O NER é uma via de reparo de DNA capaz de lidar com uma ampla variedade de lesões. Participam do NER diversas proteínas, entre elas a endonuclease XPG. Pacientes que possuem mutações no gene XPG apresentam a síndrome XP, e em alguns casos XP/CS. Investigamos a natureza genética de dois pacientes XP-G, que são irmãos e apresentam fenótipo moderado. As células destes pacientes demonstraram alta sensibilidade à luz UVC. Quando expostas a um agente oxidativo, apenas células XP-G/CS exibiram sensibilidade. Identificamos duas mutações missense no gene XPG desses pacientes, e comparamos com outras mutações existentes. Observamos que as mutações possuem um impacto negativo na funcionalidade de XPG. A proteína com a mutação p.Ala28Asp exibiu uma atividade residual em testes de complementação. Os resultados indicam que o fenótipo XP-G desses pacientes é causado por duas mutações missense em heterozigose composta, e que células portadoras dessas alterações exibem respostas diferenciadas frente aos estresses genotóxicos causados pela luz UV e pelo agente oxidativo utilizado. / NER is the most flexible of all known DNA repair mechanisms. XPG is an endonuclease that participates in the final steps of NER. Mutations in this gene may result in the human syndrome XP and, in some cases, in the XP/CS. We investigated the genetic nature in two XP-G patients, siblings and mildly affected. The cells from these patients demonstrated the high UV sensitivity typical of this syndrome. When exposed to an oxidative agent, only XP-G/CS cells exhibited sensitivity. We identified two missense mutations in the XPG gene of these patients, and a comparison with other known mutations is presented. These mutations have a negative impact in the function of XPG. The protein harboring the mutation p.Ala28Asp exhibited residual activity in complementation tests. These results indicate that the phenotype of XP-G patients is caused by two missense mutations in a compound heterozygous manner, and that the cells carrying these alterations exhibit different responses against genotoxic stress caused by the UV light and by the oxidative agent used.

Colon cancer is one of the most commonly diagnosed cancers in the US, yet small intestine cancer is a rare event. While there are many similarities between these two tissues, inherent differences such as redox status, may contribute to the variation in cancer occurrence. We examined the difference in reactive oxygen species (ROS) generation, antioxidant enzyme activity and oxidative DNA damage in the small and large intestine of rats under normal conditions and following exposure to exogenous oxidative stress. Basal ROS and antioxidant enzyme activities were greater in the colon than the small intestine, and the balance of ROS to antioxidant enzymes in the colon was more pro-oxidant than in the small intestine. During oxidative stress, ROS and oxidative DNA damage were greater in the colon than the small intestine. Thus the colon responds to oxidative stress less effectively than the small intestine, possibly contributing to increased cancer incidence at this site. We next wanted to understand how diets containing a combination of fish or corn oil and pectin or cellulose may alter the redox environment of the colon. ROS, oxidative DNA damage, antioxidant enzyme activity and apoptosis were measured in colonocytes of rats fed one of four diets containing either corn oil or fish oil and cellulose or pectin. Measurements were madein rats untreated with carcinogen and rats exposed to a chemical carcinogen and radiation. In rats not treated with a carcinogen, fish oil enhanced ROS, and fish oil/pectin suppressed antioxidant enzymes as compared to corn oil/cellulose. Oxidative DNA damage was inversely related to ROS in the fish oil/pectin diet and apoptosis was enhanced relative to other diets. In carcinogen treated and irradiated rats, a similar protective effect was seen with fish oil/pectin as evidenced by a reduction in oxidative DNA damage and enhancement of apoptosis. This suggests that a diet containing fish oil/pectin may protect against colon carcinogenesis by modulation of the redox environment to promote apoptosis and minimize oxidative DNA damage.

Research efforts to determine the causes, effects and locations of mutations within the human genome have been widely pursued due to their role in the development of various diseases. The main cause of mutations in vivo is oxidative damage to DNA via oxidants and free radical species. Numerous studies have been performed in vitro to determine how oxidative damage is induced in DNA. Most of these in vitro studies require photosensitizers to initiate the oxidative damage through various mechanisms. For the purposes of this research, all the photosensitizers that were used initiated oxidative damage in DNA through the electron transfer mechanism. In the charge transport studies, an anthraquinone photosensitizer was covalently linked to the 5 end of DNA by a short carbon tether in order to determine the pattern of damage induced along the length of the DNA. Anthraquinone preferentially damages guanine bases. Our first work sought to determine the effects of charge transport through guanine rich quadruplex DNA dimers. The dimers were formed by the combination of two hairpins with duplex overhangs extending beyond the quadruplex region. This enabled the optimal comparison of the effects of charge transport between duplex and quadruplex DNA structures. Another area of research we pursued in this area was to determine the effects of charge transport in M-DNA (a novel DNA conformation that was reported to form in the presence of zinc ions at a pH above 8). Earlier work on M-DNA suggested that it behaved like a molecular wire. Our research attempted to determine the effects of charge transport on this structure in order to show the behavior of a DNA molecular wire as compared to the standard studies performed in this area on normal B-DNA structures. Lastly, in collaboration with Dr. Ramaiah and colleagues we designed some viologen linked acridine photosensitizers which were tested for any ability to cleave GGG bulges. In preliminary studies, these viologen linked acridine derivatives showed preferential cleavage for guanine bases. They were not covalently bound to DNA, although they could potentially form non covalent interactions with DNA such as intercalation and/or groove binding. Our overall research goal was to determine the extent and overall effect of oxidative damage (using different photosensitizers) on the various DNA structures mentioned above.

