UV-induced hyperphosphorylation of replication protein a depends on DNA replication and expression of ATM protein

Exposure to DNA-damaging agents triggers signal transduction pathways that are thought to play a role in maintenance of genomic stability. A key protein in the cellular processes of nucleotide excision repair, DNA recombination, and DNA double-strand break repair is the single-stranded DNA binding protein, RPA. We showed previously that the p34 subunit of RPA becomes hyperphosphorylated as a delayed response (4-8 h) to UV radiation (10-30 J/m(2)). Here we show that UV-induced RPA-p34 hyperphosphorylation depends on expression of ATM, the product of the gene mutated in the human genetic disorder ataxia telangiectasia (A-T). UV-induced RPA-p34 hyperphosphorylation was not observed in A-T cells, but this response was restored by ATM expression. Furthermore, purified ATM kinase phosphorylates the p34 subunit of RPA complex in vitro at many of the same sites that are phosphorylated in vivo after UV radiation. Induction of this DNA damage response was also dependent on DNA replication; inhibition of DNA replication by aphidicolin prevented induction of RPA-p34 hyperphosphorylation by UV radiation. We postulate that this pathway is triggered by the accumulation of aberrant DNA replication intermediates, resulting from DNA replication fork blockage by UV photoproducts. Further, we suggest that RPA-p34 is hyperphosphorylated as a participant in the recombinational postreplication repair of these replication products. Successful resolution of these replication intermediates reduces the accumulation of chromosomal aberrations that would otherwise occur as a consequence of UV radiation.

Ataxia-telangiectasia: chronic activation of damage-responsive functions is reduced by alpha-lipoic acid

Cells from patients with the genetic disorder ataxia-telangiectasia (A-T) are hypersensitive to ionizing radiation and radiomimetic agents, both of which generate reactive oxygen species capable of causing oxidative damage to DNA and other macromolecules. We describe in A-T cells constitutive activation of pathways that normally respond to genotoxic stress, Basal levels of p53 and p21(WAF1/CIP1), phosphorylation on serine 15 of p53, and the Tyr15-phosphorylated form of cdc2 are chronically elevated in these cells. Treatment of A-T cells with the antioxidant alpha -lipoic acid significantly reduced the levels of these proteins, pointing to the involvement of reactive oxygen species in their chronic activation. These findings suggest that the absence of functional ATM results in a mild but continuous state of oxidative stress, which could account for several features of the pleiotropic phenotype of A-T.

Effects of vitamin E deficiency on fatigue and muscle contractile properties

Radical-mediated oxidative damage of skeletal muscle membranes has been implicated in the fatigue process. Vitamin E (VE) is a major chain breaking antioxidant that has been shown to reduce contraction-mediated oxidative damage. We hypothesized that VE deficiency would adversely affect Muscle contractile function, resulting in a more rapid development of muscular fatigue during exercise. To test this postulate, rats were fed either a VE-deficient (EDEF) diet or a control (CON) diet containing VE. Following a 12-week feeding period, animals were anesthetized and mechanically ventilated. Muscle endurance (fatigue) and contractile properties were evaluated using an in situ preparation of the tibialis anterior (TA) muscle. Contractile properties of the TA muscle were determined before and after a fatigue protocol. The muscle fatigue protocol consisted of 60 min of repetitive contractions (250 ms trains at 15 Hz; duty cycle = I I %) of the TA muscle. Prior to the fatigue protocol, no significant differences existed in the force-frequency curves between EDEF and CON animals. At the completion of the fatigue protocol, muscular force production was significantly (P

Linkage and Association Analysis of Radiation Damage Repair Genes XRCC3 and XRCC5 with Nevus Density in Adolescent Twins

Previous studies have shown that a deficiency in DNA damage repair is associated with increased cancer risk, and exposure to UV radiation is a major risk factor for the development of malignant melanoma. High density of common nevi (moles) is a major risk factor for cutaneous melanoma. A nevus may result from a mutation in a single UV-exposed melanocyte which failed to repair DNA damage in one or more critical genes. XRCC3 and XRCC5 may have an effect on nevus count through their function as components of DNA repair processes that may be involved directly or indirectly in the repair of DNA damage due to UV radiation. This study aims to test the hypothesis that the frequency of flat or raised moles is associated with polymorphism at or near these DNA repair genes, and that certain alleles are associated with less efficient DNA repair, and greater nevus density. Twins were recruited from schools in south eastern Queensland and were examined close to their 12th birthday. Nurses examined each individual and counted all moles on the entire body surface. A 10cM genome scan of 274 families (642 individuals) was performed and microsatellite polymorphisms in XRCC3 and adjacent to XRCC5 were also typed. Linkage and association of nevus count to these loci were tested simultaneously using a structural-equation modeling approach implemented in MX. There is weak evidence for linkage of XRCC5 to a QTL influencing raised mole count, and also weak association. There is also weak evidence for association between flat mole count and XRCC3. No tests were significant after correction for testing multiple alleles, nor were any of the tests for total association significant. If variation in XRCC3 or XRCC5 influences UV sensitivity, and indirectly affects nevus density, then the effects are small.

