The cDNA cloning and mRNA expression of a potential selenium-binding protein gene in the scallop Chlamys farreri

Selenium binding proteins (SeBP) represent a family of proteins that are believed to be involved in controlling the oxidation/reduction in many physiological processes. The cDNA of Zhikong Scallop Chlamys farreri selenium binding protein (zSeBP) was cloned by expressed sequence tag (EST) and RACE techniques. The high similarity of zSeBP deduced amino acid sequence with the SeBP in other organisms, such as bird, fish, frog, mosquito, fruit fly, mammalian, and even nematode and microorganism indicated that zSeBP should be a member of SeBP family. The temporal expression of zSeBP in the hemocytes was measured by semi-quantitative RT-PCR after scallops were stimulated by either oxidative stress or microbial challenge. The expression of zSeBP was up-regulated progressively after stimulation, and then dropped gradually to the original level. Meanwhile, malondialdehyde (MDA) measured by the colorimetric method in the microbial challenged scallops increased immediately after scallops was challenged by microbes, and was significantly higher than that in the control scallops. Results indicated that the microbial infection could incense the disorder of oxidation/reduction and may result in high MDA production. The negative correlation between the expression level of zSeBP and the MDA content suggested that zSeBP could play an important role in mediating the anti-oxidation mechanisms and immune response in marine invertebrates. (c) 2005 Published by Elsevier Ltd.

Design, synthesis and development of novel indolocarbazole derivatives as potential anti-cancer agents

This thesis describes work carried out on the design of new routes to a range of bisindolylmaleimide and indolo[2,3-a]carbazole analogs, and investigation of their potential as successful anti-cancer agents. Following initial investigation of classical routes to indolo[2,3-a]pyrrolo[3,4-c]carbazole aglycons, a new strategy employing base-mediated condensation of thiourea and guanidine with a bisindolyl β-ketoester intermediate afforded novel 5,6-bisindolylpyrimidin-4(3H)-ones in moderate yields. Chemical diversity within this H-bonding scaffold was then studied by substitution with a panel of biologically relevant electrophiles, and by reductive desulfurisation. Optimisation of difficult heterogeneous literature conditions for oxidative desulfurisation of thiouracils was also accomplished, enabling a mild route to a novel 5,6-bisindolyluracil pharmacophore to be developed within this work. The oxidative cyclisation of selected acyclic bisindolyl systems to form a new planar class of indolo[2,3-a]pyrimido[5,4-c]carbazoles was also investigated. Successful conditions for this transformation, as well as the limitations currently prevailing for this approach are discussed. Synthesis of 3,4-bisindolyl-5-aminopyrazole as a potential isostere of bisindolylmaleimide agents was encountered, along with a comprehensive derivatisation study, in order to probe the chemical space for potential protein backbone H-bonding interactions. Synthesis of a related 3,4-arylindolyl-5-aminopyrazole series was also undertaken, based on identification of potent kinase inhibition within a closely related heterocyclic template. Following synthesis of approximately 50 novel compounds with a diversity of H-bonding enzyme-interacting potential within these classes, biological studies confirmed that significant topo II inhibition was present for 9 lead compounds, in previously unseen pyrazolo[1,5-a]pyrimidine, indolo[2,3-c]carbazole and branched S,N-disubstituted thiouracil derivative series. NCI-60 cancer cell line growth inhibition data for 6 representative compounds also revealed interesting selectivity differences between each compound class, while a new pyrimido[5,4-c]carbazole agent strongly inhibited cancer cell division at 10 µM, with appreciable cytotoxic activity observed across several tumour types.

Superoxide dismutase mimics: chemistry, pharmacology, and therapeutic potential.

