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The underlying pathophysiological mechanisms of hepatic ischemia-reperfusion (I/R) injury have not been completely elucidated. However, it is well known that oxidative stress, caused by a burst of reactive oxygen species (ROS) production during the reperfusion phase, plays a crucial role. A growing body of evidence indicates that the intracellular availability of free iron represents a requirement for ROS-induced adverse effects, as iron catalyzes the generation of highly reactive free radicals. The aim of this study was to examine whether a combination of iron chelators with varying lipophilicity could offer enhanced protection against I/R by diminishing the conversion of weak oxidants, like HHepG2 cells (hepatocellular carcinoma cell line) were exposed to oxidative stress conditions after pre-treatment with the iron chelators desferrioxamine (DFO) and deferiprone (DFP) alone or in combination. Labile iron pool was estimated using the calcein-acetoxymethyl ester (calcein-AM) method and DNA damage with the comet assay. We subsequently used a rabbit model (male New Zealand white rabbits) of hepatic I/R-induced injury to investigate, by measuring biochemical (ALT, ALT, ALP, γGT) and histological parameters, whether this may be true for in vivo conditions.The combination of a membrane-permeable iron chelator (DFP) with a strong membrane-impermeable one (DFO) raises the level of protection in both hepatic cell lines exposed to oxidative stress conditions and hepatic I/R rabbit model.Our results show that combinations of iron chelators with selected lipophilicity and iron-binding properties may represent a valuable strategy to protect against tissue damage during reperfusion after a period of ischemia.

Hereditary Hemorrhagic Telangiectasia type 1 (HHT1) is an autosomal dominant inherited disease characterized by arteriovenous malformations and hemorrhage. HHT1 is caused by mutations in ENDOGLIN, which encodes an ancillary receptor for Transforming Growth Factor-β/Bone Morphogenetic Protein-9 expressed in all vascular endothelial cells. Haploinsufficiency is widely accepted as the underlying mechanism for HHT1. However, it remains intriguing that only some, but not all, vascular beds are affected, as these causal gene mutations are present in vasculature throughout the body. Here, we have examined the endoglin expression levels in the blood vessels of multiple organs in mice and in humans. We found a positive correlation between low basal levels of endoglin and the general prevalence of clinical manifestations in selected organs. Endoglin was found to be particularly low in the skin, the earliest site of vascular lesions in HHT1, and even undetectable in the arteries and capillaries of heterozygous endoglin mice. Endoglin levels did not appear to be associated with organ-specific vascular functions. Instead, our data revealed a critical endoglin threshold compatible with the haploinsufficiency model, below which endothelial cells independent of their tissue of origin exhibited abnormal responses to Vascular Endothelial Growth Factor. Our results support the development of drugs promoting endoglin expression as potentially protective.

Recent advances in induced pluripotent stem cells (iPSC) and gene editing technologies enable the development of novel human cell-based disease models for phenotypic drug discovery (PDD) programs. Although these novel devices could predict the safety and efficacy of investigational drugs in humans more accurately, their development to the clinic still strongly rely on mammalian data, notably the use of mouse disease models. In parallel to human organoid or organ-on-chip disease models, the development of relevant in vitro mouse models is therefore an unmet need for evaluating direct drug efficacy and safety comparisons between species and in vivo and in vitro conditions. Here, a vascular sprouting assay that utilizes mouse embryonic stem cells differentiated into embryoid bodies (EBs) is described. Vascularized EBs cultured onto 3D-collagen gel develop new blood vessels that expand, a process called sprouting angiogenesis. This model recapitulates key features of in vivo sprouting angiogenesis-formation of blood vessels from a pre-existing vascular network-including endothelial tip cell selection, endothelial cell migration and proliferation, cell guidance, tube formation, and mural cell recruitment. It is amenable to screening for drugs and genes modulating angiogenesis and shows similarities with recently described three-dimensional (3D) vascular assays based on human iPSC technologies.

Background: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. Methods: We took advantage of Acvrl1 +/− mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1 +/− mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. Results: Acvrl1 +/− retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial “tip cells” at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1 +/− mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1 +/− embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1 +/− (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1 +/− embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1 +/− endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1 +/− mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1 +/− tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. Conclusions: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.

