reduziu também as trocas gasosas nos genótipos de amendoim, contudo, os rizóbios mitigaram os efeitos do déficit hídrico. Houve aumento no teor de prolina nas plantas sob restrição hídrica e as enzimas antioxidantes atuaram de acordo com as necessidades dos mecanismos de defesa das plantas. Os genótipos estudados foram tolerantes ao déficit hídrico na presença das estirpes 123-10A e SEMIA 6144, sendo que 123-10A foi mais a responsiva na maioria das variáveis analisadas.
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Caracteriza??o morfol?gica, perfil prote?mico, status redox e express?o de enzimas antioxidantes em isolados de Trypanosoma cruzi (Z3), provenientes do Estado do Rio de Janeiro, Brasil / Morphological characterization, proteomic profile, redox status and expression of antioxidante enzymes in Trypanosoma cruzi isolates from the state of the Rio de Janeiro, BrazilSILVA, Cristina Santos da 31 March 2016 (has links)
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Previous issue date: 2016-03-31 / CAPES / Chagas? Disease or American trypanosomiasis is an important parasitic disease in the Americas and is still considered one of the major neglected tropical diseases affecting millions of people worldwide due to lack of effective control. Thus, it has a significant impact on human health. This disease has its epidemiology conditioned by triatomines and mammals and its etiological agent is the protozoan Trypanosoma cruzi.Investigations on the adaptation mechanisms, gene regulation and parasite interaction vs. vector evolved, which proves the need for the use of molecular tools for the study and elucidation on adaptive changes in these parasites. Recently, it was found evidence where studies indicate that the expression of T. cruzi?s antioxidant enzymes such as cytosolic peroxiredoxin (TcCPx), mitochondrial peroxiredoxin (TcMPx), (TcAPX) and trypanothione synthetase (TXNI, TXNII), superoxide dismutase (SOD A and SOD B) can be part of parasite?s protective system and indicate factors related to different levels of virulence of the etiological agent.Thus, proteomics analysis and evaluation of the expression of antioxidant enzymes from different subcellular compartments can corroborate to the studies related to virulence indices and expression of proteins involved in this process. In this sense, the objectives of this study were to evaluate the morphology of the isolated samples SMM98, SMM36 and SMM1, analyze beyond the proteomic profile, the expression of antioxidant enzymes (TcCPx; TcMPx; TcAPx; TcTXNI; TcTXNII, SOD A and SOD B) and observe the redox status expression using isolates SMM36 and SMM98 in comparison with strains TCC, Silvio and DM28c. The results showed different morphology from the isolated, high levels of virulence, varied protein profile comparison between SMM98 isolated and SMM36, when treated with NAC and Heme changes were observed in the development of the parasites, indicating the participation of the Redox Status of the Rio de Janeiro used in this study. / A doen?a de Chagas ou tripanossom?ase americana ? uma importante doen?a parasit?ria nas Am?ricas e ainda hoje ? considerada uma das principais doen?as tropicais negligenciadas acometendo milh?es de pessoas no mundo devido ? falta de controle efetivo. Desta forma, apresenta um impacto significativo sobre a sa?de humana. Esta enfermidade tem sua epidemiologia condicionada pelos triatom?neos e os mam?feros e tem como agente etiol?gico o protozo?rio Trypanosoma cruzi.Investiga??es sobre os mecanismos de adapta??o, regula??o g?nica e intera??o parasito x vetor evolu?ram, o que comprova a necessidade da utiliza??o de ferramentas moleculares para o estudo e elucida??es sobre as mudan?as adaptativas ocorridas nestes parasitos. Recentemente evid?ncias surgiram neste sentido, onde estudos indicam que a express?o de enzimas antioxidantes do T. cruzi tais como: peroxirredoxinas citos?lica (TcCPx), mitocondriais (TcMPx), (TcAPX) e tripanotiona sintetase (TXNI, TXNII), super?xido dismutase (SOD A e SOD B) podem fazer parte do sistema protetivo do parasito e indicar fatores relacionados aos diferentes n?veis de virul?ncia deste agente etiol?gico. Desta forma, an?lises prote?mica e avalia??o da express?o de enzimas antioxidantes de diferentes compartimentos subcelulares podem corroborar para com os estudos relacionados aos ?ndices de virul?ncia e express?o de prote?nas envolvidas neste processo. Neste sentido, os objetivos deste trabalho foram avaliar a morfologia dos isolados das amostras SMM98, 36 e 1, analisar al?m do perfil prote?mico, a express?o de enzimas antioxidantes (TcCPx; TcMPx; TcAPx; TcTXNI; TcTXNII, SOD A e SOD B) e observar a express?o do status redox utilizando os isolados SMM36 e SMM98 em compara??o com as cepas TCC, Silvio e DM28c. Os resultados obtidos demonstraram aspectos morfol?gicos diferenciados dentre os isolados, n?veis de virul?ncia elevados, perfil de prote?nas variados quando comparados entre os isolados SMM98 e SMM36. E, quando tratados com Heme e NAC foram observadas altera??es no desenvolvimento dos parasitos, denotando a participa??o do Status Redox frente aos isolados do Rio de Janeiro utilizados neste estudo.
