Trypanosoma cruzi PARP is enriched in the nucleolus and is present in a thread connecting nuclei during mitosis.

Poly (ADP-ribose) polymerase (PARP) is responsible for the synthesis of ADP-ribose polymers, which are involved in a wide range of cellular processes such as preservation of genome integrity, DNA damage signaling and repair, molecular switches between distinct cell death pathways, and cell cycle progression. Previously, we demonstrated that the only PARP present in T. cruzi migrates to the nucleus upon genotoxic stimulus. In this work, we identify the N-terminal domain as being sufficient for TcPARP nuclear localization and describe for the first time that TcPARP is enriched in the parasite's nucleolus. We also describe that TcPARP is present in a thread-like structure that connects two dividing nuclei and co-localizes with nucleolar material and microtubules. Furthermore, ADP-ribose polymers could also be detected in this thread during mitosis. These findings represent a first approach to new potential TcPARP functions inside the nucleus and will help understand its role well beyond the largely described DNA damage response protein in trypanosomatids.

First body of evidence suggesting a role of a tankyrase-binding motif (TBM) of vinculin (VCL) in epithelial cells.

BACKGROUND: Adherens junctions (AJ) are involved in cancer, infections and neurodegeneration. Still, their composition has not been completely disclosed. Poly(ADP-ribose) polymerases (PARPs) catalyze the synthesis of poly(ADP-ribose) (PAR) as a posttranslational modification. Four PARPs synthesize PAR, namely PARP-1/2 and Tankyrase-1/2 (TNKS). In the epithelial belt, AJ are accompanied by a PAR belt and a subcortical F-actin ring. F-actin depolymerization alters the AJ and PAR belts while PARP inhibitors prevent the assembly of the AJ belt and cortical actin. We wondered which PARP synthesizes the belt and which is the PARylation target protein. Vinculin (VCL) participates in the anchorage of F-actin to the AJ, regulating its functions, and colocalized with the PAR belt. TNKS has been formerly involved in the assembly of epithelial cell junctions. HYPOTHESIS: TNKS poly(ADP-ribosylates) (PARylates) epithelial belt VCL, affecting its functions in AJ, including cell shape maintenance. MATERIALS AND METHODS: Tankyrase-binding motif (TBM) sequences in hVCL gene were identified and VCL sequences from various vertebrates, Drosophila melanogaster and Caenorhabditis elegans were aligned and compared. Plasma membrane-associated PAR was tested by immunocytofluorescence (ICF) and subcellular fractionation in Vero cells while TNKS role in this structure and cell junction assembly was evaluated using specific inhibitors. The identity of the PARylated proteins was tested by affinity precipitation with PAR-binding reagent followed by western blots. Finally, MCF-7 human breast cancer epithelial cells were subjected to transfection with Tol2-plasmids, carrying a dicistronic expression sequence including Gallus gallus wt VCL (Tol-2-GgVCL), or the same VCL gene with a point mutation in TBM-II (Tol2-GgVCL/*TBM) under the control of a ß-actin promoter, plus green fluorescent protein following an internal ribosome entry site (IRES-GFP) to allow the identification of transfected cells without modifying the transfected protein of interest. RESULTS AND DISCUSSION: In this work, some of the hypothesis predictions have been tested. We have demonstrated that: (1) VCL TBMs were conserved in vertebrate evolution while absent in C. elegans; (2) TNKS inhibitors disrupted the PAR belt synthesis, while PAR and an endogenous TNKS pool were associated to the plasma membrane; (3) a VCL pool was covalently PARylated; (4) transfection of MCF-7 cells leading to overexpression of Gg-VCL/*TBM induced mesenchymal-like cell shape changes. This last point deserves further investigation, bypassing the limits of our transient transfection and overexpression system. In fact, a 5th testable prediction would be that a single point mutation in VCL TBM-II under endogenous expression control would induce an epithelial to mesenchymal transition (EMT). To check this, a CRISPR/Cas9 substitution approach followed by migration, invasion, gene expression and chemo-resistance assays should be performed.

Tankyrase inhibitors hinder Trypanosoma cruzi infection by altering host-cell signalling pathways.