Within the Interstitial Lung Diseases (ILD), patients with idiopathic pulmonary fibrosis (IPF) and a subset of those with non-IPF fibrotic ILD have a distinct clinical phenotype of progression despite management. This group of patients has been collectively termed the progressive fibrotic phenotype (PFP). Their early recognition may facilitate access to antifibrotic therapies to prevent or slow progression. Macrophages/monocytes within the lung orchestrate the progression and maintenance of fibrosis. A novel role for monocyte-derived macrophages during tissue damage and wound healing is the expression of collagens. We examined Collagen 1a1 expression in airway macrophages from ILD patients at diagnosis. COL1A1 mRNA levels from BAL cells were elevated in IPF and Non-IPF patients. The presence of a UIP pattern and a subsequent progressive phenotype were significantly associated with the higher BAL COL1A1 levels. In Non-IPF patients, higher COL1A1 levels were associated with a more than twofold increase in mortality. The intracellular localisation of COL1A1 in airway macrophages was demonstrated by confocal microscopy in CD45 and CD163 co-staining assays. Additionally, airway macrophages co-expressed COL1A1 with the profibrotic SPP1 gene product osteopontin. The levels of SPP1 mRNA and OPN in the BAL were significantly higher in IPF and Non-IPF patients relative to healthy. Our results suggest that profibrotic airway macrophages are increased in the BAL of patients with IPF and other ILDs and co-express COL1A1 and OPN. Importantly, COL1A1 expression by pro-fibrotic airway macrophages could be a marker of disease progression and poor survival in ILDs.

Hereditary hemorrhagic telangiectasia (HHT) is a genetic disorder characterized by multi-systemic vascular dysplasia affecting 1 in 5000 people worldwide. Individuals with HHT suffer from many complications including nose and gastrointestinal bleeding, anemia, iron deficiency, stroke, abscess, and high-output heart failure. Identification of the causative gene mutations and the generation of animal models have revealed that decreased transforming growth factor-β (TGF-β)/bone morphogenetic protein (BMP) signaling and increased vascular endothelial growth factor (VEGF) signaling activity in endothelial cells are responsible for the development of the vascular malformations in HHT. Perturbations in these key pathways are thought to lead to endothelial cell activation resulting in mural cell disengagement from the endothelium. This initial instability state causes the blood vessels to response inadequately when they are exposed to angiogenic triggers resulting in excessive blood vessel growth and the formation of vascular abnormalities that are prone to bleeding. Drugs promoting blood vessel stability have been reported as effective in preclinical models and in clinical trials indicating possible interventional targets based on a normalization approach for treating HHT. Here, we will review how disturbed TGF-β and VEGF signaling relates to blood vessel destabilization and HHT development and will discuss therapeutic opportunities based on the concept of vessel normalization to treat HHT.

Iron is a transition metal and essential constituent of almost all living cells and organisms. As component of various metalloproteins it is involved in critical biochemical processes such as transport of oxygen in tissues, electron transfer reactions during respiration in mitochondria, synthesis and repair of DNA, metabolism of xenobiotics, etc. However, when present in excess within cells and tissues, iron disrupts redox homeostasis and catalyzes the propagation of reactive oxygen species (ROS), leading to oxidative stress. ROS are critical for physiological signaling pathways, but oxidative stress is associated with tissue injury and disease. At the cellular level, oxidative stress may lead to ferroptosis, an iron-dependent form of cell death. In this review, we focus on the intimate relationship between iron metabolism and oxidative stress in health and disease. We discuss aspects of redox- and iron-mediated signaling, toxicity, ferroptotic cell death, homeostatic pathways and pathophysiological implications.