Colon cancer is one of the most commonly diagnosed cancers in the US, yet small intestine cancer is a rare event. While there are many similarities between these two tissues, inherent differences such as redox status, may contribute to the variation in cancer occurrence. We examined the difference in reactive oxygen species (ROS) generation, antioxidant enzyme activity and oxidative DNA damage in the small and large intestine of rats under normal conditions and following exposure to exogenous oxidative stress. Basal ROS and antioxidant enzyme activities were greater in the colon than the small intestine, and the balance of ROS to antioxidant enzymes in the colon was more pro-oxidant than in the small intestine. During oxidative stress, ROS and oxidative DNA damage were greater in the colon than the small intestine. Thus the colon responds to oxidative stress less effectively than the small intestine, possibly contributing to increased cancer incidence at this site. We next wanted to understand how diets containing a combination of fish or corn oil and pectin or cellulose may alter the redox environment of the colon. ROS, oxidative DNA damage, antioxidant enzyme activity and apoptosis were measured in colonocytes of rats fed one of four diets containing either corn oil or fish oil and cellulose or pectin. Measurements were madein rats untreated with carcinogen and rats exposed to a chemical carcinogen and radiation. In rats not treated with a carcinogen, fish oil enhanced ROS, and fish oil/pectin suppressed antioxidant enzymes as compared to corn oil/cellulose. Oxidative DNA damage was inversely related to ROS in the fish oil/pectin diet and apoptosis was enhanced relative to other diets. In carcinogen treated and irradiated rats, a similar protective effect was seen with fish oil/pectin as evidenced by a reduction in oxidative DNA damage and enhancement of apoptosis. This suggests that a diet containing fish oil/pectin may protect against colon carcinogenesis by modulation of the redox environment to promote apoptosis and minimize oxidative DNA damage.

Influence of stress factors on tunneling nanotubes in cultivated human retinal pigment epithelial cells (ARPE-19). The eye as one of the most important sense organs of the human body is exposed to visible light radiation and other stress factors every day. Especially the retina (of the eye) is a sensible tissue for oxidative damage (Wu et al., 2006). The retinal pigment epithelium (RPE) is an important layer of the retina, which forms the outer layer and phagocytises the shed disc membranes of the photoreceptor outer segments. Furthermore, the RPE is involved in the maintenance of the visual cycle and regulates the retinal balance (Bok, 1993). To maintain those functions, a steady communication between the RPE-cells and the adjacent neighbour cells is necessary. Tunneling nanotubes (TNTs) build a newly discovered variety of cell communication and thus establish intercellular signal transduction and transport different cell components including pathogens (Rustom et al., 2004; Onfelt et al., 2006; Sherer und Mothes, 2008; Veranic et al., 2008). The formation of TNTs in the neuron-like pheochromocytoma cell line PC12 was first reported by Rustom et al in 2004. In the following years a growing number of cell types containing TNTs were described. For example a lot of TNT-reports were found between immune cells (Onfelt et al., 2004; Sowinski et al., 2008). Chinnery et al. first described TNTs in vivo in 2008. Here they found TNTs between dendritic cells in the cornea of the mouse. An important characteristic of TNTs is that they do not attach to the substratum. They contain F-actin as a characteristic feature of there structure (Rustom et al., 2004). Our study group detected the formation of TNTs between ARPE-19-cells, a human retinal pigment epithelial cell line. They contain F-actin, but no microtubules. Further it was observed an exchange of electrical signals, small molecules and even the transfer of organelles between cells via TNTs (see publication Wittig et al., 2012). It is often described in the literature, that TNTs are very sensitive against stress factors, like prolonged light excitation, mechanical and chemical stress, which then can result in rupture of the TNTs (Rustom et al., 2004; Koyanagi et al., 2005; Gurke et al., 2008a; Pontes et al., 2008; Sowinski et al., 2008; Domhan et al., 2011; Wang und Gerdes, 2012). Up to now it is widely unclear how pathological conditions influences TNTs. There are several studies, which report an induction but also an inhibition of TNT-formation by different factors. The reaction of cell-cell-interactions between RPE cells on stress factors is not jet analysed. So our motivation was, to analyse the influence of different stress factors on the number, the morphology and formation of TNTs. ARPE-19-cells were treated with blue light, with a wavelength of 470 and 405 nm, with 3000 μM glyoxal, with 200 μM H2O2, with medium without serum as well as with cytochalasin-D and latrunculin-B. With the help of differential interference contrast (DIC) microscopy the formed TNTs were counted and the morphology was evaluated. A 24 hours cultivation of untreated ARPE-19 cells resulted in 15 TNTs per 100 cells on