Oxidative stress has become widely viewed as an underlying condition in a number of diseases, such as ischemia-reperfusion disorders, central nervous system disorders, cardiovascular conditions, cancer, and diabetes. Thus, natural and synthetic antioxidants have been actively sought. Superoxide dismutase is a first line of defense against oxidative stress under physiological and pathological conditions. Therefore, the development of therapeutics aimed at mimicking superoxide dismutase was a natural maneuver. Metalloporphyrins, as well as Mn cyclic polyamines, Mn salen derivatives and nitroxides were all originally developed as SOD mimics. The same thermodynamic and electrostatic properties that make them potent SOD mimics may allow them to reduce other reactive species such as peroxynitrite, peroxynitrite-derived CO(3)(*-), peroxyl radical, and less efficiently H(2)O(2). By doing so SOD mimics can decrease both primary and secondary oxidative events, the latter arising from the inhibition of cellular transcriptional activity. To better judge the therapeutic potential and the advantage of one over the other type of compound, comparative studies of different classes of drugs in the same cellular and/or animal models are needed. We here provide a comprehensive overview of the chemical properties and some in vivo effects observed with various classes of compounds with a special emphasis on porphyrin-based compounds.

alpha-Synuclein aggregation in neurodegenerative diseases and its inhibition as a potential therapeutic strategy

There is strong evidence for the involvement of alpha-synuclein in the pathologies of several neurodegenerative disorders, including PD (Parkinson's disease). Development of disease appears to be linked to processes that increase the rate at which alpha-synuclein forms aggregates. These processes include increased protein concentration (via either increased rate of synthesis or decreased rate of degradation), and altered forms of alpha-synuclein (such as truncations, missense mutations, or chemical modifications by oxidative reactions). Aggregated forms of the protein are toxic to cells and one therapeutic strategy would be to reduce the rate at which aggregation occurs. To this end we have designed several peptides that reduce alpha-synuclein aggregation. A cell-permeable version of one such peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with alpha-synuclein (A53T), a familial PD-associated mutation.

Heightened maternal inflammation is linked to placental oxidative and nitrosative stress associated with fetal growth restriction in the rat

Deficient trophoblast invasion and spiral artery remodeling are associated with pregnancy complications such as pre-eclampsia (PE) and fetal growth restriction (FGR). Using a model in which pregnant Wistar rats are given daily, low-dose, injections of bacterial lipopolysaccharide (LPS; 10 – 40 µg/kg) on gestational days (GD) 13.5 – 16.5, our group has shown that abnormal maternal inflammation is causally linked to shallow trophoblast invasion, deficient spiral artery remodeling, and altered utero-placental hemodynamics leading to FGR/PE; these alterations were shown to be mediated by TNF-a. The present research evaluated certain consequences of decreased placental perfusion; this was accomplished by examining placental alterations indicative of decreased placental perfusion. Additionally, the role of glyceryl trinitrate (GTN) was determined as a potential therapeutic to prevent the consequences of decreased placental perfusion. Results indicated that dams experiencing heightened maternal inflammation showed significantly greater expression of hypoxia-inducible factor-1a (HIF-1a) and nitrotyrosine, both of which are markers of decreased perfusion and oxidative/nitrosative stress. Contrary to expectations, inflammation did not appear to affect nitric oxide (NO) bioavailability, as revealed by a lack of change in placental or plasma levels of cyclic guanosine monophosphate (cGMP). However, continuous transdermal administration of GTN (25 µg/hr) on GD 12.5 – 16.5 prevented the accumulation of HIF-1a and nitrotyrosine in placentas from LPS-treated rats. These results support the concept that maternal inflammation contributes to placental hypoxia and oxidative/nitrosative stress. Additionally, they indicate that GTN has potential applications in the treatment and/or prevention of pregnancy complications associated with abnormal maternal inflammation.

