A facile approach to hydrophilic oxidized fullerenes and their derivatives as cytotoxic agents and supports for nanobiocatalytic systems

A facile, environment-friendly, versatile and reproducible approach to the successful oxidation of fullerenes (oxC60) and the formation of highly hydrophilic fullerene derivatives is introduced. This synthesis relies on the widely known Staudenmaier’s method for the oxidation of graphite, to produce both epoxy and hydroxy groups on the surface of fullerenes (C60) and thereby improve the solubility of the fullerene in polar solvents (e.g. water). The presence of epoxy groups allows for further functionalization via nucleophilic substitution reactions to generate new fullerene derivatives, which can potentially lead to a wealth of applications in the areas of medicine, biology, and composite materials. In order to justify the potential of oxidized C60 derivatives for bio-applications, we investigated their cytotoxicity in vitro as well as their utilization as support in biocatalysis applications, taking the immobilization of laccase for the decolorization of synthetic industrial dyes as a trial case.Peer ReviewedPostprint (published version

Study on the emission effects of NMR waveform frequencies of doxorubicin hydrochloride and a complex Ag(I) with organic ligands to an in vivo leiomyosarcoma model

The effects of electromagnetic waves on a biological system have guided the research toinvestigate the cancer response as a result of in vitro, in vivo and at a clinical level exposure to electromagnetic fields at low frequencies (EMF), resulting in very encouraging findingsregarding their antitumor activity. In this PhD thesis, we investigated the effects of emitted electromagnetic waveforms of frequencies, derived from the nuclear magnetic resonance spectrum (1Η-ΝΜR) of both doxorubicin hydrochloride, as well as, a complex of Ag(I) with tryphenylphosphine and 2- mercaptopyrimidine, in vitro and in vivo model of leiomyosarcoma. Specifically, we have conducted a comparative study of the effects of emitted EMF resonant frequencies of both compounds, with that of the compounds themselves, in various experimental procedures. Also, as internal control groups, we used the ham exposure and exposure to white noise. The evaluation of the cytotoxicity was conducted via MTT assay, whilst the percentage of the apoptosis-necrosis, as well as the cell cycle analysis (cell cycle arrest), were determined by flow cytometry analysis. Toxicological studies on healthy Wistar rats were conducted prior the anticancer studies. Haemotoxylin-eosin stain used for histopathological analysis. Tumor weight changes, survival days and histopatholocical profile, were used as indexes, in order to evaluate the anticancer properties in all groups of animals. The Ag(I) complex induces apoptosis on leiomyosarcoma (LMS) cells, in a dosedependent manner, and causes cell cycle arrest at S-phase, as also doxorubicin hydrochloride do, while the emitted EMF or white noise do not cause any cell death. The higher concentration of Ag(I) used in toxicological studies, caused cell death in most of the animals and histological lesions on lungs and liver. In contrast, the emission of EMF of resonant frequencies of the complex did not affect any of the organs. It has to be stated, that the 80% of the animals which were exposed to the electromagnetic waves of resonant frequencies of doxorubicin and 60% of the animals treated with the doxorubicin hydrochloride alone were totally cured and are still alive, with subsequent improvement of all anticancer indexes. A similar pattern of anticancer activity was followed by animals exposed to EMF resonant frequencies of silver complex (70%) or treated with the complex itself (50%). Also, the groups of animals exposed to EMF of both compounds manifested increased apoptotic phenomena on their tumors compared to those of animals treated with both compounds, while animals exposed to white noise or sham exposed animals did not present any difference at all in vivo procedures, compared to control group of animals. The emission of EMF resonant frequencies of both doxorubicin and silver complex induce higher anticancer activity in accordance to the increased survival levels, compared to that of animals treated with both compounds, probably due to the absence of side effects of the EMFs. Thus, it is necessary to investigate a possible mechanism of action, able to clarify the absence of side effects and simultaneously the high apoptotic levels on animal tumors, indicating strong anticancer effects.