average. After excitation of the ARPE-19-cells with blue light 470 and 405 nm the number of TNTs decreased 50 % and 28,5 % accordingly in comparison to untreated cells (100 %). Furthermore, the cell culture, which was treated with glyoxal and H2O2 resulted in a reduction of 17,5 % and 53 % TNTs in comparison to the untreated cell culture. Cells which were cultured with serum free medium had an decreased TNT-number of 56.8 % in comparison with serum containing medium. TNTs of untreated ARPE-19-cells have a diameter from 50 to 300 nm (Wittig et al., 2012). Every TNTs, which were formed under named stress factors had the same diameter like untreated cells. In this study an average TNT length of 23 +/- 16 μm was measured between cells without treatment. This correlated with the TNT-lengths of cells which excitated with blue light 405 and 470 nm with 26 +/- 13 μm and 24 +/- 14 μm. In contrast the TNT-lenghts of cells treated with glyoxal and H2O2 with 16 +/- 11 μm and 15 +/- 13 μm were less and from cells cultured without serum with 34 +/- 20 μm were above the average length of TNTs of untreated cells. TNTs of ARPE-19-cells without treatment and TNTs which were treated with stress factors contained F-actin but no microtubules. Depolymerisation of F-actin, induced by addition of cytochalasin-D or latrunculin-B, led to disappearance of TNTs. This is an evidence for the importance of F-actin as an essential component of TNTs between ARPE-19-cells. Under the influence of blue light excitation the TNTs formed as good as untreated cells after contact of migrating cells. Reason for the reduced TNT-formation under stress factors could be explained by the generation of oxidative stress due to reactive oxygen species (ROS). ROS induced under blue light- or glyoxal-treatment as well as H2O2 could influence cell function by inactivation of cell-mediated proteins or induction of F-actin oxidation with subsequent destruction of the actin-network and inhibition of the actin-polymerisation (Chen, 1993; Ballinger et al., 1999; Thornalley et al., 1999; Valen et al., 1999; Dalle-Donne et al., 2002; Nilsson et al., 2003; Shangari und O'Brien, 2004; Zhu et al., 2005; Knels et al. 2008; Roehlecke et al., 2009). The reduced actin-polymerisation as well as the disruption of the TNTs due to changes at the actin-cytoskeleton and at the membranes could explain the reduced TNT-formation (Valen et al., 1999; Dalle-Donne et al., 2002; Reber et al., 2002; Zhu et al., 2005; Knels et al., 2008). The inhibition of the cell growth under oxidative stress conditions and under nutritional deficiency by serum free medium could lead to a reduced TNT-formation too. In this study we found a reduction of TNT-number between ARPE-19-cells under different stress conditions. It is possible, that TNTs are formed between RPE- and photoreceptor-cells in vivo, where they can exchange useful or recyclable materials between cells (Wang et al., 2011; Wittig et al., 2012). Disruption of TNTs by reactive oxygen species could cause a decreased exchange of informations. It is possible, that the cells, RPE- as well as photoreceptor-cells, die due to a deficiency of nutrients. This could be another reason in the formation of age related macular degeneration, which shows a destruction of RPE-cells and secondary of the photoreceptorcells. / Das Auge ist als eines der wichtigsten Sinnesorgane des Menschen täglich sichtbarer Lichtstrahlung und weiteren Stressfaktoren ausgesetzt. Die Netzhaut des Auges ist besonders empfindlich für oxidative Schäden (Wu et al., 2006). Eine bedeutende Schicht der Netzhaut im Auge stellt das retinale Pigmentepithel (RPE) dar, welches die äußere Schicht der Retina bildet und täglich die abgeworfenen Photorezeptoraußensegmentscheiben phagozytiert. Zudem ist das RPE wesentlich am visuellen Prozess sowie der Aufrechterhaltung des retinalen Gleichgewichts beteiligt (Bok, 1993). Um diese Funktionen zu gewährleisten, ist eine ständige Kommunikation zwischen den RPEZellen sowie zu angrenzenden Nachbarzellen innerhalb der Netzhaut notwendig. So ist über Tunneling Nanotubes (TNTs), als neu entdeckte Kommunikationsform, ein interzellulärer Transport von Signalen und verschiedensten Zellkomponenten, aber auch von Pathogenen, möglich (Rustom et al., 2004; Onfelt et al., 2006; Sherer und Mothes, 2008; Veranic et al., 2008). Erstmals 2004 beschrieben Rustom et al. die Bildung von TNTs zwischen Rattennierenzellen in vitro. In den folgenden Jahren kam es zu einer Vielzahl weiterer TNT-Entdeckungen zwischen verschiedensten Zellen in vitro. So findet man zum Beispiel vermehrt TNTBeschreibungen zwischen Immunzellen (Onfelt et al., 2004; Sowinski et al., 2008). Ein erster Nachweis an TNTs in vivo erfolgte 2008 durch die Arbeitsgruppe Chinnery et al.. Hierbei fand man TNTs zwischen dendritischen Zellen in der Mauscornea. Ein wichtiges Merkmal von TNTs ist, dass sie sich als frei im Medium schwebende interzelluläre Verbindungen darstellen, ohne Kontakt zum Substrat zu haben. TNTs sind im Wesentlichen als stabilisierendes Hauptstrukturmerkmal aus Aktin aufgebaut (Rustom et al., 2004). In unserer Arbeitsgruppe wurde die Bildung von TNTs zwischen ARPE-19-Zellen, einer humanen Pigmentepithelzelllinie, entdeckt. Neben dem strukturellen Aufbau aus