Chagas disease is a potentially life-threatening protozoan infection affecting around 8 million people, for which only chemotherapies with limited efficacy and severe adverse secondary effects are available. The aetiological agent, Trypanosoma cruzi, displays varied cell invading tactics and triggers different host cell signals, including the Wnt/ß-catenin pathway. Poly(ADP-ribose) (PAR) can be synthetized by certain members of the poly(ADP-ribose) polymerase (PARP) family: PARP-1/-2 and Tankyrases-1/2 (TNKS). PAR homoeostasis participates in the host cell response to T. cruzi infection and TNKS are involved in Wnt signalling, among other pathways. Therefore, we hypothesized that TNKS inhibitors (TNKSi) could hamper T. cruzi infection. We showed that five TNKSi (FLALL9, MN64, XAV939, G007LK and OULL9) diminished T. cruzi infection of Vero cells. As most TNKSi did not affect the viability of axenically cultivated parasites, our results suggested that TNKSi were interfering with parasite­host cell signalling. Infection by T. cruzi induced nuclear translocation of ß-catenin, as well as upregulation of TNF-α expression and secretion. These changes were hampered by TNKSi. Further signals should be monitored in this model and in vivo. As a TNKSi has entered cancer clinical trials with promising results, our findings encourage further studies aiming at drug repurposing strategies.

Poly(ADP-ribose) metabolism in human parasitic protozoa.

Poly(ADP-ribosyl)ation reactions constitute a post-translational protein modification synthesized in higher eukaryotes by a family of poly(ADP-ribose)polymerases (PARP) and catabolized mainly by poly(ADP-ribose) glycohydrolase (PARG). The best understood role of PARP is the maintenance of genomic integrity via the promotion of DNA repair that leads to cell survival when low levels of genotoxic stress occur. The participation of PARP in unleashing cell death at higher levels of damage has also been broadly studied. The biology of poly(ADP-ribosyl)ation in protozoan parasites, however, still remains a mystery. This review will examine the presence of the key enzyme involved in ADP-ribose polymer (PAR) metabolism in protozoan parasites associated with human diseases. Theoretical and experimental data obtained up to date have revealed the presence of PAR metabolism only in the trypanosomatids Trypanosoma cruzi and T. brucei, the apicomplexan Toxoplasma gondii and Entamoeba histolytica. T. cruzi and T. brucei, as opposed to humans and other organisms, have only one PARP and one PARG with subcellular localizations that are distinct from the ones described for their mammalian counterparts. The topics discussed in this review describe the first studies on PAR metabolism in trypanosomatids, specially the role of PAR on DNA damage response, cell cycle progression and cell death after genotoxic stimuli. The results described show differences in some aspects of PAR metabolism in trypanosomatids in comparison to other eukaryotes. New questions about the function of this metabolic pathway in the parasites under study are open and we hope it encourages the research community to explore this signaling pathway as a new possible target of clinical relevance in these and other disease-causing parasites.

Structural and Biochemical Characterization of Poly-ADP-ribose Polymerase from Trypanosoma brucei.

Trypanosoma brucei is a unicellular parasite responsible for African trypanosomiasis or sleeping sickness. It contains a single PARP enzyme opposed to many higher eukaryotes, which have numerous PARPs. PARPs are responsible for a post-translational modification, ADP-ribosylation, regulating a multitude of cellular events. T. brucei PARP, like human PARPs-1-3, is activated by DNA binding and it potentially functions in DNA repair processes. Here we characterized activation requirements, structure and subcellular localization of T. brucei PARP. T. brucei PARP was found to be selectively activated by 5' phosphorylated and 3' phosphorylated DNA breaks. Importantly, the N-terminal region is responsible for high-affinity DNA-binding and required for DNA-dependent enzymatic activation. This module is also required for nuclear localization of the protein in response to oxidative stress. Solution structures of activating and non-activating PARP-DNA complexes were determined with small-angle X-ray scattering revealing distinct differences in their DNA-binding modes.

Poly(ADP-ribosylation) is present in murine sciatic nerve fibers and is altered in a Charcot-Marie-Tooth-1E neurodegenerative model.

