Pancreatic islets from dexamethasone-treated rats show alterations in global gene expression and mitochondrial pathways

Chronic administration of glucocorticoids (GC) leads to characteristic features of type 2 diabetes in mammals. The main action of dexamethasone in target cells occurs through modulation of gene expression, although the exact mechanisms are still unknown. We therefore investigated the gene expression profile of pancreatic islets from rats treated with dexamethasone using a cDNA array screening analysis. The expression of selected genes and proteins involved in mitochondria] apoptosis was further analyzed by PCR and immunoblotting. Insulin, triglyceride and free fatty acid plasma levels, as well as glucose-induced insulin secretion, were significantly higher in dexamethasone-treated rats compared with controls. Out of 1176 genes, 60 were up-regulated and 28 were down-regulated by dexamethasone treatment. Some of the modulated genes are involved in apoptosis, stress response, and proliferation pathways. RT-PCR confirmed the cDNA array results for 6 selected genes. Bax alpha protein expression was increased, while Bcl-2 was decreased. In vivo dexamethasone treatment decreased the mitochondrial production of NAD(P)H, and increased ROS production. Concluding, our data indicate that dexamethasone modulates the expression of genes and proteins involved in several pathways of pancreatic-islet cells, and mitochondria dysfunction might be involved in the deleterious effects after long-term GC treatment.

Association between the C242T polymorphism in the p22phox gene with arterial stiffness in the Brazilian population

de Oliveira Alvim R, Lima Santos PCJ, Goncalves Dias R, Rodrigues MV, de Sa Cunha R, Mill JG, Junior WN, Krieger JE, Pereira AC. Association between the C242T polymorphism in the p22phox gene with arterial stiffness in the Brazilian population. Physiol Genomics 44: 587-592, 2012. First published April 10, 2012; doi:10.1152/physiolgenomics.00122.2011.-NADPH oxidase p22phox subunit is responsible for the production of reactive oxygen species in the vascular tissue. The C242T polymorphism in the p22phox gene has been associated with diverse coronary artery disease phenotypes, but the findings about the protective or harmful effects of the T allele are still controversial. Our main aim was to assess the effect of p22phox C242T genotypes on arterial stiffness, a predictor of late morbidity and mortality, in individuals from the general population. We randomly selected 1,178 individuals from the general population of Vitoria City, Brazil. Genotypes for the C242T polymorphism were detected by PCR-RFLP, and pulse wave velocity (PWV) values were measured with a noninvasive automatic device Complior. p22phox and TNF-alpha gene expression were quantified by real-time PCR in human arterial mammary smooth muscle cells. In both the entire and nonhypertensive groups: individuals carrying the TT genotype had higher PWV values and higher risk for increased arterial stiffness [odds ratio (OR) 1.93, 95% confidence interval (CI) 1.27-2.92 and OR 1.78, 95% CI 1.07-2.95, respectively] compared with individuals carrying CC + CT genotypes, even after adjustment for covariates. No difference in the p22phox gene expression according C242T genotypes was observed. However, TNF-alpha gene expression was higher in cells from individual carrying the T allele, suggesting that this genetic marker is associated with functional phenotypes at the gene expression level. In conclusion, we suggest that p22phox C242T polymorphism is associated with arterial stiffness evaluated by PWV in the general population. This genetic association shed light on the understanding of the genetic modulation on vascular dysfunction mediated by NADPH oxidase.

Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src-Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day] pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs. (Hypertension. 2012; 59: 1263-1271.). Online Data Supplement

Hydroxyl scavenging activity accounts for differential antioxidant protection of Plantago major against oxidative toxicity in isolated rat liver mitochondria

Objectives The aim of this work was to study the effects of P. major against the oxidative damage of isolated rat liver mitochondria. Methods The extracts were obtained using methanol (MeOH), ethyl acetate (EAc), dichloromethane (DCM), and hexane (Hex) as solvents. Key findings Hex, DCM, and EAc totally, and MeOH partially, inhibited ROS generation and lipid peroxidation of membranes induced by Fe2+ or t-BOOH. However, only MeOH was able to prevent the t-BOOH-induced glutathione and NAD(P)H oxidation. All extracts chelated Fe2+ and reduced DPP Hradicals. EPR analysis revealed that P. major exhibited potent scavenger activity for hydroxyl radicals. Conclusions The potent antioxidant activity exhibited by P. major was able to prevent oxidative mitochondrial damage, contributing to the understanding of its hepatoprotective action against ROS-mediated toxicity.