Effect of alcohol on iron storage diseases of the liver

The consumption of excess alcohol in patients with liver iron storage diseases, in particular the iron-overload disease hereditary haemochromatosis (HH), has important clinical consequences. HH, a common genetic disorder amongst people of European descent, results in a slow, progressive accumulation of excess hepatic iron. If left untreated, the condition may lead to fibrosis, cirrhosis and primary hepatocellular carcinoma. The consumption of excess alcohol remains an important cause of hepatic cirrhosis and alcohol consumption itself may lead to altered iron homeostasis. Both alcohol and iron independently have been shown to result in increased oxidative stress causing lipid peroxidation and tissue damage. Therefore, the added effects of both toxins may exacerbate the pathogenesis of disease and impose an increased risk of cirrhosis. This review discusses the concomitant effects of alcohol and iron on the pathogenesis of liver disease. We also discuss the implications of co-existent alcohol and iron in end-stage liver disease.

Metal accumulation and oxidative stress biomarkers in octopus (Octopus vulgaris) from Northwest Atlantic

Metals are ubiquitous in the environment and accumulate in aquatic organisms and are known for their ability to enhance the production of reactive oxygen species (ROS). In aquatic species, oxidative stress mechanisms have been studied by measuring antioxidant enzyme activities and oxidative damages in tissues. The aim of this study was to apply and validate a set of oxidative stress biomarkers and correlate responses with metal contents in tissues of common octopus (Octopus vulgaris). Antioxidant enzyme activity (catalase — CAT, superoxide dismutase — SOD and glutathione S-transferases — GST), oxidative damages (lipid peroxidation — LPO and protein carbonyl content — PCO) andmetal content (Cu, Zn, Pb, Cd and As) in the digestive gland and armof octopus, collected in the NWPortuguese coast in different periods, were assessed after capture and after 14 days in captivity. CAT and SOD activitieswere highly responsive to fluctuations inmetal concentrations and able to reduce oxidative damage, LPO and PCO in the digestive gland. CAT activity was also positively correlated with SOD and GST activities, which emphasizes that the three enzymes respond in a coordinated way to metal induced oxidative stress. Our results validate the use of oxidative stress biomarkers to assess metal pollution effects in this ecological and commercial relevant species.Moreover, octopus seems to have the ability to control oxidative damage by triggering an antioxidant enzyme coordinated response in the digestive gland.

Síntese de derivados de ácidos fenólicos e estudo da sua actividade antioxidante

A peroxidação lipídica, além de causar sérios danos no corpo humano, é a principal causa da deterioração dos alimentos afectando a sua cor, aroma e valor nutricional, o que conduz a uma diminuição do seu ciclo de vida. O fenómeno de oxidação pode ser retardado com recurso a antioxidantes, de origem natural ou sintética, que inibam a formação de espécies reactivas, ou que reajam com estas, formando posteriormente radicais com menor grau de reactividade. Nesta dissertação procedeu-se à síntese e elucidação estrutural de novos antioxidantes, derivados dos ácidos 3,4-diidroxibenzóico (PCA) e 3,4-diidroxifenilacético (DOPAC), e à avaliação da sua actividade anti-radicalar e antioxidante. Os novos antioxidantes sintetizados foram caracterizados usando RMN de 1H e de 13C, FTIR e EM-IE. A avaliação da actividade antioxidante foi realizada com base no método do radical 2,2- difenil-1-picrilhidrazilo (DPPH·) e por técnicas electroquímicas (voltametria de impulso diferencial e voltametria cíclica). Os resultados obtidos permitiram concluir que a eficácia anti-radicalar (AE) é determinada por aspectos da estrutura molecular dos compostos, nomeadamente pela presença de grupos hidroxilo no anel aromático e também de grupos extensores relativamente à posição do grupo carboxílico. Os resultados permitiram verificar que o DOPAC apresenta a mais elevada eficiência anti-radicalar dos compostos em estudo, incluindo o trolox e o ácido gálhico (compostos de referência). Com base nos resultados obtidos concluiu-se que, um menor potencial de oxidação conduz a uma melhor actividade anti-radicalar dos compostos. De facto, verificou-se para o PCA e respectivos ésteres o maior potencial de oxidação e também a menor eficiência anti-radicalar. Em contrapartida, os antioxidantes com maior eficiência anti-radicalar, DOPAC, trolox e ácido gálhico, apresentaram menor potencial de oxidação.