BACKGROUND: Poly-ADP-ribose (PAR) is a polymer synthesized by poly-ADP-ribose polymerases (PARPs) as a postranslational protein modification and catabolized mainly by poly-ADP-ribose glycohydrolase (PARG). In spite of the existence of cytoplasmic PARPs and PARG, research has been focused on nuclear PARPs and PAR, demonstrating roles in the maintenance of chromatin architecture and the participation in DNA damage responses and transcriptional regulation. We have recently detected non-nuclear PAR structurally and functionally associated to the E-cadherin rich zonula adherens and the actin cytoskeleton of VERO epithelial cells. Myelinating Schwann cells (SC) are stabilized by E-cadherin rich autotypic adherens junctions (AJ). We wondered whether PAR would map to these regions. Besides, we have demonstrated an altered microfilament pattern in peripheral nerves of Trembler-J (Tr-J) model of CMT1-E. We hypothesized that cytoplasmic PAR would accompany such modified F-actin pattern. METHODS: Wild-type (WT) and Tr-J mice sciatic nerves cryosections were subjected to immunohistofluorescence with anti-PAR antibodies (including antibody validation), F-actin detection with a phalloidin probe and DAPI/DNA counterstaining. Confocal image stacks were subjected to a colocalization highlighter and to semi-quantitative image analysis. RESULTS: We have shown for the first time the presence of PAR in sciatic nerves. Cytoplasmic PAR colocalized with F-actin at non-compact myelin regions in WT nerves. Moreover, in Tr-J, cytoplasmic PAR was augmented in close correlation with actin. In addition, nuclear PAR was detected in WT SC and was moderately increased in Tr-J SC. DISCUSSION: The presence of PAR associated to non-compact myelin regions (which constitute E-cadherin rich autotypic AJ/actin anchorage regions) and the co-alterations experienced by PAR and the actin cytoskeleton in epithelium and nerves, suggest that PAR may be a constitutive component of AJ/actin anchorage regions. Is PAR stabilizing the AJ-actin complexes? This question has strong implications in structural cell biology and cell signaling networks. Moreover, if PAR played a stabilizing role, such stabilization could participate in the physiological control of axonal branching. PARP and PAR alterations exist in several neurodegenerative pathologies including Alzheimer's, Parkinson's and Hungtington's diseases. Conversely, PARP inhibition decreases PAR and promotes neurite outgrowth in cortical neurons in vitro. Coherently, the PARP inhibitor XAV939 improves myelination in vitro, ex vivo and in vivo. Until now such results have been interpreted in terms of nuclear PARP activity. Our results indicate for the first time the presence of PARylation in peripheral nerve fibers, in a healthy environment. Besides, we have evidenced a PARylation increase in Tr-J, suggesting that the involvement of cytoplasmic PARPs and PARylation in normal and neurodegenerative conditions should be re-evaluated.

Disrupted ADP-ribose metabolism with nuclear Poly (ADP-ribose) accumulation leads to different cell death pathways in presence of hydrogen peroxide in procyclic Trypanosoma brucei.

BACKGROUND: Poly(ADP-ribose) (PAR) metabolism participates in several biological processes such as DNA damage signaling and repair, which is a thoroughly studied function. PAR is synthesized by Poly(ADP-ribose) polymerase (PARP) and hydrolyzed by Poly(ADP-ribose) glycohydrolase (PARG). In contrast to human and other higher eukaryotes, Trypanosoma brucei contains only one PARP and PARG. Up to date, the function of these enzymes has remained elusive in this parasite. The aim of this work is to unravel the role that PAR plays in genotoxic stress response. METHODS: The optimal conditions for the activity of purified recombinant TbPARP were determined by using a fluorometric activity assay followed by screening of PARP inhibitors. Sensitivity to a genotoxic agent, H2O2, was assessed by counting motile parasites over the total number in a Neubauer chamber, in presence of a potent PARP inhibitor as well as in procyclic transgenic lines which either down-regulate PARP or PARG, or over-express PARP. Triplicates were carried out for each condition tested and data significance was assessed with two-way Anova followed by Bonferroni test. Finally, PAR influence was studied in cell death pathways by flow cytometry. RESULTS: Abolition of a functional PARP either by using potent inhibitors present or in PARP-silenced parasites had no effect on parasite growth in culture; however, PARP-inhibited and PARP down-regulated parasites presented an increased resistance against H2O2 treatment when compared to their wild type counterparts. PARP over-expressing and PARG-silenced parasites displayed polymer accumulation in the nucleus and, as expected, showed diminished resistance when exposed to the same genotoxic stimulus. Indeed, they suffered a necrotic death pathway, while an apoptosis-like mechanism was observed in control cultures. Surprisingly, PARP migrated to the nucleus and synthesized PAR only after a genomic stress in wild type parasites while PARG occurred always in this organelle. CONCLUSIONS: PARP over-expressing and PARG-silenced cells presented PAR accumulation in the nucleus, even in absence of oxidative stress. Procyclic death pathway after genotoxic damage depends on basal nuclear PAR. This evidence demonstrates that the polymer may have a toxic action by itself since the consequences of an exacerbated PARP activity cannot fully explain the increment in sensitivity observed here. Moreover, the unusual localization of PARP and PARG would reveal a novel regulatory mechanism, making them invaluable model systems.