Insulin alone or with captopril: effects on signaling pathways (AKT and AMPK) and oxidative balance after ischemia-reperfusion in isolated hearts

Insulin and the inhibition of the reninangiotensin system have independent benefits for ischemiareperfusion injury, but their combination has not been tested. Our aim was to evaluate the effects of insulin+captopril on insulin/angiotensin signaling pathways and cardiac function in the isolated heart subjected to ischemiareperfusion. Isolated hearts were perfused (Langendorff technique) with KrebsHenseleit (KH) buffer for 25 min. Global ischemia was induced (20 min), followed by reperfusion (30 min) with KH (group KH), KH+angiotensin-I (group A), KH+angiotensin-I+captopril (group AC), KH+insulin (group I), KH+insulin+angiotensin-I (group IA), or KH+insulin+angiotensin-I+captopril (group IAC). Group A had a 24% reduction in developed pressure and an increase in end-diastolic pressure vs. baseline, effects that were reverted in groups AC, IA, and IAC. The phosphorylation of protein kinase B (AKT) was higher in groups I and IA vs. groups KH and A. The phosphorylation of AMP-activated protein kinase (AMPK) was similar to 31% higher in groups I, IA, and IAC vs. groups KH, A, and AC. The tert-butyl hydroperoxide (tBOOH)-induced chemiluminescence was lower (similar to 2.2 times) in all groups vs. group KH and was similar to 35% lower in group IA vs. group A. Superoxide dismutase content was lower in groups A, AC, and IAC vs. group KH. Catalase activity was similar to 28% lower in all groups (except group IA) vs. group KH. During reperfusion of the ischemic heart, insulin activates the AKT and AMPK pathways and inhibits the deleterious effects of angiotensin-I perfusion on SOD expression and cardiac function. The addition of captopril does not potentiate these effects.

The role of reactive oxygen species in the modulation of the contraction induced by angiotensin II in carotid artery from diabetic rat

The modulation played by reactive oxygen species on the angiotensin II-induced contraction in type I-diabetic rat carotid was investigated. Concentration-response curves for angiotensin II were obtained in endothelium-intact or endothelium-denuded carotid from control or streptozotocin-induced diabetic rats, pre-treated with tiron (superoxide scavenger), PEG-catalase (hydrogen peroxide scavenger), dimethylthiourea (hydroxyl scavenger), apocynin [NAD(P) H oxidase inhibitor], SC560 (cyclooxygenase-1 inhibitor), SC236 (cyclooxygenase-2 inhibitor) or Y-27632 (Rho-kinase inhibitor). Reactive oxygen species were measured by flow cytometry in dihydroethidium (DHE)-loaded endothelial cells. Cyclooxygenase and AT1-receptor expression was assessed by immunohistochemistry. Diabetes increased the angiotensin II-induced contraction but reduced the agonist potency in rat carotid. Endothelium removal, tiron or apocynin restored the angiotensin II-induced contraction in diabetic rat carotid to control levels. PEG-catalase, DMTU or SC560 reduced the angiotensin II-induced contraction in diabetic rat carotid at the same extent. SC236 restored the angiotensin II potency in diabetic rat carotid. Y-27632 reduced the angiotensin II-induced contraction in endothelium-intact or -denuded diabetic rat carotid. Diabetes increased the DHE-fluorescence of carotid endothelial cells. Apocynin reduced the DHE-fluorescence of endothelial cells from diabetic rat carotid to control levels. Diabetes increased the muscular cyclooxygenase-2 expression but reduced the muscular AT1-receptor expression in rat carotid. In summary, hydroxyl radical, hydrogen peroxide and superoxide anion-derived from endothelial NAD(P) H oxidase mediate the hyperreactivity to angiotensin II in type I-diabetic rat carotid, involving the participation of cyclooxygenase-1 and Rho-kinase. Moreover, increased muscular cyclooxygenase-2 expression in type I-diabetic rat carotid seems to be related to the local reduced AT1-receptor expression and the reduced angiotensin II potency. (C) 2011 Elsevier B. V. All rights reserved.