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Effects of the Cyanobacterium Nodularia spumigena on Selected Estuarine FaunaDavies, Warren Raymond, warren.davies@optusnet.com.au January 2007 (has links)
Nodularia spumigena is an estuarine cyanobacteria that produces the toxin nodularin. This toxic cyanobacteria is known to have caused death to domestic and wild animals and is recognised as dangerous to human health. N. spumigena causes harmful algal blooms in many parts of the world including Australia. The toxic solutes of N. spumigena are potentially dangerous when contact is made to contaminated water bodies or is ingested by primary consumers. In Australia blooms of N. spumigena are common in the Gippsland Lakes in South-eastern Victoria and cause socio - economic hardships to the local communities. This PhD investigates the toxic effects of N. spumigena and its solutes to a range of aquatic life. A method known as SPME - HPLC showed promise in environmental monitoring of N. spumigena toxins by measuring nodularin from water samples. Other research presented study into the lethal and sublethal effects of on an extract from N. spumigena to aquatic fauna. Resu lts showed the N. spumigena extract was not lethal to many aquatic fauna although zooplankton from the Gippsland Lakes showed mortality at environmental relevant levels. Biochemical studies focusing on animal detoxification and antioxidation enzymes and DNA integrity showed sublethal effects to the N. spumigena extract. Results presented in this thesis show that an extract of N. spumigena elicited detoxification and antioxidation responses in animals tested. Furthermore, the use of the COMET assay showed increased damage to DNA of animals tested. Results also showed that different organs in animals tested responded differently to the aqueous extract, suggesting mode of uptake maybe important in toxicosis. Further, feeding studies with N. spumigena help elucidate mode of uptake using enzyme response biomarkers. The overall results of this research provided an assessment of the toxic affects of N. spumigena on aquatic fauna with special reference to the Gippsland Lakes, Victoria, Australia.
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Effects Of Acrylamide And Resveratrol On Rabbit Liver And Kidney Antioxidant EnzymesKalin, Cigdem 01 January 2010 (has links) (PDF)
Resveratrol is one of the promising naturally occurring polyphenolic compound found in red wine having antioxidant and anti-carcinogenic properties. However, in vivo studies investigating the effects of resveratrol on antioxidant enzymes are limited. In the present study, we investigated, for the first time, the influence of resveratrol on liver and kidney antioxidant enzymes and oxidative stress markers in acrylamide treated and control rabbits.
New Zealand male rabbits were treated with acrylamide and resveratrol, separately in two different doses and conditions. Their combined effects were also investigated. While, acrylamide treatment significantly decreased the glutathione peroxidase (GPx) activity in liver (1.24-fold), it was significantly increased (1.20 &ndash / 1.40-fold) by combined effect of resveratrol and acrylamide in liver and kidney. Furthermore, alone resveratrol administration increased (~1.37 &ndash / fold) GPx activity in kidney. Although, glutathione reductase (GR) was found to be significantly increased (~1.30-fold) in two different dose of resveratrol treated rabbit liver, it was not changed in acrylamide and their combined treatments. Despite, glutathione (GSH) content was decreased around 1.6 fold as a result of acrylamide treatment in rabbit liver and kidney cytosols, GSH level was returned to normal levels by resveratrol tretment in rabbit liver and kidney. Furthermore, acrylamide treatment significantly increased the SDH activity in blood serum (1.68-fold) and in liver (1.27-fold) with respect to control. On the other hand, resveratrol treatment brought this activity nearly normal level in acrylamide treated rabbits.. Besides, sorbitol deydrogenase (SDH) was found to be decreased (3.13-fold) significantly in rabbit liver cytosol as a result of single dose of 100 mg/kg b.w. resveratrol treatment. Moreover, catalase activity and MDA level were not affected from either resveratrol or acrylamide and with their combination effect in investigated rabbit organs.
