Changes in intracellular redox homeostasis are implicated in both normal cell signaling and as pathophysiological mechanisms contributing to a variety of age-related diseases, including diabetes, atherosclerosis, neurodegenerative conditions, and cancer. Though a variety of well described mechanisms exist to counterbalance the overproduction of cellular oxidants and maintain optimal intracellular redox poise, the understanding of the mechanism(s) through which cellular redox homeostasis regulates cell signaling functions is less well understood. Here, we demonstrate that signaling by the immediate early gene / orphan nuclear hormone receptor NGFI-B (Nur77, TR3), which functions pleiotropically in the regulation of cell growth, metabolism, differentiation and death in diverse tissues, is redox-regulated at both the level of induction and NGFI-B-dependent gene transcription. Using co-immunoprecipitation experiments in cells, we also identified a novel interaction between NGFI-B and the cytoplasmic thiol-reducing catalyst thioredoxin1 (Trx1), that, similar to DTT, blocks NGFI-B-dependent gene expression in a manner that depends on the Trx1 active site cysteines. Together these observations add NGFI-B-dependent gene expression to a growing portfolio of transcription factor pathways that are redox-regulated. NGFI-B, in addition, appears to regulate the mitochondrial membrane potential in L6 skeletal myoblasts. NGFI-B is indispensible for T-cell receptor-mediated apoptosis and induces cell death in a variety of cell types in response to diverse pro-apoptotic stimuli. Like p53, translocation of NGFI-B from the nucleus to the mitochondria may be a critical aspect of its pro-apoptotic function. Interestingly, we found that enforced NGFI-B expression in L6 skeletal muscle myoblasts led to a significant decrease of MMP that peaked 48hr after transfection and did not require a cell death-inducing stimulus. Moreover, NGFI-B transfected cells had no increase in mitochondrial cytochrome C release despite loss of MMP at 48 hr. Combined, these data suggest that loss of MMP in muscle cells may be an early event in the apoptotic process regulated by NGFI-B. This, along with the redox regulation of NGFI-B, provides unique evidence of a relationship between the mitochondria, mitochondrial by-products, ROS, and the regulation of and by the transcription factor NGFI-B. / text