Role of creatine supplementation on exercise-induced cardiovascular function and oxidative stress

Many degenerative diseases are associated with increased oxidative stress. Creatine has the potential to act as an indirect and direct antioxidant; however, limited data exist to evaluate the antioxidant capabdities of creatine supplementation within in vivo human systems. This study aimed to investigate the effects of oral creatine supplementation on markers of oxidative stress and antioxidant defenses following exhaustive cycling exercise. Following preliminary testing and two additional familiarization sessions, 18 active males repeated two exhaustive incremental cycling trials (T1 and T2) separated by exactly 7 days. The subjects were assigned, in a double-blind manner, to receive either 20 g of creatine (Cr) or a placebo (P) for the 5 days preceding T2. Breath-by-breath respiratory data and heart rate were continually recorded throughout the exercise protocol and blood samples were obtained at rest (preexercise), at the end of exercise (postexercise), and the day following exercise (post24 h). Serum hypdroperoxide concentrations were elevated at postexercise by 17 +/- 5% above preexercise values (p = 0.030). However, supplementation did not influence lipid peroxidation (serum hypdroperoxide concentrations), resistance of low density lipoprotein to oxidative stress (t(1/2max) LDL oxidation) and plasma concentrations of non-enzymatic antioxidants (retinol, alpha-carotene, beta-carotene, alpha-tocopherol, gamma-tocopherol, lycopene and vitamin Q. Heart rate and oxygen uptake responses to exercise were not affected by supplementation. These findings suggest that short-term creatine supplementation does not enhance non-enzymatic antioxidant defence or protect against lipid peroxidation induced by exhaustive cycling in healthy males.

Purple sweet potato colour - a potential therapy for galactosemia?

Galactosemia is an inherited metabolic disease in which galactose is not properly metabolised. There are various theories to explain the molecular pathology, and recent experimental evidence strongly suggests that oxidative stress plays a key role. High galactose diets are damaging to experimental animals and oxidative stress also plays a role in this toxicity which can be alleviated by purple sweet potato colour (PSPC). This plant extract is rich in acetylated anthocyanins which have been shown to quench free radical production. The objective of this Commentary is to advance the hypothesis that PSPC, or compounds therefrom, may be a viable basis for a novel therapy for galactosemia.

Inhibition of Rho-kinase protects cerebral barrier from ischaemia-evoked injury through modulations of endothelial cell oxidative stress and tight junctions

Ischaemic strokes evoke blood-brain barrier (BBB) disruption and oedema formation through a series of mechanisms involving Rho-kinase activation. Using an animal model of human focal cerebral ischaemia, this study assessed and confirmed the therapeutic potential of Rho-kinase inhibition during the acute phase of stroke by displaying significantly improved functional outcome and reduced cerebral lesion and oedema volumes in fasudil- versus vehicle-treated animals. Analyses of ipsilateral and contralateral brain samples obtained from mice treated with vehicle or fasudil at the onset of reperfusion plus 4 h post-ischaemia or 4 h post-ischaemia alone revealed these benefits to be independent of changes in the activity and expressions of oxidative stress- and tight junction-related parameters. However, closer scrutiny of the same parameters in brain microvascular endothelial cells subjected to oxygen-glucose deprivation ± reperfusion revealed marked increases in prooxidant NADPH oxidase enzyme activity, superoxide anion release and in expressions of antioxidant enzyme catalase and tight junction protein claudin-5. Cotreatment of cells with Y-27632 prevented all of these changes and protected in vitro barrier integrity and function. These findings suggest that inhibition of Rho-kinase after acute ischaemic attacks improves cerebral integrity and function through regulation of endothelial cell oxidative stress and reorganization of intercellular junctions. Inhibition of Rho-kinase (ROCK) activity in a mouse model of human ischaemic stroke significantly improved functional outcome while reducing cerebral lesion and oedema volumes compared to vehicle-treated counterparts. Studies conducted with brain microvascular endothelial cells exposed to OGD ± R in the presence of Y-27632 revealed restoration of intercellular junctions and suppression of prooxidant NADPH oxidase activity as important factors in ROCK inhibition-mediated BBB protection.

On the antioxidant properties of erythropoietin and its association with the oxidative-nitrosative stress response to hypoxia in humans

Aim: The aim of this study was to examine if erythropoietin (EPO) has the potential to act as a biological antioxidant and determine the underlying mechanisms.