Autotaxin (ATX) is a secreted glycoprotein, widely present in biological fluids including blood. ATX catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to lysophosphatidic acid (LPA), a growth factor-like, signaling phospholipid. LPA exerts pleiotropic effects mediated by its G-protein-coupled receptors that are widely expressed and exhibit overlapping specificities. Although ATX also possesses matricellular properties, the majority of ATX reported functions in adulthood are thought to be mediated through the extracellular production of LPA. ATX-mediated LPA synthesis is likely localized at the cell surface through the possible interaction of ATX with integrins or other molecules, while LPA levels are further controlled by a group of membrane-associated lipid-phosphate phosphatases. ATX expression was shown to be necessary for embryonic development, and ATX deficient embryos exhibit defective vascular homeostasis and aberrant neuronal system development. In adult life, ATX is highly expressed in the adipose tissue and has been implicated in diet-induced obesity and glucose homeostasis with multiple implications in metabolic disorders. Additionally, LPA has been shown to affect multiple cell types, including stromal and immune cells in various ways. Therefore, LPA participates in many processes that are intricately involved in the pathogenesis of different chronic inflammatory diseases such as vascular homeostasis, skeletal and stromal remodeling, lymphocyte trafficking and immune regulation. Accordingly, increased ATX and LPA levels have been detected, locally and/or systemically, in patients with chronic inflammatory diseases, most notably idiopathic pulmonary fibrosis (IPF), chronic liver diseases, and rheumatoid arthritis. Genetic and pharmacological studies in mice have confirmed a pathogenetic role for ATX expression and LPA signaling in chronic inflammatory diseases, and provided the proof of principle for therapeutic interventions, as exemplified by the ongoing clinical trials for IPF.

Iron is an essential cofactor for important biological activities and biochemical reactions, including the transport of oxygen via red blood cells and its reduction to water during respiration. While iron's bioavailability is generally limited, pathological accumulation of the metal within tissues aggravates the generation of reactive oxygen species (ROS) and elicits toxic effects, which are mainly related to oxidative stress. Here, we describe the role of iron in ROS-induced toxicity and discuss molecular mechanisms and physiological aspects of ROS- and iron-mediated signaling. In addition, we review our current understanding of the regulation of iron homeostasis at the cellular and systemic levels, and focus on the pathogenesis and management of iron overload disorders. Crit Rev Clin Lab Sci

Apoptosis has been suggested to have an important role in the pathogenesis of restenosis in addition to cell migration and proliferation. The aim of the present study was to investigate in an experimental in vivo model the occurrence of apoptosis postangioplasty and its relation to bcl-2 and peroxynitrite detection. Eighteen hypercholesterolemic rabbits underwent transluminal angioplasty of the right iliac artery. The rabbits were sacrificed on the 1st, 2nd, 3rd, 7th, 15th, and 28th day postangioplasty (3 animals per time point) and both the angioplasted and non-injured arteries were studied. Apoptosis was assessed by the terminal uridine nick-end labeling method (TUNEL). Bcl-2 and peroxynitrite were detected by immunochemistry using anti-bcl-2 and anti-nitrotyrosine antibodies. In the angioplasted arteries the number of apoptotic cells was

Apoptosis has been suggested to have an important role in the pathogenesis of restenosis in addition to cell migration and proliferation. The aim of the present study was to investigate in an experimental in vivo model the occurrence of apoptosis postangioplasty and its relation to bcl-2 and peroxynitrite detection. Eighteen hypercholesterolemic rabbits underwent transluminal angioplasty of the right iliac artery. The rabbits were sacrificed on the 1st, 2nd, 3rd, 7th, 15th, and 28th day postangioplasty (3 animals per time point) and both the angioplasted and non-injured arteries were studied. Apoptosis was assessed by the terminal uridine nick-end labeling method (TUNEL). Bcl-2 and peroxynitrite were detected by immunochemistry using anti-bcl-2 and anti-nitrotyrosine antibodies. In the angioplasted arteries the number of apoptotic cells was

Ischemia-reperfusion (IR) injury is a multifactorial process triggered when the liver or other organs are transiently subjected to reduced blood supply followed by reperfusion. It has been shown that "reactive oxygen species" (ROS) are generated during ischemia and reperfusion and may represent pivotal mediators of the ensuing pathological complications. In some cases, however, moderate production of ROS may exert protective effects, a phenomenon presumably related to "ischemic preconditioning". This review will focus mainly on: a) describing the sources and the biochemical mechanisms of ROS generation during ischemia and reperfusion, b) discussing current developments in understanding the biochemical pathways by which ROS may induce toxic or protective effects, c) critically evaluating the results of previous attempts to counteract the toxic effects of ROS by using a variety of antioxidant and transition metal-chelating agents, and d) if feasible, proposing potential new pharmaceutical agents aimed at ameliorating ROS-inducing deleterious effects during reperfusion. It is concluded that ROS are generated from different sources, at different periods during IR, and may act by a variety of not well understood biochemical mechanisms which ultimately lead to cell damage and tissue failure. Curr Pharm Des