Τις τελευταίες δεκαετίες υπάρχει ένα αυξανόμενο ενδιαφέρον για το ρόλο των ROS στην πειραματική και κλινική ιατρική (Alexandre et al., 2007; Zheleva et al., 2007; Videla et al., 2007). Οι ROS, όπως το ανιόν του υπεροξειδίου, το υπεροξείδιο του υδρογόνου (ΗΡ) και η υδροξυλική ρίζα, είναι γνωστό ότι διαδραματίζουν ένα διττό ρόλο στα βιολογικά συστήματα, όπου είναι επιβλαβείς στις υψηλές και ευεργετικές στις χαμηλές συγκεντρώσεις (Valko et al., 2004). Στις υψηλές συγκεντρώσεις οι ROS φαίνεται να διαμεσολαβούν την απόπτωση. Ενδογενή ή εξωγενή ερεθίσματα οδηγούν στη συσσώρευση των ROS, το λεγόμενο «οξειδωτικό στρες», που χαρακτηρίζεται από βλάβη των κυτταρικών δομών, των λιπιδίων, των μεμβρανών, των πρωτεϊνών και των νουκλεϊκών οξέων (Poli et al., 2004). Αντίθετα, οι χαμηλές συγκεντρώσεις των ROS μέσα στα κύτταρα δρούν ως δεύτεροι αγγελιοφόροι σε αρκετά ενδοκυτταρικά σηματοδοτικά μονοπάτια που οδηγούν στην ενεργοποίηση κινασών τυροσίνης, των ΜΑΡ κινασών, της πρωτεϊνικής κινάσης C, του υποδοχέα του επιδερμικού αυξητικού παράγοντα, πρωτεϊνικών φωσφατασών, των καναλιών καλίου και των μεταγραφικών παραγόντων AP-1 και NF-κB (Droge, 2002). Πρόσφατα έχουμε δείξει ότι υπό φυσιολογικές, μη-στρεσογόνες συνθήκες, το ΗΡ επάγει τον πολλαπλασιασμό και τη μετανάστευση στα ανθρώπινα κύτταρα καρκίνου του προστάτη LNCaP, μέσω ενεργοποίησης της έκφρασης του αυξητικού παράγοντα HARP (Polytarchou et al., 2005). Η HARP ή πλειοτροπίνη είναι ένας εκκρινόμενος αυξητικός παράγοντας 18 kDa που παρουσιάζει υψηλή συγγένεια για την ηπαρίνη. Η HARP είναι υψηλά συντηρημένη μεταξύ των διαφόρων ειδών, όπως ο άνθρωπος, το ποντίκι, ο αρουραίος, τα βοειδή, τα ψάρια, ο βάτραχος και τα έντομα, και το γονίδιό της εκφράζεται σε ένα ιδιαίτερα περιορισμένο χρονικό και χωρικό μοτίβο κατά τη διάρκεια της ανάπτυξης. Φαίνεται ότι η HARP είναι μια σημαντική πρωτεΐνη που συμβάλλει ενδεχομένως στη λειτουργία διαφόρων ρυθμιστικών συστημάτων. Αρκετά δεδομένα υποδεικνύουν ότι η HARP εμπλέκεται στον πολλαπλασιασμό, τη μετανάστευση και τη διαφοροποίηση των κυττάρων και διαδραματίζει σημαντικό ρόλο στις διάφορες κυτταρικές διαδικασίες (Kadomatsu and Muramatsu, 2004; Papadimitriou et al., 2004). Επιπλέον, η HARP ανιχνεύεται σε μια πληθώρα καρκινωμάτων λειτουργώντας ως πρωτο-ογκογονίδιο και φαίνεται να παίζει κύριο ρόλο στη φυσιολογική και την επαγόμενη από καρκινικούς όγκους αγγειογένεση (Kadomatsu and Muramatsu, 2004; Papadimitriou et al., 2004; Hatziapostolou et al., 2005;2006; Polykratis et al., 2005; Christman et al., 2005; Zhang et al., 2006). Είναι πλέον γνωστό ότι οι χαμηλές συγκεντρώσεις ROS ενεργοποιούν ενδοκυτταρικά σηματοδοτικά μονοπάτια που οδηγούν σε ενισχυμένη αγγειογένεση in vivo (Maulik and Das, 2002; Polytarchou and Papadimitriou, 2004; Ushio-Fukai, 2006; Kim et al., 2006; Chen et al., 2007) και ενεργοποίηση των ενδοθηλιακών κυττάρων in vitro (Lelkes et al., 1998; Maulik and Das, 2002; Stone and Collins, 2002; Yasuda et al., 1999; Polytarchou and Papadimitriou, 2004; Ushio-Fukai, 2006; Kim et al., 2006; Chen et al., 2007; Guo et al., 2007). Επίσης, οι ROS, όπως το ανιόν του υπεροξειδίου και το ΗΡ, φαίνεται να διαμεσολαβούν τα αγγειογενετικά σήματα που ξεκινούν από αυξητικούς παράγοντες, όπως ο VEGF (Lin et al., 2003 Ushio-Fukai et al., 2002). Προηγούμενη μελέτη της ερευνητικής μας ομάδας έδειξε ότι το ανιόν του υπεροξειδίου και το ΗΡ είναι πιθανοί επαγωγείς των λειτουργιών των ενδοθηλιακών κυττάρων in vitro, ενδεχομένως μέσω επαγωγής της ενεργοποίησης της ενδοθηλιακής συνθάσης του μονοξειδίου του αζώτου (eNOS), που οδηγεί σε αυξημένη παραγωγή του ενδογενούς μονοξειδίου του αζώτου (NO) και επακόλουθη ενεργοποίηση της διαλυτής γουανυλικής κυκλάσης (sGC) (Polytarchou and Papadimitriou, 2005). Ο κύριος στόχος της παρούσας μελέτης ήταν να διερευνηθεί η πιθανή εμπλοκή της eNOS στην επαγωγή της παραγωγής της HARP από χαμηλές συγκεντρώσεις ΗΡ. Τα αποτελέσματά μας δείχνουν ότι η ενεργοποίηση του ανθρώπινου γονιδίου της HARP επάγεται από το ΗΡ μέσω ενεργοποίησης της eNOS στα κύτταρα HUVEC και LNCaP. Όπως αναφέρθηκε και παραπάνω, περίσσεια ROS σε συνδυασμό με ανεπάρκεια των κυτταρικών αντιοξειδωτικών συστημάτων οδηγεί σε οξειδωτικό στρες (Halliwell and Whiteman, 2004) και ενεργοποίηση μονοπατιών απόπτωσης και νέκρωσης (Paraskevas et al., 2000; Fleury et al., 2002; Nakano et al., 2004). Ένα δεύτερο μέρος της παρούσας εργασίας ασχολείται με τον τρόπο που οι υψηλές συγκεντρώσεις ΗΡ επηρεάζουν την ανάπτυξη των ενδοθηλιακών κυττάρων από στεφανιαία φλέβα βοός (CVEC). / -