Aktin, konnte ein Austausch von elektrischen Signalen sowie molekularen Stoffen und der Transport von Organellen (Mitochondrien) durch TNTs zwischen ARPE-19-Zellen nachgewiesen werden (siehe Publikation Wittig et al., 2012). Wie schon mehrfach in der Literatur beschrieben, reagieren TNTs sehr sensibel auf Stressfaktoren, so zum Beispiel auf längere Lichtreizung, mechanischen und chemischen Stress, was jeweils zur Ruptur der Strukturen führen kann (Rustom et al., 2004; Koyanagi et al., 2005; Gurke et al., 2008; Pontes et al., 2008; Sowinski et al., 2008; Domhan et al., 2011; Wang und Gerdes, 2012). Weitgehend unklar ist bisher der Einfluss von pathologischen Bedingungen auf die TNTs. Es gibt mehrere Studien, in denen durch verschiedenste Faktoren über eine Induktion, aber auch über eine Hemmung der TNT-Bildung berichtet wurde. Die Reaktion von Zell-Zell-Interaktionen zwischen RPE-Zellen auf Stressfaktoren wurde bisher in wissenschaftlichen Arbeiten nicht untersucht. Dies nahmen wir zum Anlass, den Einfluss von unterschiedlichen Stressfaktoren auf die Anzahl von TNTs, ihre Morphologie und Bildung zu untersuchen. Es erfolgte eine Behandlung der ARPE-19-Zellen mit Blaulicht in den Wellenlängen 405 und 470 nm, mit 3000 μM Glyoxal, mit 200 μM H2O2, mit serumfreiem Medium sowie mit Cytochalasin D und Latrunculin B. Die gebildeten TNTs wurden anschließend mit Hilfe der Lichtmikroskopie ausgezählt sowie deren Morphologie beurteilt. So bildeten unbehandelte ARPE-19-Zellen nach 24 Stunden Kultivierung im Durchschnitt 15 TNTs pro 100 Zellen aus. Nach 24stündiger Bestrahlung der ARPE-19-Zellen mit Blaulicht 470 nm und 405 nm fiel die TNT-Anzahl auf 50 % und 28,5 % im Vergleich zu unbehandelten Zellen (100 %). Weiterhin fanden sich in den Glyoxal- und H2O2-behandelten Kulturschalen 17,5 % und 53 % TNTs verglichen mit der unbehandelten Zellkultur. In der serumfreien Kulturschale verringerten sich die TNTs 24 Stunden nach Ausplattierung der Zellen auf 56,8 % im Vergleich zu in Medium mit Serum kultivierten Zellen. TNTs unbehandelter ARPE-19-Zellen besitzen einen Durchmesser von 50 bis 300 nm (Wittig et al., 2012). Alle unter oben genannten Stressfaktoren gebildeten TNTs befanden sich in Hinblick auf ihren Durchmesser im Bereich der TNTs unbehandelter Zellen. Bei TNTs unbehandelter Zellen wurde in dieser Arbeit eine durchschnittliche Länge von 23 +/- 16 μm gemessen. Dies entsprach dem TNT-Längendurchschnitt von mit Blaulicht 405 nm und 470 nm bestrahlter ARPE-19-Zellen mit 26 +/- 13 μm und mit 24 +/- 14 μm. Unter Glyoxal und H2O2 gebildete TNTs lagen im Gegensatz dazu mit 16 +/- 11 μm und 15 +/- 13 μm unterhalb und unter serumfreier Kultivierung mit 34 +/- 20 μm über dem TNTLängendurchschnitt unbehandelter Zellen. Alle TNTs, sowohl unbehandelter als auch mit Stressfaktoren behandelter ARPE-19-Zellen, sind aus Aktin aufgebaut. Jedoch ließ sich kein Tubulin nachweisen. Nach Zugabe von Aktinpolymerisationshemmern waren keine TNTs nachweisbar, was beweist, dass F-Aktin essentieller Bestandteil von TNTs zwischen ARPE-19-Zellen ist. Unter dem Einfluss von Blaulicht 470 und 405 nm bildeten sich die TNTs, wie auch bei unbehandelten Zellen, durch ein Zusammentreffen der Zellen mit anschließendem Auseinandergleiten. Die Ursache für die verminderte Bildung an TNTs unter verschiedenen Stressfaktoren könnte in der Entstehung von oxidativem Stress durch die Ausbildung von reaktiven Sauerstoffspezies (ROS) begründet sein. So können zum Beispiel die unter Blaulicht- und Glyoxalexposition entstehenden ROS sowie H2O2, als eine Hauptform der ROS, die Zellfunktion durch Inaktivierung zellulärer Proteine beeinflussen sowie eine direkte Oxidation an Aktin hervorrufen mit folglicher Aktinnetzwerkzerstörung und Hemmung der Aktinpolymerisation (Chen, 1993; Ballinger et al., 1999; Thornalley et al., 1999; Valen et al., 1999; Dalle-Donne et al., 2002; Nilsson et al., 2003; Shangari und O'Brien, 2004; Zhu et al., 2005; Knels, Worm et al. 2008; Roehlecke et al., 2009). Die verminderte Aktinpolymerisation, aber auch die Zerreißungen der TNTs durch Veränderungen am Aktinzytoskelett sowie an den Membranen könnten zu einer verringerten TNT-Bildung führen (Valen et al., 1999; Dalle-Donne et al., 2002; Reber et al., 2002; Zhu et al., 2005; Knels et al., 2008). Auch eine Hemmung des Zellwachstums unter oxidativen Stressbedingungen sowie unter Nährstoffmangel durch Serumentzug könnte mit einer verminderten TNT-Bildung einhergehen. Wir haben in unserer Untersuchung gezeigt, dass es durch verschiedene Stresseinflüsse zu einer Reduktion der TNTs zwischen ARPE-19-Zellen kommt. Es ist denkbar, dass solche TNTs in vivo zwischen RPE- und Photorezeptorzellen ausgebildet werden, wo sie nützliches oder recycelbares Material zwischen Zellen austauschen (Wang et al., 2011; Wittig et al., 2012). Bei Zerstörung der TNTs durch zum Beispiel oxidative Faktoren könnte es zu einer Verringerung des Informationsaustausches kommen. Es ist möglich, dass durch die Minderversorgung die Zellen absterben, sowohl RPE- als auch Photorezeptorzellen. Dies könnte ein weiterer möglicher Ursachenansatz in der Entstehung der altersabhängigen Makuladegeneration sein, welche als Erkrankungserscheinung den Untergang der RPEZellen und damit sekundär der Photorezeptorzellen aufweist.