VERO cells harbor a poly-ADP-ribose belt partnering their epithelial adhesion belt.

Poly-ADP-ribose (PAR) is a polymer of up to 400 ADP-ribose units synthesized by poly-ADP-ribose-polymerases (PARPs) and degraded by poly-ADP-ribose-glycohydrolase (PARG). Nuclear PAR modulates chromatin compaction, affecting nuclear functions (gene expression, DNA repair). Diverse defined PARP cytoplasmic allocation patterns contrast with the yet still imprecise PAR distribution and still unclear functions. Based on previous evidence from other models, we hypothesized that PAR could be present in epithelial cells where cadherin-based adherens junctions are linked with the actin cytoskeleton (constituting the adhesion belt). In the present work, we have examined through immunofluorescence and confocal microscopy, the subcellular localization of PAR in an epithelial monkey kidney cell line (VERO). PAR was distinguished colocalizing with actin and vinculin in the epithelial belt, a location that has not been previously reported. Actin filaments disruption with cytochalasin D was paralleled by PAR belt disruption. Conversely, PARP inhibitors 3-aminobenzamide, PJ34 or XAV 939, affected PAR belt synthesis, actin distribution, cell shape and adhesion. Extracellular calcium chelation displayed similar effects. Our results demonstrate the existence of PAR in a novel subcellular localization. An initial interpretation of all the available evidence points towards TNKS-1 as the most probable PAR belt architect, although TNKS-2 involvement cannot be discarded. Forthcoming research will test this hypothesis as well as explore the existence of the PAR belt in other epithelial cells and deepen into its functional implications.

Host cell poly(ADP-ribose) glycohydrolase is crucial for Trypanosoma cruzi infection cycle.

Trypanosoma cruzi, etiological agent of Chagas' disease, has a complex life cycle which involves the invasion of mammalian host cells, differentiation and intracellular replication. Here we report the first insights into the biological role of a poly(ADP-ribose) glycohydrolase in a trypanosomatid (TcPARG). In silico analysis of the TcPARG gene pointed out the conservation of key residues involved in the catalytic process and, by Western blot, we demonstrated that it is expressed in a life stage-dependant manner. Indirect immunofluorescence assays and electron microscopy using an anti-TcPARG antibody showed that this enzyme is localized in the nucleus independently of the presence of DNA damage or cell cycle stage. The addition of poly(ADP-ribose) glycohydrolase inhibitors ADP-HPD (adenosine diphosphate (hydroxymethyl) pyrrolidinediol) or DEA (6,9-diamino-2-ethoxyacridine lactate monohydrate) to the culture media, both at a 1 µM concentration, reduced in vitro epimastigote growth by 35% and 37% respectively, when compared to control cultures. We also showed that ADP-HPD 1 µM can lead to an alteration in the progression of the cell cycle in hydroxyurea synchronized cultures of T. cruzi epimastigotes. Outstandingly, here we demonstrate that the lack of poly(ADP-ribose) glycohydrolase activity in Vero and A549 host cells, achieved by chemical inhibition or iRNA, produces the reduction of the percentage of infected cells as well as the number of amastigotes per cell and trypomastigotes released, leading to a nearly complete abrogation of the infection process. We conclude that both, T. cruzi and the host, poly(ADP-ribose) glycohydrolase activities are important players in the life cycle of Trypanosoma cruzi, emerging as a promising therapeutic target for the treatment of Chagas' disease.