Increased reactive oxygen species (ROS) have been reported to be at the centre of various diseases. Although several reports have implicated elevated levels of ROS in the pathogenesis of diabetes mellitus, the early detection of ROS is still not attainable. This limitation causes difficulty in the early diagnosis of ROS related disorders. The presence of high levels of ROS was reported to result in differential expression of antioxidant genes involved in protecting cells from their deleterious effects. Among the antioxidant genes that are expressed, it was postulated that expression of metallothioneins (MTs) are also induced. MTs are low molecular weight, cysteine-rich proteins involved in metal homeostasis and reported to harbour antioxidant function. The aim of this investigation was to explore MTs as biomarkers for elevated levels of ROS in whole blood of type 2 diabetic (T2D) individuals. The level of ROS in diabetic, non-diabetic as well as individuals at risk of developing T2D was determined via the use of biochemical assays. Real-Time PCR was utilised to analyse the expression of MTs and the presence of MT proteins was analysed via the ELISA. In this study it was observed that diabetic individuals had elevated levels of ROS. However, no significant difference in the expression of MTs and the presence of MT proteins between the diabetic and non-diabetic individuals was observed. In vitro experimental conditions indicated that MT expression is induced by elevated levels of ROS. In pathological conditions the ROS-dependent induction of MT expression needs to be elucidated further. It therefore can be suggested that MTs can not yet be utilised as biomarkers for the detection of elevated levels of ROS in pathological conditions with ROS aetiology. This investigation also highlights the fact that blood is not an optimal medium in which this objective can be attained. / Thesis (Ph.D. (Biochemistry))--North-West University, Potchefstroom Campus, 2005.

Septic shock is a leading cause of death in intensive care units. As part of the septic process, the endothelium becomes activated and propagates the septic condition. It has become evident that reactive oxygen species (ROS) are involved in the signaling of mediators of sepsis, such as tumor necrosis factor-alpha (TNF-alpha) and the lipopolysaccharide coating of gram-negative bacteria (LPS). An important source of these ROS is NADPH oxidase, which is a ubiquitously expressed enzyme complex that also exists in endothelial cells (EC). We showed that O2- from NADPH oxidase was important for LPS, as well as TNF-alpha, induction of two markers of an activated endothelium, interleukin-8 (IL-8) and intercellular adhesion molecule-1 (ICAM-1) in human umbilical vein endothelial cells (HUVEC). / Expression of a gene can be increased by a rise in transcription as well as post-transcriptional changes, such as mRNA stability modifications. We assessed the role of NADPH oxidase in this process and found a complex interaction. Although LPS increases IL-8 transcription, it also destabilizes IL-8 mRNA in a p38 and extracellular signal-regulated kinase (ERK) MAPK dependent manner, which was only evident after blocking NADPH oxidase. This regulation involved the mRNA de-stabilizing factor tristetraprolin (TTP). In contrast, TNF-alpha enhanced the stability of IL-8, IL-6 and ICAM-1 mRNA in a p38 MAPK dependent, but NADPH oxidase independent manner. Furthermore, LPS did not have an effect on mRNA stability of IL-6 or ICAM-1 in our system. Thus, we conclude from our studies that the NAPDH oxidase is important for the induction of inflammatory molecules in LPS and TNF-alpha treated EC and is also involved in mRNA stability regulation of these molecules in a signal and gene specific fashion.

Previous findings have indicated that oxidative stress plays a role in the development of osteoporosis and that individual polyphenols, by virtue of their antioxidant properties, may mitigate these damaging effects. Nutritional supplements, greens+ bone builderTM, containing polyphenols and other micronutrients beneficial for bone health are of recent interest as complementary strategies in the management of osteoporosis. A randomized controlled study was conducted to explore the combined effects of the nutrients found within the supplement on bone health for 8 weeks. Total polyphenol content and antioxidant capacity increased whereas oxidative stress parameters and the bone resorption marker, crosslinked C-telopeptide of type I collagen decreased after supplementation. There was no significant change in the bone formation marker, procollagen type I N-terminal propeptide. This thesis shows an association of polyphenols with other micronutrients acts through their antioxidant capacity to decrease oxidative stress parameters and bone resorption, thus potentially reducing the risk for osteoporosis.