Methods: The rate at which its recombinant form (rHuEPO) reacts with hydroxyl (HO center dot), 2,2-diphenyl-1-picrylhydrazyl (DPPH center dot) and peroxyl (ROO center dot) radicals was evaluated in-vitro. The relationship between the erythopoietic and oxidative-nitrosative stress response to poikilocapneic hypoxia was determined separately in-vivo by sampling arterial blood from eleven males in normoxia and following 12 h exposure to 13% oxygen. Electron paramagnetic resonance spectroscopy, ELISA and ozone-based chemiluminescence were employed for direct detection of ascorbate (A(center dot-)) and N-tert-butyl-a-phenylnitrone spin-trapped alkoxyl (PBN-OR) radicals, 3-nitrotyrosine (3-NT) and nitrite (NO2-).

Results: We found rHuEPO to be a potent scavenger of HO center dot (k(r) = 1.03-1.66 x 10(11) M-1 s(-1)) with the capacity to inhibit Fenton chemistry through catalytic iron chelation. Its ability to scavenge DPPH. and ROO center dot was also superior compared to other more conventional antioxidants. Hypoxia was associated with a rise in arterial EPO and free radical-mediated reduction in nitric oxide, indicative of oxidative-nitrosative stress. The latter was confirmed by an increased systemic formation of A(center dot-), PBN-OR, 3-NT and corresponding loss of NO2- (P <0.05 vs. normoxia). The erythropoietic and oxidative-nitrosative stress responses were consistently related (r =-0.52 to 0.68, P <0.05).

Conclusion: These findings demonstrate that EPO has the capacity to act as a biological antioxidant and provide a mechanistic basis for its reported cytoprotective benefits within the clinical setting.

Volatile terpenoids and C13 norisoprenoids in wines: development of rapid methods of analysis and evaluation of the sesquiterpenoids biological potential