The metabolism of 9-chloro-ß-lapachone and its effects in isolated hepatocytes. The involvement of NAD(P)H:quinone oxidoreductase 1 (NQO1).

A ß-lapachone analogue (3,4-dihydro-2,2-dimethyl-9-chloro-2H-naphtho[1,2b]pyran-5,6-dione) (9-chloro ß-lapachone), named CGQ, with antitumoral, antiviral and antitrypanocidal activities was assayed for cytotoxic effects on isolated rat hepatocytes. The incubation of hepatocytes with this o-naphthoquinone showed (a) decreased adenylate energy charge, as a result of a decrease in ATP, and an increase in AMP levels; (b) increased NADP(+) content, with a concomitant decrease of NADPH, NADH and NAD(+) content; (c) decreased GSH content, accompanied by an increase in GSSG formation; (d) stimulated oxygen uptake as well as increased superoxide anion production and hydrogen peroxide formation; (e) inhibited lipid peroxidation; (f) hepatocyte viability was not reduced unless the NQO1 inhibitor dicoumarol was present. We hypothesize that the cytotoxicity of CGQ in dicoumarol-treated hepatocytes was the result of inhibition of the NQO1 detoxification pathway, thus allowing more quinone to be metabolized towards the one-electron pathway to form reactive semiquinones and/or reactive oxygen species. The results obtained indicate a protective role of NQO1 in preventing CGQ cytotoxicity in isolated rat hepatocytes.

Poly(ADP-ribose) polymerase plays a differential role in DNA damage-response and cell death pathways in Trypanosoma cruzi.

Poly(ADP-ribosyl)ation is a post-translational modification of proteins. Poly(ADP-ribose) polymerase (PARP) and poly(ADP-ribose) glycohydrolase (PARG) are the enzymes responsible for poly(ADP-ribose) (PAR) polymer metabolism and are present in most higher eukaryotes. The best understood role of PARP is the maintenance of genomic integrity either via promotion of DNA repair at low levels of genotoxic stress or via promotion of cell death at higher levels of damage. The unicellular eukaryote Trypanosoma cruzi, as opposed to humans and other organisms, has only one PARP (TcPARP) and one PARG (TcPARG). In the present study we show that under different DNA-damaging agents (H(2)O(2) or UV-C radiation) TcPARP is activated and translocated from the cytosol to the nucleus, while TcPARG always shows a nuclear localisation. Parasites in the presence of PARP or PARG inhibitors, as well as parasites over-expressing either TcPARP or TcPARG, suggested that PAR metabolism could be involved in different phases of cell growth, even in the absence of DNA damage. We also believe that we provide the first reported evidence that different proteins could be poly(ADP-ribosyl)ated in response to different stimuli, leading to different cell death pathways.

TcPARP: A DNA damage-dependent poly(ADP-ribose) polymerase from Trypanosoma cruzi.

Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme present in most eukaryotes and has been involved in processes such as DNA repair and gene expression. The poly(ADP-ribose) polymer (PAR) is mainly catabolised by poly(ADP-ribose) glycohydrolase. Here, we describe the cloning and characterisation of a PARP from Trypanosoma cruzi (TcPARP). The recombinant enzyme (Mr=65) required DNA for catalytic activity and it was strongly enhanced by nicked DNA. Histones purified from T. cruzi increased TcPARP activity and the covalent attachment of [32P]ADP-ribose moieties to histones was demonstrated. TcPARP required no magnesium or any other metal ion cofactor for its activity. The enzyme was inhibited by 3-aminobenzamide, nicotinamide, theophylline and thymidine but not by menadione. We demonstrated an automodification reaction of TcPARP, and that the removal of attached PAR from this protein resulted in an increase of its activity. The enzyme was expressed in all parasite stages (amastigotes, epimastigotes and trypomastigotes). When T. cruzi epimastigotes were exposed to DNA-damaging agents such as hydrogen peroxide or beta-lapachone, PAR drastically increased in the nucleus, thus confirming PAR synthesis in vivo and suggesting a physiological role for PARP in trypanosomatid DNA repair signalling.