Azione antiossidante del sulforafane: analisi in cellule cardiache in coltura ed in un modello animale di esercizio fisico

Oxidative stress is considered to be of major relevance for a variety of pathological processes. Thus, it is valuable to identify compounds, which might act as antioxidants, i.e. compounds that antagonize the deleterious action of reactive oxygen species (ROS) on biomolecules. The mode of action of these compounds could be either to scavenge ROS directly or to trigger protective mechanisms inside the cell, thereby resulting in improved defense against ROS. Sulforaphane (SF) (1-isothiocyanato-(4R)-(methylsulfinyl)butane) is a naturally occurring cancer chemopreventive agent found as a precursor glucosinolate in Cruciferous vegetables like broccoli. Although SF is not a direct-acting antioxidant, there is substantial evidence that SF acts indirectly to increase the antioxidant capacity of animal cells and their abilities to cope with oxidative stress. Induction of phase 2 enzymes is one means by which SF enhances the cellular antioxidant capacity. Enzymes induced by SF include Glutathione S-transferases (GST) and NAD[P]H:quinone oxidoreductase (NQO1) which can function as protectors against oxidative stress. To protect themselves from oxidative stress, cells are equipped with reducing buffer systems including the GSH and thioredoxin (Trx) reductase. GSH is an important tripeptide thiol which in addition to being the substrate for GSTs maintains the cellular oxidation– reduction balance and protects cells against free radical species. Aim of the first part of this thesis was to investigate the ability of SF to induce the expression and the activity of different phase 2 and antioxidant enzymes (such as GST, GR, GPx, NQO1, TR, SOD, CAT) in an in vitro model of rat cardiomyocytes, and also to define if SF treatment supprts cells in counteracting oxidative stress induced by H2O2 It is well known that acute exhaustive exercise causes significant reactive oxygen species generation that results in oxidative stress, which can induce negative effects on health and well being. In fact, increased oxidative stress and biomarkers (e.g., protein carbonyls, MDA, and 8- hydroxyguanosine) as well as muscle damage biomarkers (e.g. plasmatic Creatine cinase and Lactate dehydrogenase) have been observed after supramaximal sprint exercises, exhaustive longdistance cycling or running as well as resistance-type exercises, both in trained and untrained humans. Markers of oxidative stress also increase in rodents following exhaustive exercise. Moreover, antioxidant enzyme activities and expressions of antioxidant enzymes are known to increase in response to exhaustive exercise in both animal and human tissues. Aim of this project was to evaluate the effect of SF supplementation in counteracting oxidative stress induced by physical activity through its ability to induce phase 2, and antioxidant enzymes in rat muscle. The results show that SF is a nutraceutical compound able to induce the activity of different phase 2 and antioxidant enzymes in both cardiac muscle and skeletal muscle. Thanks to its actions SF is becoming a promising molecule able to prevent cardiovascular damages induced by oxidative stress and muscle damages induced by acute exhaustive exercise.