Vitis vinifera L., the most widely cultivated fruit crop in the world, was the starting point for the development of this PhD thesis. This subject was exploited following on two actual trends: i) the development of rapid, simple, and high sensitive methodologies with minimal sample handling; and ii) the valuation of natural products as a source of compounds with potential health benefits. The target group of compounds under study were the volatile terpenoids (mono and sesquiterpenoids) and C13 norisoprenoids, since they may present biological impact, either from the sensorial point of view, as regards to the wine aroma, or by the beneficial properties for the human health. Two novel methodologies for quantification of C13 norisoprenoids in wines were developed. The first methodology, a rapid method, was based on the headspace solid-phase microextraction combined with gas chromatography-quadrupole mass spectrometry operating at selected ion monitoring mode (HS-SPME/GC-qMS-SIM), using GC conditions that allowed obtaining a C13 norisoprenoid volatile signature. It does not require any pre-treatment of the sample, and the C13 norisoprenoid composition of the wine was evaluated based on the chromatographic profile and specific m/z fragments, without complete chromatographic separation of its components. The second methodology, used as reference method, was based on the HS-SPME/GC-qMS-SIM, allowing the GC conditions for an adequate chromatographic resolution of wine components. For quantification purposes, external calibration curves were constructed with β-ionone, with regression coefficient (r2) of 0.9968 (RSD 12.51 %) and 0.9940 (RSD of 1.08 %) for the rapid method and for the reference method, respectively. Low detection limits (1.57 and 1.10 μg L-1) were observed. These methodologies were applied to seventeen white and red table wines. Two vitispirane isomers (158-1529 L-1) and 1,1,6-trimethyl-1,2-dihydronaphthalene (TDN) (6.42-39.45 μg L-1) were quantified. The data obtained for vitispirane isomers and TDN using the two methods were highly correlated (r2 of 0.9756 and 0.9630, respectively). A rapid methodology for the establishment of the varietal volatile profile of Vitis vinifera L. cv. 'Fernão-Pires' (FP) white wines by headspace solid-phase microextraction combined with comprehensive two-dimensional gas chromatography with time-of-flight mass spectrometry (HS-SPME/GCxGC-TOFMS) was developed. Monovarietal wines from different harvests, Appellations, and producers were analysed. The study was focused on the volatiles that seem to be significant to the varietal character, such as mono and sesquiterpenic compounds, and C13 norisoprenoids. Two-dimensional chromatographic spaces containing the varietal compounds using the m/z fragments 93, 121, 161, 175 and 204 were established as follows: 1tR = 255-575 s, 2tR = 0,424-1,840 s, for monoterpenoids, 1tR = 555-685 s, 2tR = 0,528-0,856 s, for C13 norisoprenoids, and 1tR = 695-950 s, 2tR = 0,520-0,960 s, for sesquiterpenic compounds. For the three chemical groups under study, from a total of 170 compounds, 45 were determined in all wines, allowing defining the "varietal volatile profile" of FP wine. Among these compounds, 15 were detected for the first time in FP wines. This study proposes a HS-SPME/GCxGC-TOFMS based methodology combined with classification-reference sample to be used for rapid assessment of varietal volatile profile of wines. This approach is very useful to eliminate the majority of the non-terpenic and non-C13 norisoprenic compounds, allowing the definition of a two-dimensional chromatographic space containing these compounds, simplifying the data compared to the original data, and reducing the time of analysis. The presence of sesquiterpenic compounds in Vitis vinifera L. related products, to which are assigned several biological properties, prompted us to investigate the antioxidant, antiproliferative and hepatoprotective activities of some sesquiterpenic compounds. Firstly, the antiradical capacity of trans,trans-farnesol, cis-nerolidol, α-humulene and guaiazulene was evaluated using chemical (DPPH• and hydroxyl radicals) and biological (Caco-2 cells) models. Guaiazulene (IC50= 0.73 mM) was the sesquiterpene with higher scavenger capacity against DPPH•, while trans,trans-farnesol (IC50= 1.81 mM) and cis-nerolidol (IC50= 1.48 mM) were more active towards hydroxyl radicals. All compounds, with the exception of α-humulene, at non-cytotoxic levels (≤ 1 mM), were able to protect Caco-2 cells from oxidative stress induced by tert-butyl hydroperoxide. The activity of the compounds under study was also evaluated as antiproliferative agents. Guaiazulene and cis-nerolidol were able to more effectively arrest the cell cycle in the S-phase than trans,trans-farnesol and α-humulene, being the last almost inactive. The relative hepatoprotection effect of fifteen sesquiterpenic compounds, presenting different chemical structures and commonly found in plants and plant-derived foods and beverages, was assessed. Endogenous lipid peroxidation and induced lipid peroxidation with tert-butyl hydroperoxide were evaluated in liver homogenates from Wistar rats. With the exception of α-humulene, all the sesquiterpenic compounds under study (1 mM) were effective in reducing the malonaldehyde levels in both endogenous and induced lipid peroxidation up to 35% and 70%, respectively. The developed 3D-QSAR models, relating the hepatoprotection activity with molecular properties, showed good fit (R2LOO > 0.819) with good prediction power (Q2 > 0.950 and SDEP < 2%) for both models. A network of effects associated with structural and chemical features of sesquiterpenic compounds such as shape, branching, symmetry, and presence of electronegative fragments, can modulate the hepatoprotective activity observed for these compounds. In conclusion, this study allowed the development of rapid and in-depth methods for the assessment of varietal volatile compounds that might have a positive impact on sensorial and health attributes related to Vitis vinifera L. These approaches can be extended to the analysis of other related food matrices, including grapes and musts, among others. In addition, the results of in vitro assays open a perspective for the promising use of the sesquiterpenic compounds, with similar chemical structures such as those studied in the present work, as antioxidants, hepatoprotective and antiproliferative agents, which meets the current challenges related to diseases of modern civilization.