Uric acid is the final product of purine metabolism in humans. The final two reactions of its production catalyzing the conversion of hypoxanthine to xanthine and the latter to uric acid are catalysed by the enzyme xanthine oxidoreductase, which may attain two inter-convertible forms, namely xanthine dehydrogenase or xanthine oxidase. The latter uses molecular oxygen as electron acceptor and generates superoxide anion and other reactive oxygen products. The role of uric acid in conditions associated with oxidative stress is not entirely clear. Evidence mainly based on epidemiological studies suggests that increased serum levels of uric acid are a risk factor for cardiovascular disease where oxidative stress plays an important pathophysiological role. Also, allopurinol, a xanthine oxidoreductase inhibitor that lowers serum levels of uric acid exerts protective effects in situations associated with oxidative stress (e.g. ischaemia-reperfusion injury, cardiovascular disease). However, there is increasing experimental and clinical evidence showing that uric acid has an important role in vivo as an antioxidant. This review presents the current evidence regarding the antioxidant role of uric acid and suggests that it has an important role as an oxidative stress marker and a potential therapeutic role as an antioxidant. Further well designed clinical studies are needed to clarify the potential use of uric acid (or uric acid precursors) in diseases associated with oxidative stress.

Uric acid is the final product of purine metabolism in humans. The final two reactions of its production catalyzing the conversion of hypoxanthine to xanthine and the latter to uric acid are catalysed by the enzyme xanthine oxidoreductase, which may attain two inter-convertible forms, namely xanthine dehydrogenase or xanthine oxidase. The latter uses molecular oxygen as electron acceptor and generates superoxide anion and other reactive oxygen products. The role of uric acid in conditions associated with oxidative stress is not entirely clear. Evidence mainly based on epidemiological studies suggests that increased serum levels of uric acid are a risk factor for cardiovascular disease where oxidative stress plays an important pathophysiological role. Also, allopurinol, a xanthine oxidoreductase inhibitor that lowers serum levels of uric acid exerts protective effects in situations associated with oxidative stress (e.g. ischaemia-reperfusion injury, cardiovascular disease). However, there is increasing experimental and clinical evidence showing that uric acid has an important role in vivo as an antioxidant. This review presents the current evidence regarding the antioxidant role of uric acid and suggests that it has an important role as an oxidative stress marker and a potential therapeutic role as an antioxidant. Further well designed clinical studies are needed to clarify the potential use of uric acid (or uric acid precursors) in diseases associated with oxidative stress. Curr Pharm Des

1. Apart from ‘no-reflow’ phenomenon/microvascular injury and myocardial injury/stunning, both of which undoubtedly constitute the MRI, one cannot ignore the reperfusion arrhythmias that considerably contribute to myocardial dysfunction observed in this setting [2]. 2. It is prudent to acknowledge that various problems in the experimental study of MRI still exist and sometimes provoke confusion [3,4]. The duration of ischemia, the timing of drug administration, the drug delivery method and the selection of end-points for cardioprotection, are some of them. For example, it is highly possible that intravenous (and not intracoronary) administration of drugs during ischemia may not reach the ischemic area in the absence of collaterals. 3. It is well established that repetitive short periods of ischemia may be cardioprotective due to induction of ischemic preconditioning [5]. On the other hand, repetitive stunning may lead to chronic stunning which is believed to be a form of myocardial hibernation [5,6]. Consequently, the concept of MRI has currently become more complex and subtle manipulations are needed in order to alleviate its detrimental effects and potentate its potential favorable effects.