Alimenti Funzionali e Componenti Nutraceutici come Biomodulatori

Recent knowledge supports the hypothesis that, beyond meeting nutrition needs, diet may modulate various functions in the body and play beneficial roles in some diseases. Research on functional foods is addressing the physiologic effects and health benefits of foods and food components, with the aim of authorizing specific health claims. The recognition that oxidative stress plays a major role in the pathophysiology of cardiac disorders has led to extensive investigations of the protective effects of exogenous antioxidants, but results are controversial. A promising strategy for protecting cardiac cells against oxidative damage may be through the induction of endogenous phase 2 enzymes with the enhancement of cellular antioxidant capacity. Sulforaphane (SF), a naturally occurring isothiocyanate abundant in Cruciferous vegetables, has gained attention as a potential chemopreventive compound thanks to its ability to induce several classes of genes implicated in reactive oxygen species (ROS) and electrophiles detoxification. Antioxidant responsive element (ARE)-mediated gene induction is a pivotal mechanism of cellular defence against the toxicity of electrophiles and ROS. The transcription factor NF-E2-related factor-2 (Nrf2), is essential for the up-regulation of these genes. We investigated whether SF could exert cardioprotective effects against oxidative stress and elucidated the mechanisms underpinning these effects. Accordingly, using cultured rat neonatal cardiomyocytes as a model system, we evaluated the time-dependent induction of gene transcription, the corresponding protein expression and activity of various antioxidant and phase 2 enzymes (catalase, superoxide dismutase, glutathione and related enzymes glutathione reductase, glutathione peroxidase and glutathione S-transferase, NAD(P)H: quinone oxidoreductase 1 and thioredoxine reductase) elicited by SF. The results were correlated to intracellular ROS production and cell viability after oxidative stress generated by H2O2, and confirmed the ability of SF to exert cytoprotective effects acting as an indirect antioxidant. Furthermore, to get better insight into SF mechanism of action, we investigated the effect of SF treatment on Nrf2 and the upstream signalling pathways MAPK ERK1/2 and PI3K/Akt, known to mediate a pro survival signal in the heart. The use of specific inhibitors of ERK1/2 and Akt phosphorylation demonstrated their involvement in phase 2 enzymes induction. The concentration of SF tested in this study is comparable to peak plasma concentration achieved after dietary exposure giving clear relevance to our data to support dietary intake of Cruciferous vegetables in cytoprotection against oxidative stress, a common determinant of many cardiovascular diseases.

Zuckertransport und Regulation des Hexosestoffwechsels bei Oenococcus oeni

Das Milchsäurebakterium Oenococcus oeni, welches für den biologischen Säureabbau im Wein eingesetzt wird, verstoffwechselt Hexosen über den Phosphoketolaseweg. Dabei können beträchtliche Mengen Acetat entstehen. Die Ursachen dafür wurden untersucht, insbesondere der Fructosestoffwechsel. Außerdem wurde der Hexosetransport untersucht, über den bei O. oeni noch nichts bekannt war. Die Aufnahme von Hexosen in die Zelle erfolgt mit hoher Affinität (KM=10 µM) über einen Symport mit H+, aber mit sehr niedriger spezifischer Aktivität (Vmax=9 U / g TG). Zusätzlich werden Hexosen mit ausreichender Aktivität über (vermutlich erleichterte) Diffusion in die Zelle transportiert, allerdings nur bei hohen Hexosekonzentrationen. Es wurden Gene gefunden, die für ein Hexose- Phosphotransferasesystem kodieren, welches in O. oeni keine bedeutende Rolle beim Transport spielt, aber vermutlich eine regulative Funktion hat. Zur Bildung von Essigsäure tragen verschiedene Faktoren bei: Der Ethanolweg, der in der heterofermentativen Milchsäuregärung die Reoxidation von NAD(P)H bewerkstelligt, ist durch die niedrige spezifische Aktivität der Acetaldehyddehydrogenase limitiert. Diese Limitierung wird noch verstärkt, wenn die zellulären Gehalte von Coenzym A aufgrund von Pantothensäuremangel niedrig sind. O. oeni umgeht durch Bildung von Erythrit die Limitierung, und Acetylphosphat wird nicht zu Ethanol reduziert, sondern als Acetat ausgeschieden. Bei Cofermentation von Hexosen mit externen Elektronenakzeptoren, wie Fructose, Pyruvat oder Sauerstoff, werden letztere zur Reoxidation von NAD(P)H genutzt, und als Folge wird Acetat ausgeschieden. Der Fluss von Fructose in den Phosphoketolaseweg wird durch das Enzym Phosphoglucoseisomerase verhindert, wenn dieses durch 6-Phosphogluconat gehemmt wird. Als Konsequenz wird Fructose im Mannitweg reduziert, was die Bildung von Essigsäure im Phosphoketolaseweg fördert. Bei niedrigen Wachstums- und Stoffwechselraten, z.B. bei C-Limitierung, ist der Ethanolweg nicht limitierend für den Stoffwechsel, und Hexosen werden über heterofermentative Milchsäuregärung umgesetzt, ohne daß Acetat entsteht. Pyruvat kann gleichzeitig als Elektronenakzeptor und als Energiequelle dienen: O. oeni ist in der Lage, Pyruvat mittels Disproportionierung zu Lactat und Acetat+CO2 zu fermentieren, und dabei Energie zu konservieren (0,5 ATP / Pyruvat).