An important liver damage marker enzyme other than ALT and AST, SDH was characterized in terms of substrate, cofactor and enzyme concentration in rabbits which have been not investigated before and found to be 200 mM, 141 µ / M and 0.5 µ / L, respectively in rabbit liver. Furthermore, the Km value was first calculated in liver of New Zealand rabbits as 55,5 mM.
In addition to these, in vitro effects of resveratrol on GST activity was also studied throughout this study. Resveratrol was shown to be a noncompetitive inhibitor for liver cytosolic GST against substrate CDNB with Ki of 175 µ / M. On the other hand, resveratrol was shown to be a competitive inhibitor for liver cytosolic GST against substrate GSH with Ki of 55 µ / M.
The results of the present study have demonstrated for the first time that resveratrol induced some of the antioxidant enzyme activities and as well nonenzymatic antioxidants in rabbit liver and kidney. The results of GPx, GR, SDH activities and GSH level have also suggested that resveratrol may have protective effects on acrylamide induced hepatoxicity and renal toxicity. Therefore, it may be a therapeutic approach for the oxidative stress-related diseases such as cancer. However, further in vivo studies are required to clarify the effect of resveratrol on both acrylamide-induced toxicity and bioavailability in the body.
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The Effects Of Antioxidants On Some Rat Tissues And MembranesGorgulu, Guvenc 01 April 2004 (has links) (PDF)
High blood glucose levels induce metabolic disorders that initiate a sequence of events including renal, arterial, cardiac and retinal disorders. Diabetes mellitus increases oxidative stress in tissues of animals including humans. The resulting oxidative stress might play role in the development of diabetic complications.
In the present study, 36 male Wistar rats (250-300g) were divided into 5 groups as Control (n=6), Diabetic (n=7), Diabetic + Vit C (n=7), Diabetic + & / #945 / -Lipoic acid (n=6) and Diabetic + Combination of Vit C and & / #945 / -Lipoic acid (n=10).
From the livers of all groups cytoplasmic and microsomal membrane fractions were prepared from liver and antioxidant enzymes namely, superoxide dismutase, glutathione peroxidase, catalase and glutathione S-transferase activities were measured. Microsomal lipid peroxidation, total lipid, total protein, reduced glutathione levels of each group was determined and compared. Microsomal fractions were also analyzed by FT-IR spectroscopy.
The total protein levels of diabetic rats were found to be decreased significantly (p< / 0.05) compared to controls and the & / #945 / -lipoic acid and vitamin C supplemented groups tend to compensate the decreased levels of total proteins. Decreased catalase activity in diabetic group compared to control was restored by & / #945 / -lipoic acid, vitamin C treatment and/or combination of both. Increased glutathione peroxidase activity was decreased to control levels by the treatement of both & / #945 / -lipoic acid and vitamin C. Superoxide dismutase activities of diabetic rats were increased (p< / 0.05) compared to control group. Whereas glutathione S-transferase activities though showed some fluctuations, the results were not statistically significant. Total glutathione levels decreased in all groups significantly (p< / 0.0.5) compared to control group but any of the agent failed to compensate the reduced levels of glutathione. As an index of lipid peroxidation, TBA-reactivity (MDA) levels increased significantly in all diabetic groups and only combination group&rsquo / s TBARS levels decreased significantly compared to diabetic group.
FT-IR study of rat liver microsomal membranes was carried out in order to understand the effects of diabetes on membrane order, dynamics and lipid peroxidation status.
For this purpose CH2 symmetric wavenumber, CH2 antisymmetric bandwidth, =CH olefinic band area were compared. In temperature dependent FT-IR studies microsomal membrane phase behavior, order and dynamics were analyzed. Diabetic samples showed apparent decrease in both frequency and bandwidth. =CH olefinic band integrated area was increased for diabetic samples compared to controls. Alpha-lipoic acid and vitamin C supplemented groups showed similar effects. They tend to restore decreased levels of band frequency and bandwidth. Additive effect between & / #945 / -lipoic acid and vitamin C was seen in some cases that only the combination group achieved to restore control values while & / #945 / -lipoic acid and vitamin C were failed to restore alone.
In conclusion, STZ-induced diabetes mainly caused an increase in antioxidant enzyme activities. Also, increase in lipid peroxidation caused a decrease in the fluidity and order of the membrane resulting in more rigid membrane structures. The loss of cooperation between the antioxidant network may play a role in the secondary complications of diabetes.