Οι επιδράσεις των ηλεκτρομαγνητικών κυμάτων σε ένα βιολογικό σύστημα έχουν οδηγήσειτην έρευνα στην μελέτη της απόκρισης του καρκίνου, κατόπιν της in vitro, in vivo και κλινικού επιπέδου έκθεσής του, σε ηλεκτρομαγνητικά πεδία χαμηλών συχνοτήτων (EMF), με συνέπεια την εξαγωγή πολύ ενθαρρυντικών συμπερασμάτων όσον αφορά στην αντικαρκινική τους δράση. Στην παρούσα διατριβή, μελετήθηκαν οι επιδράσεις εκπομπής ηλεκτρομαγνητικών κυμάτων συχνοτήτων πυρηνικού μαγνητικού συντονισμού της υδροχλωρικής δοξορουβικίνης και ενός συμπλόκου αργύρου Ag(I) με τριφαινυλοφωσφίνη και 2-μερκαπτοπυριμιδίνη, σε in vitro και in vivo μοντέλα λειομυοσαρκώματος. Οι συχνότητες συντονισμού προέρχονται από το 1Η-ΝΜR φάσμα των δύο χημικών μορίων. Συγκεκριμένα, πραγματοποιήθηκε συγκριτική μελέτη των επιδράσεων της εκπομπής των EMF συχνοτήτων συντονισμού και των δύο χημικών ενώσεων, με αυτές των ίδιων των χημικών ενώσεων, σε όλες τις ακόλουθες πειραματικές διαδικασίες. Επίσης για τον έλεγχο των αποτελεσμάτων χρησιμοποιήθηκε εικονική έκθεση, και εκπομπή λευκού θορύβου. Η εκτίμηση της κυτταροτοξικότητας έγινε μέσω της μεθόδου ΜΤΤ (MTT assay) ενώ η εκτίμηση των ποσοστών απόπτωσης-νέκρωσης καθώς και της αναστολής του κυτταρικού κύκλου, έγινε μέσω της κυτταρομετρίας ροής. Για την διασφάλιση των αντικαρκινικών μελετών, προηγήθηκαν οι μελέτες τοξικότητας σε υγιείς επίμυες, και η ιστοπαθολογική ανάλυση των οργάνων, με χρώσεις αιματοξυλίνης-ηωσίνης. Ακολούθησαν οι αντικαρκινικές μελέτες, κατά τις οποίες εκτιμήθηκαν οι μεταβολές των όγκων των επιμύων, μέσω ειδικών δεικτών, οι ημέρες επιβίωσης, καθώς το ιστοπαθολογικό προφίλ. Το σύμπλοκο του Ag(I) επάγει αποπτωτικό κυτταρικό θάνατο, στα λειομυοσαρκωματικά (LMS) κύτταρα, κατά δοσοεξαρτώμενο τρόπο, και αναστέλλει τον κυτταρικό κύκλο των LMS κυττάρων, στην S φάση, όπως και η υδροχλωρική δοξορουβικίνη ενώ η εκπομπή των EMF δεν επιφέρει κυτταρικό θάνατο, όπως επίσης και η εκπομπή λευκού θορύβου. Η υψηλότερη δόση του συμπλόκου Ag(I) προκάλεσε το θάνατο στο μεγαλύτερο ποσοστό των επιμύων, και επέφερε ιστολογικές αλλοιώσεις, στον πνεύμονα και στο ήπαρ, αλλά δεν επηρέασε τα υπόλοιπα όργανα, εν αντιθέσει με την εκπομπή των ηλεκτρομαγνητικών κυμάτων, που δεν επηρέασαν κανένα σύστημα. Η εκπομπή των ηλεκτρομαγνητικών κυμάτων συχνοτήτων συντονισμού της δοξορουβικίνης, επέφερε πλήρη ίαση στο 80% των πειραματοζώων ενώ η δοξορουβικίνη στο 60%, με ταυτόχρονη πτώση των δεικτών μεταβολής των όγκων. Επίσης η εκπομπή τωνηλεκτρομαγνητικών κυμάτων συχνοτήτων συντονισμού του συμπλόκου Ag(I), επέφερε ίασηστο 70% των ζώων, ενώ το σύμπλοκο Ag(I), στο 50%. Η ιστοπαθολογική εικόνα παρουσίασεαυξημένη απόπτωση στους όγκους που υποβλήθηκαν στα EMF, σε σχέση με τα θεραπευτικά σχήματα χορήγησης της δοξορουβικίνης και του συμπλόκου του Ag(I). Οι ομάδες των πειραματοζώων της εικονικής έκθεσης στα ηλεκτρομαγνητικά κύματα και της εκπομπής λευκού θορύβου δεν παρουσίασαν καμία διαφορά σε σχέση με την ομάδα ελέγχου, σε όλες τις πειραματικές διαδικασίες. Η εκπομπή ηλεκτρομαγνητικών κυμάτων συχνοτήτων συντονισμού της δοξορουβικίνης και του συμπλόκου Ag(I), επιφέρει καλύτερη in vivo αντικαρκινική δράση σε συνδυασμό με τα υψηλά ποσοστά επιβίωσης, σε σχέση με τις δύο κυτταροτοξικές ουσίες, ίσως λόγω της απουσίας τοξικολογικών επιδράσεων από την εκπομπή των EMF. Επίσης είναι αναγκαίο να διερευνηθεί ο μηχανισμός δράσης, ο οποίος να εξηγεί μεν, την έλλειψη κυτταροτοξικής δράσης των EMF in vitro, αλλά και την ισχυρή αποπτωτική τους επίδραση in vivo