Ageing research in Greece is well established. Research groups located in universities, research institutes or public hospitals are studying various and complementary aspects of ageing. These research activities include (a) functional analysis of Clusterin/Apolipoprotein J, studies in healthy centenarians and work on protein degradation and the role of proteasome during senescence at the National Hellenic Research Foundation; (b) regulation of cell proliferation and tissue formation, a nationwide study of determinants and markers of successful ageing in Greek centenarians and studies of histone gene expression and acetylation at the National Center for Scientific Research, Demokritos; (c) work on amyloid precursor protein and Presenilin 1 at the University of Athens; (d) oxidative stress-induced DNA damage and the role of oncogenes in senescence at the University of Ioannina; (e) studies in the connective tissue at the University of Patras; (f) proteomic studies at the Biomedical Sciences Research Center Alexander Fleming; (g) work on Caenorhabditis elegans at the Foundation for Research and Technology; (h) the role of ultraviolet radiation in skin ageing at Andreas Sygros Hospital; (i) follow-up studies in healthy elderly at the Athens Home for the Aged; and (j) socio-cultural aspects of ageing at the National School of Public Health. These research activities are well recognized by the international scientific community as it is evident by the group's very good publication records as well as by their direct funding from both European Union and USA. This article summarizes these research activities and discuss future directions and efforts towards the further development of the ageing field in Greece. Exp Gerontol

Metals are necessary for the normal functioning of cells and the survival of organisms. However, exposure to higher than the physiological levels of several metals may lead to tumor development. Although the exact molecular mechanism(s) of metal-induced carcinogenesis is not clear, a vast body of evidence indicates that metal-induced generation of reactive oxygen species (ROS) may play a central role in this process. Two main pathways of ROS-induced effects are discussed in this chapter: (i) increased DNA damage induced either directly or indirectly by impeding DNA repair, and (ii) modulation of nuclear transcriptional factor activities, such as NF-kappaB and AP-1, through mitogen-activated protein kinases signal transduction mechanisms. Crit Rev Oncol Hematol

The addition of linoleic acid (18:2) to a solution containing oxymyoglobin (Mb II O 2 ), metmyoglobin (Mb III ), or metmyoglobin-azide complex (Mb III -N 3 − ) resulted in the formation of a common complex with identical absorption spectral properties. The addition of H 2 O 2 to a Mb III /linoleic acid mixture revealed a spectral profile with λ max at 530 nm and different from that observed in the reaction of Mb III with H 2 O 2 and identical to that of ferrylmyoglobin. This was accompanied by a progressive decrease in the absorption in the visible region, indicating heme degradation during the lipid peroxidation process. The oxidation products of linoleic acid during the Mb III /18:2/H 2 O 2 interaction were assessed by HPLC under anaerobic and aerobic conditions. In both instances, the chromatograms at λ 234nm revealed the formation of a main peak with a retention time of 11.1 min, which cochromatographed with a standard of 9-hydroperoxide of linoleic acid. The latter adduct was not degraded by the oxoferryl complex of myoglobin. The conclusions originating from this research are twofold. On the one hand, the identical spectral properties exhibited by the product originating from the reaction of either Mb II O 2 or Mb III with linoleic acid bridge the apparent discrepancy between the different reactivities of Mb II O 2 and Mb III toward H 2 O 2 and their ability to promote lipid peroxidation. On the other hand, the pattern of oxidation products of linoleic acid observed during the Mb III /H 2 O 2 interaction, i.e., the formation of a 9-hydroperoxide adduct as a major product, points to a specific binding character and a regioselectivity of the oxoferryl complex in the oxidation of unsaturated fatty acids or a catalytic preference for decomposition of the various isomeric hydroperoxides over that of the 9-hydroperoxide.

Alterations in cellular GSH and its compartmentation were investigated as a possible mechanism of toxicity of the anthracycline derivative daunorubicin in neonatal heart cells. Cultured beating heart cells from neonatal rats were exposed to daunorubicin at therapeutically relevant concentrations and the resulting changes in cellular GSH as well as cytosolic and mitochondrial pools of GSH were determined. Toxicity was estimated as an increased permeability of the plasma membrane to cytosolic enzymes, e.g., lactate dehydrogenase. Control heart cells were found to contain 12.2 +/- 1.8 nmoles GSH/10(6) cells. Daunorubicin caused a rapid initial decrease followed by a transient increase in cellular GSH. The extent of the latter increase was dependent on the concentration of daunorubicin. High concentrations of daunorubicin gave only a slight increase followed by a pronounced decrease in cellular GSH. By applying a digitonin-based method the effect of daunorubicin on the cytosolic and mitochondrial pools of GSH were separated. The concentration of cytosolic and mitochondrial reduced GSH was estimated to be 8.9 +/- 1.5 nmoles/10(6) cells and 3.3 +/- 0.6 nmoles/10(6) cells, respectively. The results indicate that daunorubicin caused a decrease of cytosolic GSH and, after a short lag period, a release of alctate dehydrogenase. No decrease of mitochondrial GSH occurred under these conditions indicating that daunorubicin influences selectively cytosolic GSH. No lipid peroxidation products were detected in DRB-treated cells under conditions when lactate dehydrogenase was released. Likewise, addition of the iron-chelator desferrioxamin did not influence the release of lactate dehydrogenase, whereas dithiothreitol offered partial protection.(ABSTRACT TRUNCATED AT 250 WORDS) Free Radic Res Commun