Lycopene is a potent carotenoid antioxidant shown to decrease the risk of chronic diseases associated with oxidative stress and has recently begun to be studied in relation to osteoporosis. However, studies specifically associating intervention with lycopene and a decreased risk for osteoporosis have not yet been conducted, and the mechanisms by which lycopene affects bone have yet to be elucidated. The purpose of this thesis was to explore the hypotheses that supplementation with lycopene would increase antioxidant capacity while decreasing oxidative stress parameters; subsequently decreasing bone turnover markers, and thus the risk of osteoporosis in postmenopausal women. Specifically, experiments were designed to determine whether lycopene acts in its antioxidant capacity to improve bone health, and to delineate the mechanisms of these effects. These hypotheses were investigated through a cross-sectional study, a randomized controlled clinical study, and in vitro studies on human osteoblast cells. The results presented in this thesis demonstrate that intervention with the potent antioxidant lycopene significantly increased concentrations of the 5-cis isomer and resulted in significantly decreased oxidative stress parameters in postmenopausal women. This decrease in oxidative stress parameters resulted in significantly decreased concentrations of the bone resorption marker crosslinked N-telopeptides of type I collagen (NTx). The typical diet of participants included a relatively low intake of lycopene, and the corresponding serum lycopene concentrations were not as effective in decreasing biomarkers of oxidative stress and bone resorption as those obtained from supplementation with lycopene to increase 5-cis serum lycopene. Studies on the paraoxonase enzyme suggest that lycopene is most effective in quenching oxidative stress to decrease bone turnover markers when the internal antioxidant defenses are insufficient or decremented. Mechanisms demonstrated by the in vitro findings suggest that cis lycopene is capable of both preventing and repairing the damaging effects of oxidative stress in osteoblasts. Overall, this thesis provides evidence that lycopene acts through its antioxidant capacity to decrease oxidative stress parameters and bone turnover markers, and may, therefore, reduce the risk for osteoporosis. Based on these findings, the consumption of lycopene by women to improve overall bone health should be considered.

Previous findings have indicated that oxidative stress plays a role in the development of osteoporosis and that individual polyphenols, by virtue of their antioxidant properties, may mitigate these damaging effects. Nutritional supplements, greens+ bone builderTM, containing polyphenols and other micronutrients beneficial for bone health are of recent interest as complementary strategies in the management of osteoporosis. A randomized controlled study was conducted to explore the combined effects of the nutrients found within the supplement on bone health for 8 weeks. Total polyphenol content and antioxidant capacity increased whereas oxidative stress parameters and the bone resorption marker, crosslinked C-telopeptide of type I collagen decreased after supplementation. There was no significant change in the bone formation marker, procollagen type I N-terminal propeptide. This thesis shows an association of polyphenols with other micronutrients acts through their antioxidant capacity to decrease oxidative stress parameters and bone resorption, thus potentially reducing the risk for osteoporosis.

Selection for large body size can result in the development of obesity, which in mink females is associated with poor reproduction and metabolic diseases. Caloric restriction is effective in diminishing oxidative stress and delaying aging-related diseases. This study investigated the effects of moderate diet restriction (MDR) during the fall on body condition, health, and reproductive success of mink (Neovison vison) breeder females. The 100 control (CTRL) females were fed according to normal farm feeding practice and the 100 sister-pair MDR females were fed about 20% less. In the fall, more ideal body weights and body condition scores (BCS) were seen in the MDR females, and they produced larger litter sizes. In addition, the MDR females exhibited less deoxyribonucleic acid (DNA) damage than the CTRL females. Telomeres were elongated in both groups. This dietary management practice is anticipated to result in significant advancement in the fur industry, both economically and welfare-wise.

Reactive oxygen species (ROS) have a role in cardiovascular health and disease. This study was undertaken to determine if ROS formation is influenced by either common genetic variations in p22phox, a subunit of the ROS generating enzyme NOX1, or by natural plant compounds with cardiovascular benefits. Hydrogen peroxide production was measured using Amplex Red, and superoxide generation was measured using NBT and MCLA. Each of seven p22phox variants supported ROS generation by NOX1. No differences were found in the rate of ROS production; however, unequal transfer of the p22phox gene may be a confounding factor. A variation in the 3’UTR of the p22phox gene led to lower p22phox protein levels, whereas none of the other variations affected mRNA or protein expression. The natural compound resveratrol acts as an antioxidant towards hydrogen peroxide, but not superoxide. Resveratrol does not inhibit NOX1 activity.

The development of cellular senescence is not consistent in cultured bovine fibroblasts. Numerous factors may be contributing to the variable onset of cellular senescence, including oxidative stress, telomere shortening and DNA damage. Recent results indicate that cellular senescence is also associated with markers of DNA replication. This suggests that cells may be more likely to enter a senescent state depending on their cell cycle progression. The purpose of this study was to determine the effect of cell cycle phase on the development of stress-induced premature cellular senescence (SIPS). Bovine fibroblasts were synchronized at various cell cycle phases, followed by treatment with increasing doses of H2O2. Senescent cells were detected using SA-β-galactosidase staining assay. As H2O2 dosage increased, the amount of cell death by necrosis increased in both unsynchronized and cell cycle synchronized groups, while the amount of senescence varied depending on cell cycle phase. Our results suggest that the S phase of the cell cycle is the most resistant to oxidative damage, the G2/M phase is the most susceptible, and the G1/S phase is the most likely to enter senescence as a protective measure following low doses of H2O2. Increased senescence also resulted from an increased recovery time post-H2O2 treatment, and gene expression studies suggest SIPS bovine fibroblasts senesce via the p53-independent pathway. An improved understanding of SIPS has important implications for improving cloning efficiency and understanding age-related diseases.