Asbestos Induces Oxidative Stress and Activation of Nrf2 Signaling in Murine Macrophages: Chemopreventive Role of the Synthetic Lignan Secoisolariciresinol Diglucoside (LGM2605)

The interaction of asbestos fibers with macrophages generates harmful reactive oxygen species (ROS) and subsequent oxidative cell damage that are key processes linked to malignancy. Secoisolariciresinol diglucoside (SDG) is a non-toxic, flaxseed-derived pluripotent compound that has antioxidant properties and may thus function as a chemopreventive agent for asbestos-induced mesothelioma. We thus evaluated synthetic SDG (LGM2605) in asbestos-exposed, elicited murine peritoneal macrophages as an in vitro model of tissue phagocytic response to the presence of asbestos in the pleural space. Murine peritoneal macrophages (MFs) were exposed to crocidolite asbestos fibers (20 µg/cm2) and evaluated at various times post exposure for cytotoxicity, ROS generation, malondialdehyde (MDA), and levels of 8-iso Prostaglandin F2α (8-isoP). We then evaluated the ability of LGM2605 to mitigate asbestos-induced oxidative stress by administering LGM2605 (50 µM) 4-h prior to asbestos exposure. We observed a significant (p < 0.0001), time-dependent increase in asbestos-induced cytotoxicity, ROS generation, and the release of MDA and 8-iso Prostaglandin F2α, markers of lipid peroxidation, which increased linearly over time. LGM2605 treatment significantly (p < 0.0001) reduced asbestos-induced cytotoxicity and ROS generation, while decreasing levels of MDA and 8-isoP by 71%–88% and 41%–73%, respectively. Importantly, exposure to asbestos fibers induced cell protective defenses, such as cellular Nrf2 activation and the expression of phase II antioxidant enzymes, HO-1 and Nqo1 that were further enhanced by LGM2605 treatment. LGM2605 boosted antioxidant defenses, as well as reduced asbestos-induced ROS generation and markers of oxidative stress in murine peritoneal macrophages, supporting its possible use as a chemoprevention agent in the development of asbestos-induced malignant mesothelioma

The Flaxseed-Derived Lignan Phenolic Secoisolariciresinol Diglucoside (SDG) Protects Non-Malignant Lung Cells from Radiation Damage

Plant phenolic compounds are common dietary antioxidants that possess antioxidant and anti-inflammatory properties. Flaxseed (FS) has been reported to be radioprotective in murine models of oxidative lung damage. Flaxseed’s protective properties are attributed to its main biphenolic lignan, secoisolariciresinol diglucoside (SDG). SDG is a free radical scavenger, shown in cell free systems to protect DNA from radiation-induced damage. The objective of this study was to investigate the in vitro radioprotective efficacy of SDG in murine lung cells. Protection against irradiation (IR)-induced DNA double and single strand breaks was assessed by γ-H2AX labeling and alkaline comet assay, respectively. The role of SDG in modulating the levels of cytoprotective enzymes was evaluated by qPCR and confirmed by Western blotting. Additionally, effects of SDG on clonogenic survival of irradiated cells were evaluated. SDG protected cells from IR-induced death and ameliorated DNA damage by reducing mean comet tail length and percentage of γ-H2AX positive cells. Importantly, SDG significantly increased gene and protein levels of antioxidant HO-1, GSTM1 and NQO1. Our results identify the potent radioprotective properties of the synthetic biphenolic SDG, preventing DNA damage and enhancing the antioxidant capacity of normal lung cells; thus, rendering SDG a potential radioprotector against radiation exposure