Ruolo delle specie reattive dell'ossigeno e di caveole/raft lipidici nella biosegnalazione in linee cellulari umane

Membrane lipid rafts are detergent-resistant microdomains containing glycosphingolipids, cholesterol and glycosylphosphatidylinositol-linked proteins; they seem to be actively involved in many cellular processes including signal transduction, apoptosis, cell adhesion and migration. Lipid rafts may represent important functional platforms where redox signals are produced and transmitted in response to various agonists or stimuli. In addition, a new concept is emerging that could be used to define the interactions or amplification of both redox signalling and lipid raft-associated signalling. This concept is characterized by redox-mediated feed forward amplification in lipid platforms. It is proposed that lipid rafts are formed in response to various stimuli; for instance, NAD(P)H oxidase (Nox) subunits are aggregated or recruited in these platforms, increasing Nox activity. Superoxide and hydrogen peroxide generation could induce various regulatory activities, such as the induction of glucose transport activity and proliferation in leukaemia cells. The aim of our study is to probe: i) the involvement of lipid rafts in the modulation of the glucose transporter Glut1 in human acute leukemia cells; ii) the involvement of plasma membrane caveolae/lipid rafts in VEGF-mediated redox signaling via Nox activation in human leukemic cells; iii) the role of p66shc, an adaptor protein, in VEGF signaling and ROS production in endothelial cells (ECs); iv) the role of Sindecan-2, a transmembrane heparan sulphate proteoglycan, in VEGF signaling and physiological response in ECs and v) the antioxidant and pro-apoptotic activities of simple dietary phenolic acids, i. e. caffeic, syringic and protocatechuic acids in leukemia cells, characterized by a very high ROS content. Our results suggest that the role played by NAD(P)H oxidase-derived ROS in the regulation of glucose uptake, proliferation and migration of leukaemia and endothelial cells could likely occur through the control of lipid raft-associated signalling.

Identifizierung von Hexosecarriern und Regulation des Phosphoketolasewegs in Oenococcus oeni

Oenococcus oeni ist ein heterofermentatives Milchsäurebakterium, das Hexosen über den Phosphoketolaseweg zu Lactat, Acetyl-P und CO2 umsetzt. In Anhängigkeit von der C-Quelle werden Ethanol, Acetat, Mannit und geringe Mengen an Erythrit gebildet. Im Genom von O. oeni PSU-1 wurden neun Gene für sekundäre Carrier identifiziert, die aufgrund ihrer Sequenz und Lage im Genom als Kandidaten für Hexosecarrier in Frage kommen. In Stamm O. oeni B1 wurde die Expression der Gene OEOE_0819 und OEOE_1574 stark durch Glucose induziert. Die Gene konnten jedoch Hexosetransport-Defektmutanten von E. coli nicht komplementieren. Die deutliche Induktion der Gene deutet auf ihre Funktion im Hexosetransport hin. Bei den Carriern 819 und 1574 handelt es sich vermutlich um früher identifizierte Δp-getriebene sekundäre Hexosetransporter. Die Expression der Kandidaten zeigte teilweise Unterschiede bei verschiedenen O. oeni Stämmen. Auch die Funktion von Phosphotransferasesystemen bei der Aufnahme von Hexosen wurde durch Expressionsmessungen untersucht. Einige Systeme weisen Ähnlichkeit zu Glucose- und Fructose-transportierenden Systemen auf und kommen als Aufnahmesysteme für Hexosen in Frage. Das Phosphotransferasesystem OEOE_0464-0466 wird besonders durch Fructose induziert. O. oeni bildet in Abhängigkeit von der C-Quelle unterschiedliche Wege zur Reoxidation des im zentralen Phosphoketolaseweg entstandenen NAD(P)H aus. Die Regulation des Zentralstoffwechsels und des peripheren Stoffwechsels wurde durch Bestimmung der relativen Genexpression und durch Proteomanalysen untersucht. Die Anwesenheit von Citrat und Pyruvat ändert die Expression der Gene deutlich. Bei Zucht mit Ribose, Glucose oder Fructose jedoch bleibt die der meisten Gene konstant. Am stärksten wird der Ethanolweg reguliert, der hauptsächlich nach Zucht mit Glucose zur Reoxidation des NAD(P)H genutzt wird. Die Expression des adhE-Gens der Acetaldehyd- und Alkohol-Dehydrogenase nimmt bei Zucht auf anderen C-Quellen stark ab. Pyruvat ist ein zentrales Intermediat des Energiestoffwechsels und Ausgangsprodukt für Reaktionen des Bausstoffwechsels. Die Expression einiger Pyruvat-liefernder und Pyruvat-verbrauchender Reaktionen wurde bestimmt, um so die Entstehung und den Verbrauch des Pyruvats abhängig von der Stoffwechselsituation beurteilen zu können.