Purification and properties of poly(ADP-ribose)polymerase from Crithidia fasciculata. Automodification and poly(ADP-ribosyl)ation of DNA topoisomerase I.

Poly(ADP-ribose)polymerase has been purified more than 160000-fold from Crithidia fasciculata. This is the first PARP isolated to apparent homogeneity from trypanosomatids. The purified enzyme absolutely required DNA for catalytic activity and histones enhanced it 2.5-fold, when the DNA:histone ratio was 1:1.3. The enzyme required no magnesium or any other metal ion cofactor. The apparent molecular mass of 111 kDa, determined by gel filtration would correspond to a dimer of two identical 55-kDa subunits. Activity was inhibited by nicotinamide, 3-aminobenzamide, theophylline, thymidine, xanthine and hypoxanthine but not by adenosine. The enzyme was localized to the cell nucleus. Our findings suggest that covalent poly(ADP-ribosyl)ation of PARP itself or DNA topoisomerase I resulted in the inhibition of their activities and provide an initial biochemical characterization of this covalent post-translational modification in trypanosomatids.

Citotoxicidad de la ß-Lapachona: una O-Naftoquinona con posibles usos terapéuticos / Citotoxicity of ß-Lapachone: an O-naphthoquinone with possible therapeutic uses

La ß-lapachona (ß-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida, del carcinosarcoma de Walker 256 y del Trypanosoma cruzi. La ß-lap genera productos reactivos del oxígeno (EROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. ß-lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de las topoisomerasas I y II de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la reparación y mantenimiento de la estructura del ADN. Se investigó la citotoxicidad de ß-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y diferentes formas de leucemia aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de ß-lap y su relación con los procesos de apoptosis y necrosis. Entre esos mecanismos se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN), caspasas y nucleasas, que finalmente degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la ß-lap es su reducción enzimática, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de EROS. La acción de ß-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres generados por la ß-lap. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer. (AU)

Citotoxicidad de la ß-Lapachona: una O-Naftoquinona con posibles usos terapéuticos / Citotoxicity of ß-Lapachone: an O-naphthoquinone with possible therapeutic uses

La ß-lapachona (ß-lap) es una o-naftoquinona extraída de la madera del lapacho. Las observaciones iniciales mostraron su acción inhibidora del crecimiento del sarcoma de Yoshida, del carcinosarcoma de Walker 256 y del Trypanosoma cruzi. La ß-lap genera productos reactivos del oxígeno (EROS: anión superóxido, radical hidroxilo y peróxido de hidrógeno) a los que inicialmente se atribuyó su citotoxicidad. ß-lap resultó un potente inhibidor de la síntesis de ADN en T. cruzi, de las topoisomerasas I y II de la poli(ADP-ribosa) polimerasa (PARP) de diferentes orígenes, enzimas responsables de la reparación y mantenimiento de la estructura del ADN. Se investigó la citotoxicidad de ß-lap en células de cáncer epidermoide de laringe, melanoma, cáncer de ovario, de mama, de próstata, de pulmón, adenocarcinoma de colon y diferentes formas de leucemia aportando un mejor conocimiento de los mecanismos moleculares involucrados en la acción de ß-lap y su relación con los procesos de apoptosis y necrosis. Entre esos mecanismos se comprobó la activación de la calpaina, proteasa cuya actividad depende de tioles, seguida por la activación de quinasas (c-JUN), caspasas y nucleasas, que finalmente degradan al ADN y a las proteínas celulares. Una reacción importante para la actividad de la ß-lap es su reducción enzimática, especialmente por la diaforasa y la NAD(P)H-quinona reductasa, que inician la producción de EROS. La acción de ß-lap sobre células tumorales resultaría de la inhibición directa de enzimas como las topoisomerasas, PARP y el factor TNF, sumada a la acción de radicales libres generados por la ß-lap. Los efectos citostáticos de ß-lap han abierto interesantes perspectivas para la quimioterapia del cáncer.