Synthetic Secoisolariciresinol Diglucoside (LGM2605) Protects Human Lung in an Ex Vivo Model of Proton Radiation Damage

Radiation therapy for the treatment of thoracic malignancies has improved significantly by directing of the proton beam in higher doses on the targeted tumor while normal tissues around the tumor receive much lower doses. Nevertheless, exposure of normal tissues to protons is known to pose a substantial risk in long-term survivors, as confirmed by our work in space-relevant exposures of murine lungs to proton radiation. Thus, radioprotective strategies are being sought. We established that LGM2605 is a potent protector from radiation-induced lung toxicity and aimed in the current study to extend the initial findings of space-relevant, proton radiation-associated late lung damage in mice by looking at acute changes in human lung. We used an ex vivo model of organ culture where tissue slices of donor living human lung were kept in culture and exposed to proton radiation. We exposed donor human lung precision-cut lung sections (huPCLS), pretreated with LGM2605, to 4 Gy proton radiation and evaluated them 30 min and 24 h later for gene expression changes relevant to inflammation, oxidative stress, and cell cycle arrest, and determined radiation-induced senescence, inflammation, and oxidative tissue damage. We identified an LGM2605-mediated reduction of proton radiation-induced cellular senescence and associated cell cycle changes, an associated proinflammatory phenotype, and associated oxidative tissue damage. This is a first report on the effects of proton radiation and of the radioprotective properties of LGM2605 on human lung

Novel Double-Hit Model of Radiation and Hyperoxia-Induced Oxidative Cell Damage Relevant to Space Travel

Spaceflight occasionally requires multiple extravehicular activities (EVA) that potentially subject astronauts to repeated changes in ambient oxygen superimposed on those of space radiation exposure. We thus developed a novel in vitro model system to test lung cell damage following repeated exposure to radiation and hyperoxia. Non-tumorigenic murine alveolar type II epithelial cells (C10) were exposed to >95% O2 for 8 h only (O2), 0.25 Gy ionizing γ-radiation (IR) only, or a double-hit combination of both challenges (O2 + IR) followed by 16 h of normoxia (ambient air containing 21% O2 and 5% CO2) (1 cycle = 24 h, 2 cycles = 48 h). Cell survival, DNA damage, apoptosis, and indicators of oxidative stress were evaluated after 1 and 2 cycles of exposure. We observed a significant (p < 0.05) decrease in cell survival across all challenge conditions along with an increase in DNA damage, determined by Comet analysis and H2AX phosphorylation, and apoptosis, determined by Annexin-V staining, relative to cells unexposed to hyperoxia or radiation. DNA damage (GADD45α and cleaved-PARP), apoptotic (cleaved caspase-3 and BAX), and antioxidant (HO-1 and Nqo1) proteins were increased following radiation and hyperoxia exposure after 1 and 2 cycles of exposure. Importantly, exposure to combination challenge O2 + IR exacerbated cell death and DNA damage compared to individual exposures O2 or IR alone. Additionally levels of cell cycle proteins phospho-p53 and p21 were significantly increased, while levels of CDK1 and Cyclin B1 were decreased at both time points for all exposure groups. Similarly, proteins involved in cell cycle arrest was more profoundly changed with the combination challenges as compared to each stressor alone. These results correlate with a significant 4- to 6-fold increase in the ratio of cells in G2/G1 after 2 cycles of exposure to hyperoxic conditions. We have characterized a novel in vitro model of double-hit, low-level radiation and hyperoxia exposure that leads to oxidative lung cell injury, DNA damage, apoptosis, and cell cycle arrest