Studio degli effetti funzionali e tossici di derivati di Brassicaceae in modelli sperimentali

I vegetali appartenenti alla famiglia delle Brassicaceae, sono ricchi di molecole biologicamente attive note per le numerose proprietà salutari. L’effetto di un estratto di germogli di cavolo nero toscano (TBCSE) è stato investigato, in termini chemiopreventivi, sugli enzimi epatici del metabolismo degli xenobiotici e antiossidanti, in ratti trattati con TBCSE. I risultati hanno mostrato un complesso pattern di modulazione, con una prevalente inibizione, del sistema citocromo P450-dipendente, e induzioni significative degli enzimi di fase II (glutatione transferasi e glucuronosiltransferasi) e antiossidanti (catalasi, NAD(P)H:chinone reduttasi, glutatione reduttasi e perossidasi). Successivamente, l’effetto di TBCSE è stato studiato nei confronti delle alterazioni provocate da un’alimentazione iperlipidica nel ratto. Il trattamento si è dimostrato efficace nel contrastare gli effetti deleteri dei grassi presenti nella dieta, come l’iperlipidemia, l’aumento del peso corporeo e del fegato, l’indebolimento delle attività degli enzimi antiossidanti e del potenziale detossificante a livello epatico. Complessivamente, TBCSE emerge essere un promettente prodotto nutraceutico con potenziali effetti chemiopreventivi, e da impiegare come strategia alimentare per contrastare gli effetti correlati ad una dieta iperlipidica. Il consumo di dosi sovralimentari di molecole isolate dalle Brassicaceae, tramite per esempio integratori dietetici, come strategia alimentare preventiva, potrebbe tuttavia rappresentare un rischio per la salute. La potenziale tossicità del sulforafane, glucorafanina, indolo-3-carbinolo, e 3,3'-diindolimetano, è stata valutata in epatociti primari di ratto. La citotossicità e l’induzione di stress ossidativo, osservate a concentrazioni non lontane da quelle che potrebbero essere raggiunte in vivo, insieme ad una forte modulazione dell’espressione genica, riguardante principalmente il metabolismo degli xenobiotici, risposte ad alterazioni dello stato ossidoredutivo, eventi di riparazione del DNA e di proteine, induzione dell’apoptosi, e meccanismi (co)cancerogeni, sottolineano la potenzialità di queste molecole di determinare un rischio tossicologico, in seguito ad un’assunzione prolungata e ad alte dosi.

Ursachen der Anthrazyklin-induzierten Kardiotoxizität : Einfluss der individuellen genetischen Ausstattung, des oxidativen Stresses und der Metabolisierung