Inhibición de la lipoperaxidación en microsomas de hígado por nifurtimox y nitrofurantoina / Inhibition of lipoperoxidation in liver microsomes by nifurtimox and nitrofurantoin

El nifurtimox y la nitrofurantoína, dos nitrofuranos, inhibieron la formación de malondialdehído (MDA) por microsomas de hígado de rata incubados durante una hora con un sistema generador de NADPH (NADP+, glucosa-6-P, glucosa-6-P deshidrogenasa y Cl2 Mg), ADP y Fe**3+. Las drogas ensayadas se disolvieron en dimetilformamida previo a su agregado al medio de incubación. El efecto del nifurtimox se manifestó en función del tiempo de incubación, de la concentración de droga y disminuyó significativamente cuando se omitió la adición de ADP-Fe**3+. Al tiempo de inhibir la formación de MDA, el nifurtimox inhibió la destrucción de los ácidos grasos polietilénicos microsomales. Este efecto se expresó por las variaciones de la relación araquidónico/oleico, docosahexanoico/oleico y el índice de dobles ligaduras. El nifurtimox inhibió también la destrucción del citocromo P-450, correlacionada con la lipoperoxidación. El solvente utilizado como vehículo del nifurtimox fue crítico para la inhibición, pues con DMSO el nifurtimox estimuló la formación de MDA, no así con DMFA, el solvente que se utilizó en los experimentos descriptos. Con los sistemas peroxidantes ascorbato-Fe e hidroperóxido de t-butilo-Fe, que inducen la lipoperoxidación sin involucrar la NADPH-citocromo P-450 reductasa, el efecto inhibidor del nifurtimox fue relativamente menor comparado con la inhibición observada con el sistema NADPH-Fe. En esa forma, los resultados con ascorbato-Fe y peróxido de t-butilo-Fe indican inhibición de la iniciación de la lipoperoxidación. Sin embargo, cuando se agregó NADPH en concentración catalítica, la inhibición de la lipoperoxidación inducida por el hidroperóxido de t-butilo-Fe aumentó significativamente, sugiriendo la... (AU)

Inhibición de la lipoperaxidación en microsomas de hígado por nifurtimox y nitrofurantoina / Inhibition of lipoperoxidation in liver microsomes by nifurtimox and nitrofurantoin

El nifurtimox y la nitrofurantoína, dos nitrofuranos, inhibieron la formación de malondialdehído (MDA) por microsomas de hígado de rata incubados durante una hora con un sistema generador de NADPH (NADP+, glucosa-6-P, glucosa-6-P deshidrogenasa y Cl2 Mg), ADP y Fe**3+. Las drogas ensayadas se disolvieron en dimetilformamida previo a su agregado al medio de incubación. El efecto del nifurtimox se manifestó en función del tiempo de incubación, de la concentración de droga y disminuyó significativamente cuando se omitió la adición de ADP-Fe**3+. Al tiempo de inhibir la formación de MDA, el nifurtimox inhibió la destrucción de los ácidos grasos polietilénicos microsomales. Este efecto se expresó por las variaciones de la relación araquidónico/oleico, docosahexanoico/oleico y el índice de dobles ligaduras. El nifurtimox inhibió también la destrucción del citocromo P-450, correlacionada con la lipoperoxidación. El solvente utilizado como vehículo del nifurtimox fue crítico para la inhibición, pues con DMSO el nifurtimox estimuló la formación de MDA, no así con DMFA, el solvente que se utilizó en los experimentos descriptos. Con los sistemas peroxidantes ascorbato-Fe e hidroperóxido de t-butilo-Fe, que inducen la lipoperoxidación sin involucrar la NADPH-citocromo P-450 reductasa, el efecto inhibidor del nifurtimox fue relativamente menor comparado con la inhibición observada con el sistema NADPH-Fe. En esa forma, los resultados con ascorbato-Fe y peróxido de t-butilo-Fe indican inhibición de la iniciación de la lipoperoxidación. Sin embargo, cuando se agregó NADPH en concentración catalítica, la inhibición de la lipoperoxidación inducida por el hidroperóxido de t-butilo-Fe aumentó significativamente, sugiriendo la...