Observational studies have indicated that fruit and vegetables, and dietary antioxidants may play an important role in reducing the risk of chronic diseases, potentially by affecting pathogenic mechanisms such as oxidative stress and inflammation. Clinical trials investigating the effects of supplementation with single or a few antioxidants in high doses have, however, shown inconsistent results and thus have not been able to support the observational findings. It was therefore hypothesised that a supplement, containing a combination of antioxidants mainly extracted from fruit and vegetables, and supplied at moderate doses, might act more beneficially than single antioxidants given at pharmacological doses. The effects of such a supplement were investigated in two interventional studies described in this thesis. The effects on antioxidant status, metabolic control, oxidative stress and inflammation were investigated in overweight men and in patients with type 2 diabetes, subjects that could be expected to have elevated levels of oxidative stress and inflammatory activity. The results of the studies did not support the hypothesis that supplementation with antioxidants from fruit and vegetables may have beneficial effects by counteracting oxidative stress and inflammation, despite markedly increased plasma antioxidant concentrations. However, interesting associations were observed in diabetes patients at baseline between intake of antioxidant rich food as well as levels of antioxidants in plasma, and markers of oxidative stress and inflammation. These associations are compatible with the hypothesis that a high intake of fruit and vegetables and dietary antioxidants decrease oxidative stress levels, have anti-inflammatory effects and a beneficial influence on glycaemic control. The results also indicated that glycaemic control may affect the level of oxidative stress. The absence of beneficial effects from antioxidants might to some extent be explained by the initial levels of oxidative stress and inflammation and by the antioxidative status in the subjects included in the studies. Since the levels generally were comparable with those observed in healthy subjects, this might have decreased the ability to observe any beneficial effects of supplementation with additional antioxidants. Continued investigations are needed to characterise the individuals who potentially might benefit from antioxidant supplementation. In view of apparent positive effects from a high intake of fruit and vegetables found in observational studies and until more knowledge is available from interventional trials about possible benefits and potential risks of antioxidant supplementation it still seems reasonable to recommend a diet rich in fruit and vegetables.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / Anabolic androgenic steroids (AAS) are synthetic substances derived from testosterone that promote greater muscle mass and strenght. Thus, they are used illegally to improve athletic performance of horses, dogs or athletes or to improve meat production. The doses, ranging from 10 to100 times the therapeutic recommendation, enhances the deleterious effects on various organs. The objective of this study was to evaluate the effects of different protocols (P1, P2 and P3) of boldenone undecylenate (BU) and stanozolol (ST) on markers of liver and kidney function and variables of oxidative stress in these organs. For this, 54 male Wistar rats were divided into nine groups of six animals each. Each animal received intramuscularly 5.0 mg kg-1 of BU or ST once a week for four weeks (P1); 2.5 mg kg-1 of BU or ST once a week for eight weeks (P2); and 1.25 mg kg-1 of BU or ST once a week for 12 weeks (P3). For each protocol, a control group was used (CG), and they received 0.1 ml of olive oil intramuscularly. Blood, and fragments of liver and kidney were collected for alanine aminotransferase activity (ALT), alkaline phosphatase (ALP), albumin, creatinine, cholesterol, total protein, triglycerides, urea, reactive oxygen species (ROS), thiobarbituric acid reactive substances (TBARS), total thiols (T-SH), and glutathione (GSH) evaluation. Seric ALT activity and cholesterol concentration were significantly (p<0.05) higher compared to CG when BU of protocol P1 was used. ALT activity was significantly higher (p<0.05) compared to the CG in protocol P2 when ST was used. Liver samples showed higher levels (p<0.05) of ROS and TBARS in protocols P1 and P3 when BU was used, and lower GSH activity (p<0.05) on group treated with protocol P3. Rats that have received ST under protocol P1 and P3 showed higher levels (p<0.05) of ROS, as well as increased TBARS levels in P3 but lower GSH activity in P3 (p<0.05) when compared to the CG. In the liver, the T-SH concentration was lower (p<0.05) in P2 when compared BU and ST of the CG. In renal tissues, ROS and TBARS levels were significantly higher (p<0.05) in animals that received BU under protocols P1 and P2; and GSH activity and T-SH levels were reduced in the three protocols (P1, P2 and P3). In addition, animals treated with ST occurred showed reduced renal levels of GSH levels (p<0.05) in P2 and P3. The treatment with ST also led to higher ROS levels (p<0.05) in P2 and P3, and TBARS levels in P3, but reduced concentration (p<0.05) of GSH levels in P2 and P3, and T-SH in P2 and P3. In conclusion, anabolic steroids are harmful even when used in low doses or in a few applications, since