Doxorubizin (Dox) gehört zur Gruppe der Anthrazykline, welche seit mehreren Jahrzehnten erfolgreich gegen ein breites Spektrum an Tumoren eingesetzt wird. Neben der guten Wirksamkeit besitzt Dox jedoch auch ein sehr hohes Nebenwirkungspotential. Die wohl folgenschwerste Nebenwirkung stellt die irreversible Schädigung des Herzens dar. Zahlreiche Faktoren, wie zum Beispiel die kumulative Dox-Dosis konnten bereits mit einer erhöhten Inzidenz an kardialen Schäden in Verbindung gebracht werden. Bislang ungeklärt war jedoch die Frage, warum Patienten unterschiedlich sensibel auf die Verabreichung von Dox reagierten. rnAn dem Patientenkollektiv der Ricover60-Studie wurde der Einfluss der individuellen genetischen Ausstattung auf die Entstehung der Anthrazyklin-induzierten Herzschädigung untersucht. Alle Patienten mit Dox-induzierten Herzschäden wurden identifiziert und auf das Vorhandensein von genetischen Polymorphismen der NAD(P)H-Oxidase (CYBA, RAC2 und NCF4) und der Anthrazyklin-Transporter (MRP1 und MRP2) untersucht. Sowohl für CYBA als auch für RAC2 konnte eine Anreicherung bestimmter Genotypen (CYBA: CT/TT; RAC2: TA/AA) in der Gruppe der herzgeschädigten Patienten nachgewiesen werden. In der Multivariaten Analyse von RAC2 erreichte diese Anreicherung ein signifikantes Niveau (p=0.028). Damit konnte für diesen Polymorphismus die klinische Relevanz bestätigt werden.rnDie Ursachen der Dox-induzierten Toxizität wurden außerdem an verschiedenen Mäusestämmen und Zelllinien untersucht. Balb/c- und C57BL/6-Mäuse, die bekanntermassen unterschiedlich sensibel auf Dox reagierten, wurden mit Dox behandelt. Anschliessend wurden die Organe Herz, Leber und Blut via HPLC untersucht. Es konnte gezeigt werden, dass sich 1. die Hauptanreicherungsorte für Dox und Doxol (Balb/c: Herz und Blut versus C57BL/6: Leber), 2. die nachgewiesenen Gesamtmengen an Dox+Doxol+Doxon in den drei Organen (MengeC57BL/6 > MengeBalb/c) sowie 3. die An- und Abflutungsgeschwindigkeiten von Dox zwischen den beiden Mäusestämmen unterscheiden. Schlussendlich konnte im Vergleich zu den Balb/c-Mäusen, bei den C57BL/6-Mäusen eine stärkere kardiale Anreicherung von Dox nach der mehrmaligen Dox-Injektion nachgewiesen werden. Somit scheinen der deutlich höhere Dox-Gehalt und die längere Verweilzeit in den Herzen für die stärkere kardiale Schädigung der C57BL/6-Mäuse verantwortlich zu sein. Hingegen verlief die Art der Dox-Metabolisierung in beiden Mäusestämmen ähnlich. rnBei der Betrachtung des oxidativen Stresses konnte gezeigt werden, dass in den Herzen der C57BL/6-Mäusen ein gröβerer oxidativer Stress vorlag, als bei den Balb/c-Mäusen. Ähnlich wie bei der Ricover60-Studie ließ sich auch bei den Mäusen eine Beteiligung der NAD(P)H-Oxidase am Dox-induzierten oxidativen Stress nachweisen. rnMit der HTETOP-Zelllinie konnte gezeigt werden, dass Dox unter physiologischen Bedingungen oxidativen Stress auslösen kann. Die Art und die Konzentration der gebildeten ROS waren abhängig von der Dox-Konzentration, der Einwirkzeit und der Kompensationsfähigkeit der Zellen. Durch die Gabe von Dex ließ sich das Ausmaß des oxidativen Stresses lediglich in den Mäuseherzen reduzieren. In den HTETOP-Zellen zeigte Dex selbst stressauslösende Eigenschaften. Durch die Behandlung mit Dex / DOXY konnte gezeigt werden, dass die Hemmung der Topo IIα selbst oxidativen Stress in den HTETOP-Zellen auslöst. Jedoch scheint weder die Topo IIalpha-Hemmung, noch der Dox-induzierte oxidative Stress bei physiologischen Dox-Konzentrationen (< 1 µM) eine entscheidende Rolle für die Toxizität zu spielen. rnIn der Mikroarray-Analyse der HTETOP-Zellen konnten verschiedene Gene identifiziert werden, die in den oxidativen Stress involviert sind und die durch die Gabe von Dox differentiell reguliert werden. Durch die Komedikation mit Dex / DOXY ließen sich diese Veränderungen teilweise modulieren. rn