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ICP-MS determination of Zn, Cu, Fe and Mn in muscle cells as potential markers of oxidative stressFagieh, Taghreed M. January 2017 (has links)
Oxidative stress is imbalance between oxidant and antioxidant levels in living systems. Human cells are protected from reactive oxygen species by endogenous enzymatic antioxidants. Most of these compounds require particular redox metals in their structures as cofactors to allow them to scavenge the free radicals such as Cu, Zn-SOD, Mn-SOD and catalase (Fe). The aim of this study was to quantify these metals in human cells to evaluate their effectiveness as novel biomarkers for measuring oxidative stress. The metals (Zn, Cu, Fe, Mn) were measured in vitro in skeletal muscle cells (C2C12) which were incubated under hypoxia/hyperoxia conditions generated by varying oxygen level from 1%-60% for 24 and 48 hours. Two methods were used to perform the analysis. ICP-MS was applied to liquid samples to quantify Zn, Cu, Fe and Mn in cell populations. And LA-ICP-MS was employed to solid samples to measure their intensity in individual cells. The data acquired from both techniques are positively correlated confirming the reliability of the two approaches. All elements of interest were successfully measured except Mn which was not detected in single cells using LA-ICP-MS due to the limit of detection. Interestingly, the results showed that their concentration increased dramatically in cells grown at 25%-60% O2, the most significant increase was in Cu at 60%O2. None showed any increase at 5%-15% O2 indicating normoxia states. At 1%O2, all elements except Fe showed a significant increase and the most remarkable growth was in Mn. More interestingly, increasing incubation to 48 hours for liquid samples had differing effects on the elements. Zn and Cu concentrations were unaffected by increasing incubation time except at 60%O2 where they showed further growth. In contrast, Mn concentration grew sharply over oxygen levels of 30%-50% with no further effect at 1%, while Fe concentration decreased at 1%O2 and grew steadily over oxygen levels of 5%-60%. It can be concluded that all four elements were significantly affected by stress conditions applied to cells, but at different rates. Importantly, a novel analytical method was introduced in this current study since there have been no previous reported investigations measuring changes in concentration of redox-active elements in human cells subjected to different controlled oxidative stress conditions in vitro.
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The safety and toxicity of MPA-CdTe quantum dots in legume plantsOmar, Zaahira January 2017 (has links)
Magister Scientiae - MSc (Biotechnology) / The expansion of nanotechnology, resulting in multitudes of consumer and industrial
products, causes concern amongst the scientific community regarding the risks associated
with the release of nanomaterials into the environment and its subsequent effects on plants.
Therefore, the focus of this study was aimed at investigating the effects of MPA-capped CdTe
and carbon QDs on legumes plants namely P. vulgaris and G. max. Fluorescent imaging
revealed that QDs were translocated from the roots to the aerial parts of the plant and
accumulated in the edible parts of P. vulgaris. Subsequent physiological and biochemical
tests revealed that both QD types induced oxidative stress as biological markers for stress
including lipid peroxidation and cell death were elevated. In addition, carbon QDs displayed
lower toxicity in comparison to MPA-CdTe QDs, but still possessed the ability to induce
oxidative stress in plant cells. However, the effects were more pronounced in G. max in
comparison to P. vulgaris; and more so with MPA-CdTe QDs than carbon QDs. Furthermore,
MPA-CdTe and carbon QDs altered the concentrations and translocation of essential macro
and microelements that are required for plant growth and development. This may have
detrimental effects on crop productivity and yield, with negative implications on food quality
and food security. / 2021-08-31
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Vliv metribuzinu na oxidativní stres a antioxidační enzymy raka signálního / The effect of metribuzine on oxidative stress and antioxidant enzymes of signal crayfishLIDOVÁ, Jaroslava January 2015 (has links)
The aim of this study was to investigate effects of the triazine herbicide metribuzine on oxidative stress level and antioxidant enzymes activity in gills, muscle and hepatopancreas of signal crayfish (Pacifastacus leniusculus Dana) and also extension of knowledge about effect of metribuzine on the environment. The experiment took up 60 days. Crayfish were exposured to metribuzine concentrations of 0.52 micrograms.l-1 (real environmental concentration) and 3.06 mg.l-1 (10% 96hLC50) for the first 30 days. Then a second phase followed depuration without metribuzine (30 days). Changes in the oxidative stress level (TBARS), superoxiddismutase (SOD) activity and catalase (CAT) activity were observed in all examined tissues. Changes in glutathionreductase (GR) activity were observed only in hepatopancreas. Chronic exposure of metribuzine demonstrated an oxidative damage of cell lipids, proteins and also changes in antioxidant activity in examined crayfish tissues.The results of this study suggest that crayfish are a very suitable organisms for toxicological tests and simultaneously extend knowledge about effect of metribuzine on the environment.