in all evaluated protocols was possible to observe changes in the redox balance in the liver and kidneys. / Esteroides anabólicos androgênicos (EAA) são substâncias sintéticas derivadas da testosterona que promovem crescimento muscular e ganho de força. Devido a isso, são utilizadas ilegalmente para melhora da performance atlética de equinos, caninos ou atletas ou para maior produção de carne. As doses variam de 10 a 100 vezes a recomendação terapêutica, o que potencializa os efeitos deletérios em diversos órgãos. O objetivo deste trabalho foi avaliar os efeitos de diferentes protocolos de administração (P1, P2 e P3) de undecilenato de boldenona (UB) e estanozolol (EST) em indicadores da função e lesão, bem como, parâmetros oxidativos hepático e renal. Para isso, foram utilizados 54 ratos Wistar, machos, distribuídos em nove grupos com seis animais cada que receberam por via intramuscular 5 mg/kg de UB ou EST uma vez por semana durante 4 semanas (P1); 2,5mg/kg de UB ou EST uma vez por semana durante 8 semanas (P2) e 1,25mg/kg de UB ou EST uma vez por semana durante 12 semanas (P3). Cada protocolo teve um grupo controle (GC) que recebeu 0,1 mL de azeite de oliva intramuscular. Posteriormente a eutanásia, realizada uma semana após o último tratamento, foi avaliada a atividade sérica da alanina aminotransferase (ALT) e fosfatase alcalina (FA), os níveis séricos de albumina, creatinina, colesterol, proteínas totais, triglicerídeos, ureia, espécies reativas de oxigênio (ERO), substâncias reativas ao ácido tiobarbitúrico (TBARS), glutationa reduzida (GSH) e tiois totais (T-SH). No protocolo P1 obteve-se atividade sérica de ALT e concentração de colesterol significativamente (p<0,05) maiores comparando-se o grupo UB com o grupo GC. O grupo EST obteve aumento significativo (p>0,05) da ALT em relação ao grupo controle no protocolo P2. No tecido hepático, comparando-se os grupos UB e GC, obteve-se níveis maiores (p<0,05) de ERO e TBARS em P1 e P3 e concentração menor (p<0,05) de GSH em P3. O grupo EST apresentou valores maiores (p<0,05) de ERO no P1 e P3, de TBARS no P3 e níveis menores (p<0,05) de GSH no P3 quando comparado ao grupo GC. A concentração hepática de T-SH foi menor (p<0,05) no P2 comparando UB e EST ao grupo GC. No tecido renal, ao comparar o grupo UB com o grupo GC obteve-se níveis significativamente maiores (p<0,05) de ERO e TBARS nos protocolos P1 e P2 e menores (p<0,05) de GSH e T-SH nos protocolos P1, P2 e P3. Comparando-se os grupos EST e GC os níveis de GSH foram menores (p<0,05) no P2 e P3. O grupo EST apresentou níveis maiores (p<0,05) de ERO nos protocolos P2 e P3, de TBARS no P3, concentração menor (p<0,05) de GSH no P2 e P3 e níveis menores de T-SH no P2 e P3 quando comparado ao grupo GC. Neste estudo foi possível concluir que os anabolizantes são prejudicias mesmo quando utilizados em baixas doses ou em poucas aplicações, visto que em todos os protocolos avaliados foi possível observar alterações do balanço redox no fígado e rins dos ratos.

Made available in DSpace on 2014-12-17T14:16:28Z (GMT). No. of bitstreams: 1 NairaJNB_DISSERT.pdf: 5441332 bytes, checksum: 777fb7ac704e3cf8d4b43b60ea801b57 (MD5) Previous issue date: 2009-03-26 / Recently, it has been a increasing interest in the antioxidative role of natural products to aid the endogenous protective biological systems against the deleterious effects of oxygen (ROS) and nitrogen (RNS) reactive species. Many antioxidant compounds, naturally occurring from plant sources. Natural antioxidants can protect and prevent the human body from oxidative stress and retard the progress of many diseases in which free radical are involved. Several plants used in the folk medicine to treat certain disorders that are accompanied by inflammation and other pharmacological properties have been proved their attributed properties, such antioxidant activity. Turnera ulmifolia Linn. var. elegans (Turneraceae), frequently employed by population as a medicinal plant, demonstrated antioxidant activity by in vitro and in vivo assays, using its leaf hydroethanolic extract (10%) he in vitro DPPH radical-scanvenging activity showed a strong antioxidant activity (86.57% ? 0.14), similar to Carduus marianus and catequine effects. For the in vivo assays, adult female Wistar rats (n=48) with carbon tetrachloride hepatic injury induced (2,5mL/kg i.p.) were used, Six groups or rats were uses (n=8) [G1 = control (1,25 mL/kg i.p. vehicle); G2 = CCl4 (2,5 mL/kg i.p.); G3 = CCl4 + extract 7 days (500 mg/kg p.o.); G4 = CCl4 + Legalon? 7 days (50 mg/kg p.o.), G5 = CCl4 + extract 21 days (500 mg/kg p.o.) e G6 = CCl4 + Legalon? 21 days (50 mg/kg p.o.)]