Photodynamic inactivation of bacteria by cationic porphyrins : their cellular targets and potential environmental applications

Photodynamic inactivation (PDI) is defined as the process of cell destruction by oxidative stress resulting from the interaction between light and a photosensitizer (PS), in the presence of molecular oxygen. PDI of bacteria has been extensively studied in recent years, proving to be a promising alternative to conventional antimicrobial agents for the treatment of superficial and localized infections. Moreover, the applicability of PDI goes far beyond the clinical field, as its potential use in water disinfection, using PS immobilized on solid supports, is currently under study. The aim of the first part of this work was to study the oxidative modifications in phospholipids, nucleic acids and proteins of Escherichia coli and Staphylococcus warneri, subjected to photodynamic treatment with cationic porphyrins. The aims of the second part of the work were to study the efficiency of PDI in aquaculture water and the influence of different physicalchemical parameters in this process, using the Gram-negative bioluminescent bacterium Vibrio fischeri, and to evaluate the possibility of recycling cationic PS immobilized on magnetic nanoparticles. To study the oxidative changes in membrane phospholipids, a lipidomic approach has been used, combining chromatographic techniques and mass spectrometry. The FOX2 assay was used to determine the concentration of lipid hydroperoxides generated after treatment. The oxidative modifications in the proteins were analyzed by one-dimensional polyacrylamide gel electrophoresis (SDS-PAGE). Changes in the intracellular nucleic acids were analyzed by agarose gel electrophoresis and the concentration of doublestranded DNA was determined by fluorimetry. The oxidative changes of bacterial PDI at the molecular level were analyzed by infrared spectroscopy. In laboratory tests, bacteria (108 CFU mL-1) were irradiated with white light (4.0 mW cm-2) after incubation with the PS (Tri-Py+-Me-PF or Tetra-Py+-Me) at concentrations of 0.5 and 5.0 μM for S. warneri and E. coli, respectively. Bacteria were irradiated with different light doses (up to 9.6 J cm-2 for S. warneri and up to 64.8 J cm-2 for E. coli) and the changes were evaluated throughout the irradiation time. In the study of phospholipids, only the porphyrin Tri-Py+-Me-PF and a light dose of 64.8 J cm-2 were tested. The efficiency of PDI in aquaculture has been evaluated in two different conditions: in buffer solution, varying temperature, pH, salinity and oxygen concentration, and in aquaculture water samples, to reproduce the conditions of PDI in situ. The kinetics of the process was determined in realtime during the experiments by measuring the bioluminescence of V. fischeri (107 CFU mL-1, corresponding to a level of bioluminescence of 105 relative light units). A concentration of 5.0 μM of Tri-Py+-Me-PF was used in the experiments with buffer solution, and 10 to 50 μM in the experiments with aquaculture water. Artificial white light (4.0 mW cm-2) and solar irradiation (40 mW cm-2) were used as light sources.

Searching biocompounds in marine natural extracts with potential use in tumor prevention and treatment

Cancer is a multistage process characterized by three stages: initiation, promotion and progression; and is one of the major killers worldwide. Oxidative stress acts as initiator in tumorigenesis; chronic inflammation promotes cancer; and apoptosis inactivation is an issue in cancer progression. In this study, it was investigated the antioxidant, antiinflammatory and antitumor properties of hexane, ether, chloroform, methanol and water extracts of five species of halophytes: A. macrostachyum, P. coronopus, J. acutus, C. edulis and A. halimus. Antioxidant activity was assessed by DPPH• and ABTS•+ methods, and the total phenolics content (TPC) was evaluated by the Folin-Ciocalteau method. The anti-inflammatory activity of the extracts was determined by the Griess method, and by evaluating the inhibition of NO production in LPS-stimulated RAW- 264.7 macrophages. The cytotoxic activity of the extracts against HepG2 and THP1 cell lines was estimated by the MTT assay, and the results obtained were further compared with the S17 non-tumor cell line. The induction of apoptosis of J. acutus ether extract was assessed by DAPI staining. The highest antioxidant activities was observed in C. edulis methanol and the J. acutus ether extracts against the DPPH• radical; and J. acutus ether and A. halimus ether extracts against the ABTS•+ radical. The methanol extracts of C. edulis and P. coronopus, and the ether extract of J. acutus revealed a high TPC. Generally the antioxidant activity had no correlation with the TPC. The A. halimus chloroform and P. coronopus hexane extracts demonstrated ability to reduce NO production in macrophages (> 50%), revealing their anti-inflammatory capacity. The ether extract of J. acutus showed high cytotoxicity against HepG2 cancer cells, with reduced cellular viability even at the lowest concentrations. This outcome was significantly lower than the obtained with the non-tumor cells (S17). This result was complemented by the induction of apoptosis.