The objective of this study was to elucidate the factors involved in the accumulation of lipofuscin in post-mitotic cells. The hypothesis that oxidative stress accelerates the rate of lipofuscin accumulation was tested by examining the effects of 5%, 20%, and 40% ambient oxygen concentration on lipofuscin content in cultured rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry at 7 and 12 days of in vitro age. Lipofuscin-emitted yellow autofluorescence increased in direct relationship to ambient oxygen concentration with age. Transmission electron microscopic examination of the cells after 3, 8, and 12 days in culture indicated a progressive time and oxygen dependent increase in the frequency and size of lipofuscin organelles. The results are interpreted to suggest that oxidative stress is one of the causal factors in the accumulation of lipofuscin.

Various aspects of the cardiotoxicity of the anthracycline derivative and antineoplastic drug daunorubicin were investigated using isolated and cultured cells from neonatal rat hearts as a model system. Treatment of the cells with concentrations of daunorubicin of the same order of magnitude as those used in chemotherapy was accompanied by marked toxic effects, e.g. a decreased or abolished contraction, and release of lactate dehydrogenase, pyruvate and oxidized glutathione to the medium. A decreased frequency of contraction appeared to be the most sensitive probe of daunorubicin toxicity, followed by release of pyruvate and oxidized glutathione/lactate dehydrogenase. Daunorubicin and/or its metabolites also bound to cellular protein and DNA. Exposure to daunorubicin was shown to be accompanied by a rapid induction of primarily DT-diaphorase and a slower induction of glutathione transferase. The latter observations are interpreted to indicate a protective role of quinone- and peroxide-metabolizing enzymes, respectively, and support the hypothesis that daunorubicin toxicity involves generation of free radical derivatives, which initiate lipid peroxidation. This conclusion is further substantiated by the demonstration that addition of daunorubicin leads to an increased oxygen consumption. Biochem Pharmacol

Adrenodoxin reductase from bovine adrenal cortex mitochondria was purified to homogeneity by affinity chromatography on 2',5'-ADP-Sepharose 4B. The purified adrenodoxin reductase was reconstitutively active in the presence of NADPH and adrenodoxin and with either cytochrome c or cytochrome P-450 as electron acceptor. In sonicated cytochrome P-45-containing milochondrial membranes reconstituted with adrenodoxin reductase and adrenodoxin. the content of cytochrome P-450 and level of reduction of cytochrome P-450 was not altered as compared to intact mitochondria. In contrast, hydroxylation of deoxycorticosteron was inhibited, suggesting that a component of cytochrome P-450 or an hypothetical factor required for hydroxylation of steroids by membrane-bound cytochrome P-450, was impaired.

Binding of 99Tcm-gluconate on the subcellular level in myocardial infarct tissue in the dog was investigated by means of a fractionated centrifugation technique. High isotope activity/g protein was recorded in the mitochondrial fraction indicating the 99Tcm-gluconate binds preferentially to a protein structure localized in the mitochondria.

The present communication describes improved methods for isolating and plating beating heart cells from neonatal rats using collagenase and collagen-coated petri dishes. The amplitude and frequency of contraction are continuously and simultaneously measured under well defined conditions and during prolonged periods of time with a highly sensitive and thermostated instrument. Additions, e.g. drugs and toxic agents, are made through an accessory pump system that involves extensive dilution of the added compound with medium; aliquots of medium can be withdrawn for estimation of metabolites. The system described is reliable and relatively inexpensive and allows a more extensive use of isolated heart cells, especially in studies of heart functions where small changes in amplitude and frequency of beating during prolonged periods of time are important. J Biochem Biophys Methods