N-acetyl-β -d-glucosaminidase activity in feral Carcinus maenasexposed to cadmium

Cadmium is a priority hazardous substance, persistent in the aquatic environment, with the capacity to interfere with crustacean moulting. Moulting is a vital process dictating crustacean growth, reproduction and metamorphosis. However, for many organisms, moult disruption is difficult to evaluate in the short term, what limits its inclusion in monitoring programmes. N-acetyl-β-d-glucosaminidase (NAGase) is an enzyme acting in the final steps of the endocrine-regulated moulting cascade, allowing for the cast off of the old exoskeleton, with potential interest as a biomarker of moult disruption. This study investigated responses to waterborne cadmium of NAGase activity of Carcinus maenas originating from estuaries with different histories of anthropogenic contamination: a low impacted and a moderately polluted one. Crabs from both sites were individually exposed for seven days to cadmium concentrations ranging from 1.3 to 2000 μg/L. At the end of the assays, NAGase activity was assessed in the epidermis and digestive gland. Detoxification, antioxidant, energy production, and oxidative stress biomarkers implicated in cadmium metabolism and tolerance were also assessed to better understand differential NAGase responses: activity of glutathione S-transferases (GST), glutathione peroxidase (GPx) glutathione reductase (GR), levels of total glutathiones (TG), lipid peroxidation (LPO), lactate dehydrogenase (LDH), and NADP+-dependent isocitrate dehydrogenase (IDH). Animals from the moderately polluted estuary had lower NAGase activity both in the epidermis and digestive gland than in the low impacted site. NAGase activity in the epidermis and digestive gland of C. maenas from both estuaries was sensitive to cadmium exposure suggesting its usefulness for inclusion in monitoring programmes. However, in the digestive gland NAGase inhibition was found in crabs from the less impacted site but not in those from the moderately contaminated one. Altered glutathione levels were observed in cadmium-treated crabs from the contaminated site possibly conferring enhanced tolerance to these animals through its chelator action. Investigation of enhanced tolerance should thus be accounted for in monitoring programmes employing NAGase as biomarker to avoid data misinterpretation.

Modulatory activity of antioxidants against the toxicity of Rifampicin in vivo

The World Health Organization (WHO) has shown concern about the burden of tuberculosis in the developing countries. Even though rifampicin is an effective drug in the management of tuberculosis, it has been documented to have some toxic effects in humans. Therefore, this study intends to investigate the modulatory effect of vitamins C and E on the hepatotoxicity, sperm quality and brain toxicity of Rifampicin. Forty Wistar albino rats were used, 10 animals per group. Group 1 animals received 0.3 mL of distilled water, the Group 2 animals received the therapeutic dose of rifampicin, Group 3 animals received therapeutic doses of rifampicin plus vitamin E, while Group 4 received therapeutic doses of rifampicin and vitamin C. The administration was performed orally during three months; the animals were sacrificed by cervical dislocation at the end of that period. Blood samples were collected and liver function and lipid profile was analyzed using fully automated clinical chemistry device. The liver, brain and reproductive organs underwent histopathological examination. Sperm samples were collected from the epididymis to achieve count and motility and morphological analysis. Results showed rifampicin alone to raise (p < 0.05) liver function enzymes (Aspartate amino transferase [AST], Serum alanine amino transferase [ALT] and Total Bilirubin) when compared with controls. While the vitamin E treated group showed remarkable protection, the vitamin C treated group showed questionable protection against the rifampicin induced liver damage. Sperm count results showed an important (p < 0.05) increase in the sperm quality in vitamin E and C treated groups. However, the vitamin E plus Rifampicin treated group showed increased lipid peroxidation. The histopathological findings revealed structural damages by rifampicin in liver, brain and epididymis while some remarkable architectural integrity was observed in the antioxidant-treated groups. It can be concluded that vitamin E or C improved sperm quality and protected against the brain damage caused by rifampicin. Moreover, vitamin E demonstrated remarkable hepatoprotection against rifampicin induced damage while vitamin C shows a questionable hepatoprotection.