. The hepatic oxidative injury was evaluated through biochemical parameters [alanine amino transferase (ALT), aspartate amino transferase (AST)] histopathological study, while thiobarbituric acid reactive products (TBAR), glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx) levels were used to evaluate proantioxidant parameters. The plant extract tested was found effective as hepatoprotective as evidenced by a decreasing in the ALT and AST activities (p<0.001) and TBAR (plasma, p<0.001 and liver, p<0.001). Levels of GSH (blood, p<0.001 and liver, p<0.001) and antioxidant enzymes [CAT erythrocyte (p<0.05) and hepatic (p<0.01); SOD erythrocyte (p<0.001) and hepatic (p<0.001); GPx erythrocyte (p<0.001) and hepatic (p<0.001)] were also significantly increased. Histopathological changes induced by CCl4 were significantly reduced by the extract treatment. The data obtained were comparable to that of Legalon?, a reference hepatoprotective drug. The results showed that T. ulmifolia leaf extract protects against CCl4 induced oxidative damage. Therefore, this effect must be associated to its antioxidant activity, attributed to the phenolic compounds, present in these extract, which can act as free radical scavengers / Atualmente existe um crescente interesse no estudo dos antioxidantes, classe de subst?ncias que protegem os sistemas biol?gicos dos efeitos delet?rios das esp?cies reativas de oxig?nio (ERO) e de nitrog?nio (ERN). Muitos desses antioxidantes s?o mol?culas de origem vegetal que contribuem para a preven??o e para o tratamento de doen?as nas quais o estresse oxidativo est? envolvido. Na medicina popular, plantas com comprovada atividade antiinflamat?ria, apresentam diversas atividades farmacol?gicas, dentre elas, a antioxidante. Turnera ulmifolia Linn. var. elegans (Turneraceae), uma planta medicinal, largamente utilizada pela popula??o, apresentou atividade antioxidante quando avaliada in vitro e in vivo, utilizando-se o extrato hidroetan?lico (10 %) obtidos das folhas dessa planta. A capacidade antioxidante in vitro desse extrato foi avaliada atrav?s do seq?estro do radical 1,1-difenil-2- picril-hydrazyl (DPPH ). O resultado apresentou um marcado potencial antioxidante ao seq?estrar 86,57%?0,14 do radical DPPH , compar?vel ao obtido com as subst?ncias de refer?ncia. No ensaio in vivo, empregando-se modelo experimental murino (48 ratas Wistar), inj?ria hep?tica foi induzida com CCl4 (2,5 mL/kg i.p. dose ?nica) e o dano oxidativo hep?tico foi avaliado atrav?s da atividade das transaminases (ALT e AST) e pelo exame histopatol?gico, enquanto que as subst?ncias reativas ao ?cido tiobarbit?rico (SRAT), conte?do de glutationa reduzida (GSH) e atividade das enzimas catalase (CAT), super?xido dismutase (SOD) e glutationa peroxidase (GPx) foram os par?metros pr?-antioxidante avaliados. Os animais foram divididos em seis grupos (n=8) [G1 = controle (1,25 mL/kg i.p. de ve?culo); G2 = CCl4 (2,5 mL/kg i.p.); G3 = CCl4 + extrato 7 dias (500 mg/kg p.o.); G4 = CCl4 + Legalon? 7 dias (50 mg/kg p.o.), G5 = CCl4 + extrato 21 dias (500 mg/kg p.o.) e G6 = CCl4 + Legalon? 21 dias (50 mg/kg p.o.)]. O p?s-tratamento com o extrato provocou um decr?scimo na atividade da ALT e AST (p<0,001) e de SRAT (plasma, p<0,001 e f?gado, p<0,001), aumentando o conte?do da GSH (sangue, p<0,001 e em f?gado, p<0,001), bem como a atividade das enzimas antioxidantes, [CAT eritrocit?ria (p<0,05) e hep?tica (p<0,01); SOD eritrocit?ria (p<0,001) e hep?tica (p<0,001); GPx eritrocit?ria (p<0,001) e hep?tica (p<0,001)]. A an?lise histopatol?gica mostrou uma redu??o do dano hep?tico causado pelo CCl4. Os resultados obtidos com o extrato foram an?logos aqueles do tratamento com Legalon?, padr?o antioxidante, utilizado in vivo. Com base nesses dados, conclui-se que o extrato de folhas de T. ulmifolia apresenta capacidade hepatoprotetora diante do quadro de estresse oxidativo induzido pelo CCl4. Efeitos estes, que, provavelmente, est?o associados ? atividade antioxidante detectada, atribu?das ?s subst?ncias fen?licas presentes no extrato, que devem agir como seq?estradores de radicais livres, bloqueando as rea??es em cadeias

O NER é uma via de reparo de DNA capaz de lidar com uma ampla variedade de lesões. Participam do NER diversas proteínas, entre elas a endonuclease XPG. Pacientes que possuem mutações no gene XPG apresentam a síndrome XP, e em alguns casos XP/CS. Investigamos a natureza genética de dois pacientes XP-G, que são irmãos e apresentam fenótipo moderado. As células destes pacientes demonstraram alta sensibilidade à luz UVC. Quando expostas a um agente oxidativo, apenas células XP-G/CS exibiram sensibilidade. Identificamos duas mutações missense no gene XPG desses pacientes, e comparamos com outras mutações existentes. Observamos que as mutações possuem um impacto negativo na funcionalidade de XPG. A proteína com a mutação p.Ala28Asp exibiu uma atividade residual em testes de complementação. Os resultados indicam que o fenótipo XP-G desses pacientes é causado por duas mutações missense em heterozigose composta, e que células portadoras dessas alterações exibem respostas diferenciadas frente aos estresses genotóxicos causados pela luz UV e pelo agente oxidativo utilizado. / NER is the most flexible of all known DNA repair mechanisms. XPG is an endonuclease that participates in the final steps of NER. Mutations in this gene may result in the human syndrome XP and, in some cases, in the XP/CS. We investigated the genetic nature in two XP-G patients, siblings and mildly affected. The cells from these patients demonstrated the high UV sensitivity typical of this syndrome. When exposed to an oxidative agent, only XP-G/CS cells exhibited sensitivity. We identified two missense mutations in the XPG gene of these patients, and a comparison with other known mutations is presented. These mutations have a negative impact in the function of XPG. The protein harboring the mutation p.Ala28Asp exhibited residual activity in complementation tests. These results indicate that the phenotype of XP-G patients is caused by two missense mutations in a compound heterozygous manner, and that the cells carrying these alterations exhibit different responses against genotoxic stress caused by the UV light and by the oxidative agent used.