J Steroid Biochem

99mTc-gluconate has previously been shown to bind to isolated rat heart mitochondria. In the presence of potassium cyanide this binding is enhanced several fold, an effect which has been shown to be correlated to the efficiency of the in vivo binding of the isotope complex in ischemic dog hearts. The present investigation shows that the potassium cyanide-induced binding was mainly localized to the mitochondrial inner membrane; in the presence of cyanide other organelles, e.g., nuclei and liver microsomes also showed some binding. Boiling, lipid extraction, or addition of N-ethylmaleimide caused a variable inhibition of the binding of 99mTc-gluconate to rat heart mitochondria, which also was markedly influenced by temperature, pH, time, and concentrations of protein and isotope complex, but not by conditions affecting energy levels or calcium transport. Fractionation of submitochondrial particles exposed to 99mTc-gluconate in the presence of potassium cyanide indicated that the 99mTc-gluconate-binding component cofractionated with cytochrome oxidase. It is postulated that a protein component localized in the mitochondrial inner membrane, possibly cytochrome oxidase, is responsible for the binding of 99mTc-gluconate. Eur J Nucl Med

The addition of ascorbate to ischemic rat hearts prevents the myocardial damage associated with reoxygenation. H2O2 oxidizes myoglobin (Mb+2) to higher oxidation states (Mb+4 and Mb+5) which are rapidly reduced by ascorbate. It is proposed that the operation of a myoglobin redox cycle, in which H2O2 causes the two-electron oxidation of myoglobin, is a critical determinant of reperfusion injury. Conversely, the reduction of myoglobin, in one-electron steps, may represent an essential protective mechanism against such injury in the heart. Biochem Biophys Res Commun

The cardiotoxic effect of daunorubicin was investigated using cultured heart cells from neonatal rat hearts. Exposure of the cells to low but toxic concentrations of daunorubicin led to drastically altered permeability properties of the plasma membrane as measured by release of lactate dehydrogenase. Simultaneously, 99Tcm-gluconate, an isotope complex used in scintigraphic imaging of myocardial infarcts, was accumulated in the cells. The results suggest that daunorubicin-induced cardiac damage may be estimated by isotope uptake. Acta Radiol Oncol

The effect of the antineoplastic agent daunorubicin on beating heart cells from neonatal rats was investigated with respect to cell damage and induction of enzymes possibly involved in drug metabolism. Of the enzymes assayed DT-diaphorase and glutathione-S-transferase showed a two-to-four fold increase in activity: higher concentrations of daunorubicin inactivated glutathione-S-transferase. Daunorubicin toxicity increased in the presence of dicoumarol, a specific inhibitor of DT-diaphorase. These results indicate that both DT-diaphorase and glutathione-S-transferase may be involved in the metabolism of daunorubicin. Biochem Biophys Res Commun

The objective of this study was to elucidate the factors involved in the accumulation of lipofuscin in post-mitotic cells. The hypothesis that oxidative stress accelerates the rate of lipofuscin accumulation was tested by examining the effects of 5%, 20%, and 40% ambient oxygen concentration on lipofuscin content in cultured rat cardiac myocytes. Lipofuscin was quantified by microspectrofluorometry at 7 and 12 days of in vitro age. Lipofuscin-emitted yellow autofluorescence increased in direct relationship to ambient oxygen concentration with age. Transmission electron microscopic examination of the cells after 3, 8, and 12 days in culture indicated a progressive time and oxygen dependent increase in the frequency and size of lipofuscin organelles. The results are interpreted to suggest that oxidative stress is one of the causal factors in the accumulation of lipofuscin. Free Radic Biol Med

Binding of 99Tcm-gluconate on the subcellular level in myocardial infarct tissue in the dog was investigated by means of a fractionated centrifugation technique. High isotope activity/g protein was recorded in the mitochondrial fraction indicating the 99Tcm-gluconate binds preferentially to a protein structure localized in the mitochondria. Acta Radiol Oncol

The cardiotoxic effect of daunorubicin was investigated using cultured heart cells from neonatal rat hearts. Exposure of the cells to low but toxic concentrations of daunorubicin led to drastically altered permeability properties of the plasma membrane as measured by release of lactate dehydrogenase. Simultaneously, 99Tcm-gluconate, an isotope complex used in scintigraphic imaging of myocar-dial infacts, was accumulated in the cells. The results suggest that daunorubicin-induced cardiac damage may be estimated by isotope uptake.