Characterization of the toxic potential of nanomaterials using in vitro cell models

Os nanomateriais são estruturas com uma ou mais dimensões inferiores a 100 nanómetros. Devido à sua pequena dimensão, as nanopartículas apresentam atributos únicos, tais como a sua elevada área superficial relativamente à sua massa, reactividade ou força tênsil. Estas características influenciam grandemente algumas das propriedades dos nanomateriais, como a sua hidrofobicidade, carga ou toxicidade. As propriedades das nanopartículas tornam-nas também muito úteis para o Homem, sendo aplicadas em medicina, farmácia, electrónica, cosmética, vestuário e biotecnologia, entre outras. O aumento de produção e utilização de nanomateriais tem vindo a aumentar também a possibilidade de exposição humana a este tipo de partículas, levando a preocupações relativas ao risco de toxicidade aguda ou crónica. A exposição humana pode ocorrer por diversas vias, sendo as mais relevantes a via inalatória, ingestão ou contacto com a pele. Dependendo do material e do órgão-alvo, a exposição a nanomateriais pode conduzir a diferentes consequências biológicas: a nível dos órgãos, os nanomateriais podem levar a inflamação ou a supressão do sistema imunitário e, a nível celular e molecular, a perturbações na estrutura e integridade do genoma, assim como a interacções com moléculas biológicas e inibição da actividade proteica, entre outras consequências. Um dos nanomateriais mais utilizados são os nanotubos de carbono. Estes são constituídos por grafite cilíndrica disposta numa única camada (designados nanotubos de carbono de parede simples) ou em várias (nanotubos de carbono de parede múltipla). Os nanotubos de carbono apresentam propriedades como resistência e condutividade que os tornam muito úteis em aplicações como aparelhos electrónicos, vestuário ou biomedicina; cada vez mais, portanto, se torna provável a exposição ocupacional ou ambiental a este material. A semelhança estrutural destas partículas com fibras de amianto conduziu a questões relativas à sua segurança, pelo que já foram elaborados diversos estudos relativos aos seus efeitos biológicos. Alguns trabalhos sugerem que os nanotubos de carbono têm a capacidade de produzir toxicidade associada a lesões físicas, à produção de danos oxidativos por interacção com mecanismos celulares, ou a morte celular. Outros trabalhos defendem que estas partículas não causam toxicidade relevante. O projecto de dimensão europeia “NANoREG” surgiu da necessidade de ser desenvolvida legislação e regulamentação apoiadas em conhecimento científico e adequadas à produção e ao uso actual de nanomateriais. Este trabalho teve como objectivos principais a determinação do potencial cito- e genotóxico de um conjunto de nanotubos de carbono de parede múltipla (designados NM-400 a NM-403), e a consequente tentativa de associar este potencial às características físico-químicas dos nanomateriais. Com este objectivo, a exposição por via inalatória foi analisada, pelo uso de duas linhas celulares in vitro provenientes de tecidos do tracto respiratório: epitélio pulmonar (células A549) e epitélio brônquico (células BEAS-2B). A citotoxicidade dos nanotubos de carbono foi analisada com base em três parâmetros. Em primeiro lugar, as células foram contadas após a exposição aos nanomateriais utilizando o corante azul de tripanao para excluir as células inviáveis; a contagem foi realizada 3 e 24 horas após a exposição das células aos nanotubos. Os resultados deste ensaio apontam para a ausência de citotoxicidade após a exposição mais curta, e dados inconsistentes após a mais longa. Em segundo lugar, foi realizado o ensaio clonogénico, que se baseia na capacidade das células de se dividirem após a exposição ao agente em estudo. Este ensaio só foi realizado nas células A549 pois as BEAS-2B não permitem a formação de colónias. Os resultados apontam para uma citotoxicidade após a exposição a todos os nanomateriais, cuja intensidade se relaciona directamente com o tamanho das partículas, assim como ao seu diâmetro e área de superfície. Em terceiro lugar, foram