Hierarchical accumulation of RyR post-translational modifications drives disease progression in dystrophic cardiomyopathy

AIMS:Duchenne muscular dystrophy (DMD) is a muscle disease with serious cardiac complications. Changes in Ca(2+) homeostasis and oxidative stress were recently associated with cardiac deterioration, but the cellular pathophysiological mechanisms remain elusive. We investigated whether the activity of ryanodine receptor (RyR) Ca(2+) release channels is affected, whether changes in function are cause or consequence and which post-translational modifications drive disease progression. METHODS AND RESULTS:Electrophysiological, imaging, and biochemical techniques were used to study RyRs in cardiomyocytes from mdx mice, an animal model of DMD. Young mdx mice show no changes in cardiac performance, but do so after ∼8 months. Nevertheless, myocytes from mdx pups exhibited exaggerated Ca(2+) responses to mechanical stress and 'hypersensitive' excitation-contraction coupling, hallmarks of increased RyR Ca(2+) sensitivity. Both were normalized by antioxidants, inhibitors of NAD(P)H oxidase and CaMKII, but not by NO synthases and PKA antagonists. Sarcoplasmic reticulum Ca(2+) load and leak were unchanged in young mdx mice. However, by the age of 4-5 months and in senescence, leak was increased and load was reduced, indicating disease progression. By this age, all pharmacological interventions listed above normalized Ca(2+) signals and corrected changes in ECC, Ca(2+) load, and leak. CONCLUSION:Our findings suggest that increased RyR Ca(2+) sensitivity precedes and presumably drives the progression of dystrophic cardiomyopathy, with oxidative stress initiating its development. RyR oxidation followed by phosphorylation, first by CaMKII and later by PKA, synergistically contributes to cardiac deterioration.

Mitochondrial dysfunction leads to NOX activation: A novel mechanism to maintain high glycolysis in cancer cells

Increased glycolysis and oxidative stress are common features of cancer cells. These metabolic alterations are associated with mitochondrial dysfunction and can be caused by mitochondrial DNA (mtDNA) mutations, oncogenic signals, loss of tumor suppressor, and tumor tissue hypoxia. It is well established that mitochondria play central roles in energy metabolism, maintenance of redox balance, and regulation of apoptosis. However, the biochemical and molecular mechanisms that maintain high glycolysis in cancer cells (the Warburg effect) with mitochondrial dysfunction and oxidative stress remain to be determined. The major goals of this study were to establish a unique experimental system in which the mitochondrial respiratory function can be regulated as desired, and to use this system to investigate the mechanistic link between mitochondrial dysfunction and the Warburg effect along with oxidative stress in cancer cells. To achieve these goals, I have established a tetracycline-inducible system in which a dominant negative form of mitochondrial DNA polymerase y (POLGdn) expression could be regulated by tetracycline; thus controlling mitochondrial respiratory function. Using this cell system, I demonstrated that POLGdn expression resulted in mitochondrial dysfunction through decreasing mtDNA content, depletion of mtDNA encoded mRNA and protein expression. This process was mediated by TFAM proteasome degradation. Mitochondrial dysfunction mediated by POLGdn expression led to a significant increase in cellular glycolysis and oxidative stress. Surprisingly, mitochondrial dysfunction also resulted in increased NAD(P)H oxidase (NOX) enzyme activity, which was shown to be essential for maintaining high glycolysis. Chemical Inhibition of NOX activity by diphenyliodonium (DPI) preferentially impacted the survival of mitochondrial defective cells. The colon cancer HCT116-/- cells that have lost transcriptional regulation of the mitochondrial assembling enzyme SCO2, leading to compromised mitochondrial respiratory function, were found to have increased NOX activity and were highly sensitive to DPI treatment. Ovarian epithelial cells with Ras transformation also exhibited an increase in NOX gene expression and NOX enzyme activity, rendering the cells sensitive to DPI inhibition especially under hypoxic condition. These data together suggest that NOX plays a novel role in maintaining high glycolysis in cancer cells with mitochondrial defects, and that NOX may be a potential target for cancer therapy. ^