Effects of vitamin C supplementation on acute phase Chagas disease in experimentally infected mice with Trypanosoma cruzi QM1 strain

The tissue changes that occur in Chagas disease are related to the degree of oxidative stress and antioxidant capacity of affected tissue. Studies with vitamin C supplementation did not develop oxidative damage caused by Chagas disease in the host, but other studies cite the use of peroxiredoxins ascorbate - dependent on T. cruzi to offer protection against immune reaction. Based on these propositions, thirty "Swiss" mice were infected with T. cruzi QM1 strain and treated with two different vitamin C doses in order to study the parasitemia evolution, histopathological changes and lipid peroxidation biomarkers during the acute phase of Chagas disease. The results showed that the parasite clearance was greater in animals fed with vitamin C overdose. There were no significant differences regarding the biomarkers of lipid peroxidation and inflammatory process or the increase of myocardium in animals treated with the recommended dosage. The largest amount of parasite growth towards the end of the acute phase suggests the benefit of high doses of vitamin C for trypomastigotes. The supplementation doesn't influence the production of free radicals or the number of amastigote nests in the acute phase of Chagas disease.

Integration of sediment contamination with multi–biomarker responses in a novel bioindicator candidate (Sepia officinalis) for risk assessment in impacted estuaries

Dissertação para obtenção do Grau de Mestre em Engenharia do Ambiente

New mechanisms that regulate the expression of genes implicated in the process of ketogenesis

Dissertação para obtenção do Grau de Mestre em Biotecnologia

EFFECTS OF VITAMIN C SUPPLEMENTATION ON THE CHRONIC PHASE OF CHAGAS DISEASE

Introduction:In order to examine the effectiveness of vitamin C (ascorbic acid) in combating the oxidative insult caused by Trypanosoma cruzi during the development of the chronic phase of Chagas disease, Swiss mice were infected intraperitoneally with 5.0 × 104 trypomastigotes of T. cruzi QM1strain.Methods:Mice were given supplements of two different doses of vitamin C for 180 days. Levels of lipid oxidation (as indicated by thiobarbituric acid reactive substances-TBARS), total peroxide, vitamin C, and reduced glutathione were measured in the plasma, TBARS, total peroxide and vitamin C were measured in the myocardium and histopathologic analysis was undertaken in heart, colon and skeletal muscle.Results:Animals that received a dose equivalent to 500 mg of vitamin C daily showed increased production of ROS in plasma and myocardium and a greater degree of inflammation and necrosis in skeletal muscles than those that received a lower dose or no vitamin C whatsoever.Conclusion:Although some research has shown the antioxidant effect of vitamin C, the results showed that animals subject to a 500 mg dose of vitamin C showed greater tissue damage in the chronic phase of Chagas disease, probably due to the paradoxical actions of the substance, which in this pathology, will have acted as a pro-oxidant or pro-inflammatory.

Paracoccidioidomycosis: no genetic damage in human peripheral blood cells of patients assessed by single-cell gel (comet) assay

Paracoccidioidomycosis is a systemic fungal infection caused by Paracoccidioides brasiliensis. As infectious diseases can cause DNA damage, the authors aimed at analyzing DNA breakage in peripheral blood cells of patients with paracoccidioidomycosis by using the comet assay. The results suggested that paracoccidioidomycosis does not cause genotoxicity.

Natural gaps associated with oxidative stress in Willisornis poecilinotus (Aves: Thamnophilidae) in a tropical forest

Natural disturbances in tropical forests modify the availability and quality of resources and alter the patterns of bird distribution. These environmental changes increase the metabolic rate and disrupt the redox balance promoting oxidative stress. This study aimed to compare the abundance of Willisornis poecilinotus between gaps and the understory of a forest with undisturbed canopy at Caxiuanã National Forest. The abundance was correlated with vegetation heights. The oxidative stress and the stress promoting factors were determined in both sites of sampling. We captured 81 specimens of W. poecilinotus. The number of captures was high in gaps. The specimens sampled at gaps showed high levels of oxidative stress. The biomarkers of oxidative stress were significantly correlated in gaps. The variability of oxidative stress and oxidative damage were explained only by site of sampling. These results suggest that gaps are stressors sites to W. poecilinotus, which probably can be due to an increase of metabolic rate to deal with new flight strategies of foraging and avoid predation