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Vliv chronicky podávaných subletálních dávek parakvatu na délku telomer a rezistenci vůči oxidačnímu stresu drozofilyTOMÁŠKOVÁ, Jindřiška January 2016 (has links)
As the most widely dispersed fauna around the world, insects are exposed to a range of stresses within their environments. Oxidative stress causes a disturbance of the balance between production of free radicals and antioxidant response, which leads to various physiological changes in an organism. Despite this, there are several of defense mechanisms, which include in particular the main antioxidant enzymes AKH. In this thesis, I tried to contribute especially to understand the physiological nature of telomere elongation after exposure to free radicals.
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Avaliação do estresse oxidativo em cérebro de ratas reprodutoras ao longo do envelhecimentoHeemann, Fernanda Maciel January 2015 (has links)
A reprodução é uma fase crítica e exigente na vida dos animais. Nos mamíferos, as fêmeas costumam investir muito mais no cuidado parental do que os machos e a lactação é o período mais exigente em termos energéticos da vida da fêmea. Aqui, testamos se o estresse oxidativo é uma consequência da reprodução em ratas Wistar. Foram avaliadas as atividades da glutationa peroxidase, glutationa S-transferase, superóxido dismutase, o consumo de peróxido de hidrogênio, carbonilação de proteínas, peroxidação lipídica, níveis de nitrito e nitrato, glutationa total, níveis de vitamina C, bem como os níveis de estradiol no tecido cerebral em 3 , 6, 12, e 24 meses de idade. Os animais foram agrupados de acordo com a experiência reprodutiva: reprodutores ou não reprodutores. A maioria dos parâmetros estudados mostrou uma diferença entre animais não reprodutores e reprodutores de 12 e 24 meses. Aos 24 meses de idade animais reprodutores apresentaram maior atividade de superóxido dismutase, consumo de peróxido de hidrogênio, glutationa peroxidase e carbonilação de proteínas do que os animais não reprodutores. Aos 6 meses de idade, durante o período que representaria o pico da atividade reprodutiva, animais não reprodutores apresentaram níveis mais altos de malondealdeído. Em animais não reprodutores aos 12 meses de idade observou-se níveis mais altos de estrogênio, vitamina C, consumo de peróxido de hidrogênio e atividades de superóxido dismutase e glutationa peroxidase em relação aos animais reprodutores. Demonstramos que o processo de envelhecimento induz a uma elevação no dano oxidativo e também nas defesas antioxidantes em cérebro de ratas reprodutoras, sendo de alguma forma, a reprodução um processo custoso. Este estudo mostra que existe um forte potencial para a investigação do custo reprodutivo e estresse oxidativo. / Reproduction is a critical and demanding phase of the animals’ life. In mammals, females usually invest much more in parental care than males and lactation is the most energetically demanding period of a female’s life. In this work, we tested whether oxidative stress is a consequence of reproduction in female Wistar rats. We evaluated the activities of glutathione peroxidase, glutathione S-transferase, superoxide dismutase, consumption of hydrogen peroxide, protein carbonylation, lipid peroxidation, nitrite and nitrate levels, total glutathione, vitamin C levels, as well as sex hormone levels in brain tissue at 3, 6, 12, and 24 months of age. Animals were grouped according to reproductive experience: breeders or non-breeders. The parameters studied showed a difference between non-breeders and breeders animals at 12 and 24 months. At 24 months of age breeders animals showed higher superoxide dismutase activity, consumption of hydrogen peroxide, glutathione peroxidase and carbonyl level than non-breeders animals. At 6 months of age, during the period that represents peak reproductive activity, non-breeders animals showed higher levels of malondialdehyde. In non-breeders animals at 12 months of age we observed a higher level of estrogen, vitamin C, consumption of hydrogen peroxide, superoxide dismutase and glutathione peroxidase activities than breeders animals. Finally, we demonstrated that the aging process causes higher oxidative damage and higher antioxidant defenses in brain of breeders female rats, being the reproduction process costly somehow. This study shows that there is strong potential for research linking the cost of reproduction and oxidative stress.
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