calculados dois índices de viabilidade no ensaio dos Micronúcleos, cujo objectivo é avaliar se as células se dividiram durante a exposição aos nanomateriais em comparação com o controlo, e cujos resultados apresentam incoerências em relação aos outros já referidos. Estes dados podem ser justificados pelas diferenças existentes entre os ensaios, como o tempo de exposição ou a densidade celular. Os efeitos genotóxicos dos nanomateriais foram avaliados com recurso aos ensaios do cometa e dos micronúcleos. O primeiro detecta lesões pequenas e reversíveis nas cadeias de DNA, ao passo que o segundo detecta efeitos irreversíveis ao nível cromossómico, tais como quebras ou perdas de cromossomas. Os resultados do ensaio do cometa sugerem que nenhum dos nanomateriais testados é genotóxico, uma vez que em ambas as linhas celulares e em ambos os tempos de exposição, os resultados são negativos. O ensaio dos micronúcleos, por outro lado, aponta para existência de genotoxicidade de dois dos nanomateriais (NM-401 e NM-402) nas células A549, mas não em células BEAS-2B. Uma possível explicação para estes dados aparentemente contraditórios pode residir na hipótese de estes nanotubos de carbono serem compostos com efeitos aneugénicos, mas não clastogénicos: o ensaio dos micronúcleos permite a detecção de ambos os mecanismos de acção, ao passo que o ensaio do cometa só revela a quebra de cadeias de DNA. Outra justificação para os resultados é a possível influência da perda de viabilidade das células analisadas. Com base nos dados do ensaio clonogénico, estas partículas apresentam elevada citotoxicidade, pelo que os resultados dos ensaios de genotoxicidade, em particular do Ensaio do Cometa, poderão ser afectados por estes efeitos. O meio de cultura usado para expor as células aos nanomateriais também é um parâmetro muito relevante na sua toxicidade. Neste trabalho, foram usados meios de cultura com proteínas, que podem ser adsorvidas pelas partículas e formar uma “corona” em seu redor; este processo pode alterar propriedades importantes dos nanomateriais, entre os quais o seu potencial efeito biológico. Também o método usado para conseguir uma dispersão homogénea de nanomateriais pode conduzir a diferenças nos resultados dos ensaios de toxicidade. Neste estudo, foram observados alguns problemas relativos à perda de homogeneidade das dispersões de nanotubos de carbono, o que pode ter conduzido a que as células fossem expostas a massas de partículas de grandes dimensões conjuntamente com partículas individualizadas. O período durante o qual as células são expostas ao nanomaterial é também um aspecto essencial na produção de efeitos tóxicos. Resumindo, este projecto forneceu informações relativas à toxicidade dos nanotubos de carbono que, complementadas pelas conclusões dos restantes parceiros do projecto europeu, poderão contribuir significativamente para a avaliação de risco e criação de legislação relativamente à utilização de nanomateriais. Na linha celular BEAS-2B, nenhum destes nanomateriais parece produzir efeitos tóxicos, quer a nível de célula, quer a nível de genoma, nas condições experimentais utilizadas. Nas células A549, por outro lado, os três nanomateriais testados parecem ser acentuadamente citotóxicos, e dois deles (NM-401 e NM-402) são também genotóxicos. Em relação a perspectivas futuras, pode-se concluir que nem todos os ensaios de toxicidade existentes actualmente são adequados à análise de nanopartículas, pelo que novas metodologias devem ser desenvolvidas e complementadas por ensaios in vivo. Todos os estudos envolvendo nanomateriais deverão também descrever as características físico-químicas dos materiais usados, de forma a se poderem comparar os resultados com os de outros trabalhos.

Evaluation of neuroprotective potential of plyphenols derived from Portuguese native plants: Juniperus sp. and Rubus sp.

Dissertation presented to obtain the Ph.D degree in Biochemistry

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: Therapeutic potential of mitochondrially targeted antioxidants.

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.