Hyperthyroidism may lead to an increase in oxidative stress (OS) in testis, may cause male reproductive disorders, among them, the loss in the sperm quality. The effect of resveratrol (RSV) on sperm motility and on OS parameters in testis of euthyroid and hyperthyroid rats was investigated. Hyperthyroidism was induced by daily intraperitoneal injection (i.p.) of triiodothyronine (T3) (100 μg/kg, i.p.) for six weeks. After two weeks, concomitantly to T3 treatment, animals received daily injections of RSV at dose of 1 mg/kg e 10 mg/kg (i.p.) during four weeks, totaling six experimental weeks. At the end of experimental period animals were euthanized for removal of organs. Sperm of cauda epididymal was collected for sperm motility and morphology analysis Testis were homogenized for determination of lipoperoxidation by lipid hydroperoxides and thiobarbituric reactive substances (TBARS) levels; activity of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione-S-transferase (GST); and non-enzimatic antioxidant glutathione (GSH). Hyperthyroid rats presented lower sperm motility, higher lipid hydroperoxides and TBARS levels, lower CAT and GPx activities and higher GST activity in testis than animals of control groups. RSV treatment at doses of 1 mg/kg and 10 mg/kg was able to prevent the loss on sperm motility induced by hyperthyroidism. In addition, RSV decreased lipid hydroperoxides and TBARS levels; reversed the decrease in CAT and GPx activities; and also prevented the increase in GST activity caused by hyperthyroidism in adult rat testis. Together these data show the protective effect of RSV in the testis, preserving sperm motility, and protecting testis against oxidative damage caused by hyperthyroidism, which suggests the RSV as a possible target of studies in the search for therapeutic strategies in order to preserve testicular function. / O hipertireoidismo pode levar ao aumento do estresse oxidativo (EO) nos testículos, podendo causar desordens na função reprodutiva masculina, entre elas, a perda na qualidade espermática. Este estudo avaliou o efeito do resveratrol (RSV) na qualidade espermática e em parâmetros de EO no testículo de ratos eutireóideos e hipertireóideos. O hipertireoidismo foi induzido pela injeção intraperitoneal (i.p) diária de triiodotironina (T3) (100 μg/kg) durante seis semanas. Após duas semanas do início do experimento, concomitante ao tratamento com T3, os animais começaram a receber injeções diárias de RSV nas doses de 1 mg/kg ou 10 mg/kg (i.p.) por quatro semanas, totalizando as seis semanas de tratamento. No final do período experimental, os animais foram eutanasiados para retirada dos órgãos. Os espermatozóides da cauda do epidídimo foram coletados para análise de motilidade e morfologia. Os testículos foram homogeneizados para determinação da lipoperoxidação através dos níveis de hidroperóxidos lipídicos e de substâncias que reagem ao ácido tiobarbitúrico (TBARS); da atividade das enzimas antioxidantes superóxido dismutase (SOD), catalase (CAT), glutationa peroxidase (GPx) e glutationa-S-transferase (GST); e dos níveis do antioxidante não-enzimático glutationa (GSH). Os ratos hipertireóideos apresentaram menor motilidade espermática, maiores níveis de hidroperóxidos lipídicos e de TBARS, menor atividade da CAT e da GPx e maior atividade da GST do que os animais dos grupos controle. O tratamento com RSV, nas doses de 1 mg/kg e 10 mg/kg, foi capaz de prevenir a perda de motilidade espermática induzida no hipertireoidismo. Em adição, o RSV também diminuiu níveis de hidroperóxidos lipídicos e de TBARS; reverteu a diminuição na atividade da CAT e da GPx; além de prevenir o aumento na atividade da GST causado pelo modelo de hipertireoidismo em testículos de ratos adultos. Em conjunto estes dados mostram o efeito protetor do RSV no testículo, preservando a motilidade espermática e protegendo contra danos oxidativos causados pelo hipertireoidismo, o que sugere o RSV como um possível alvo de estudos na busca de estratégias terapêuticas com o objetivo de preservar a função testicular.