Biomedical subjects

U T Brunk

Publications and source records attributed to U T Brunk.

At least 73 records · Page 4Linked to original sources

PubMed1992

Mitochondrial production of pro-oxidants and cellular senescence.

Mitochondria are the major intracellular producers of O2- and H2O2. The level of oxidative stress in cells, as indicated by the in vivo exhalation of alkanes and the concentration of molecular products of oxy-radical reactions, increases during aging in mammals as well as insects. In this paper, we discuss the relationship between mitochondrial generation of O2- and H2O2, and the aging process. The rate of mitochondrial O2- and H2O2 generation increases with age in houseflies and the brain, heart and liver of rat. This rate has been found to correspond to the life expectancy of flies and to the maximum life span potential (MLSP) of six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig and cow. In contrast, the level of antioxidant defenses provided by activities of superoxide dismutase, catalase, glutathione peroxidase and glutathione concentration neither uniformly declines with age nor corresponds to variations in MLSP of different mammalian species. It is argued that the rate of mitochondrial O2- and H2O2 generation rather than the antioxidant level may act as a longevity determinant.

Aging↗

PubMed1992

A novel hypothesis of lipofuscinogenesis and cellular aging based on interactions between oxidative stress and autophagocytosis.

Based on a series of experiments, using cultured postmitotic neonatal rat cardiac myocytes as a model system, we present a novel hypothesis of lipofuscin formation. This hypothesis proposes that lipofuscin is formed within secondary lysosomes due to an interplay of two processes, the production of partially reduced oxygen species by mitochondria and the autophagocytotic degradation within secondary lysosomes. Specifically, it is proposed that H2O2 generated by mitochondria and other organelles permeates into the lumen of secondary lysosomes, which contain iron derived from cellular structures undergoing intralysosomal degradation. The interaction between reactive ferrous iron and H2O2 results, via Fenton-type mechanisms, in the generation of hydroxyl free radicals (OH), inducing lipid peroxidation and eventually leading to intermolecular cross-linking and lipofuscin formation. Additionally, mitochondria undergoing intralysosomal decomposition might continue for a certain period to produce superoxide anion radicals (O2-) and thus also H2O2. This model of lipofuscinogenesis could satisfactorily explain the variations observed in the rates of lipofuscinogenesis among different postmitotic cell types in various species. Such variations might arise from a variety of factors including differences in the efficiency of the 'anti-oxidative shield', rate of H2O2 generation, amount of chain-breaking antioxidants, mode of intralysosomal iron chelation, rate of autophagocytosis as well as degree of efficiency of the intralysosomal hydrolytic enzymes.

Animals↗

PubMed1992

Alloxan cytotoxicity involves lysosomal damage.

The cytotoxic effects of alloxan are not understood in any great detail, although they are considered to involve reactions mediated by oxygen-derived free radicals. These reactive species may form extra-or intracellularly following alloxan reduction, and result in cell damage through a number of complex interactions with a variety of macromolecules. The purpose of the present study was to elucidate further the early intracellular effects of alloxan on a model system of macrophage-like cells in culture. Addition of alloxan (15 mM), without reducing agents, to the medium surrounding the cells (phosphate-buffered saline, PBS, 37 degrees C, pH 7.4) resulted in rapid lysosomal damage (disappearance of the proton gradient over the membrane) followed by severe cellular degeneration (swelling and blebbing) and 50% cell death (trypan blue dye exclusion test) within fifty min. Cells pretreated with the gamma-glutamyl cysteine synthetase-inhibiting agent BSO, to decrease levels of intracellular glutathione, showed enhanced sensitivity to alloxan. The results are interpreted as indicating the cytotoxicity to result from intracellular formation of superoxide radicals, hydrogen peroxide and hydroxyl radicals, the latter within secondary lysosomes containing trace amounts of reactive iron (inducing Fenton reactions). The ensuing lysosomal membrane damage may result in leakage of lysosomal hydrolases and further cellular degeneration.

Alloxan↗

PubMed1992

Extracellular reduction of alloxan results in oxygen radical-mediated attack on plasma and lysosomal membranes.

Alloxan participation in extracellular redox processes results in the formation of the reactive oxygen species (ROS) superoxide anions (O2-), hydroxyl radical (OH.) and hydrogen peroxide (H2O2), causing cell damage through a number of complex interactions probably involving several different cellular structures. These involve the plasma membrane, and we have recently presented evidence for lysosomal interference. The present study elucidates the early (within 15 min) events in a model system of macrophage-like cells (J-774) in culture. Addition of 2 mM alloxan and 1 mM cysteine to the medium surrounding the cells (phosphate-buffered saline, PBS, 37 degrees C, pH 7.4) resulted in rapid lysosomal membrane damage with disappearance of the proton gradient as visualized by acridine orange relocalization, as well as plasma membrane alterations leading to increased leakage of fluorescein after fluorescein diacetate staining. These events were later (greater than 30 min) followed by cellular degeneration in the form of blebbing. Mitochondrial damage (rhodamine 123 relocalization) was a late event. Cells pretreated with desferrioxamine (Des) and superoxide dismutase (SOD) or Des, SOD and catalase (CAT) to induce partial (H2O2 formation only) or almost full protection (no ROS formation) showed about the same reactions as when cells were exposed to alloxan and cysteine without scavengers (O2-, H2O2 and OH. formation) or with PBS only, respectively. The results are interpreted as indicating that the cytotoxicity is a consequence mainly of H2O2 involvement and probably of lysosomal influx of H2O2 with ensuing OH.formation within secondary lysosomes containing trace amounts of reactive iron. It is suggested that the resultant lysosomal membrane damage is followed by leakage of lysosomal hydrolases and ensuing cellular degeneration.

Acridine Orange↗

PubMed1991

Oxidized ascorbic acid and reaction products between ascorbic and amino acids might constitute part of age pigments.

Fluorescence and non-enzymatic browning were observed in reactions between ascorbic acid (AH2) and amino acids (AA) as well as in reactions involving AH2 autoxidation and/or polymerization in the presence of trace amounts of adventitious iron (less than or equal to 10 microM). These reaction products exhibited fluorescent spectra (400-490 nm) akin to those of extracts from lipofuscin-rich tissues. Lengthy incubation of AH2 at 37 degrees C in phosphate buffer (pH 7.0) caused a concentration- and time-dependent increase in absorbance at 412 nm, paralleling increase in 377/440 nm fluorescence, due to formation of autoxidation/polymerization products. The fluorescences of these substances were increased by acidity and quenched at alkaline conditions but restored by neutralization. The reactions between AH2 (0.1-2.0 mM) and a number of AA (1.0-4.0 mM) were also found to result in products with blue fluorescence. Following TBA test, the AH2 autoxidation/polymerization products and AH2/AA reaction products showed only moderate and slight absorbance at 535 nm, respectively, indicating a little or minute formation of aldehydes with MDA-like reactivity. The findings in this study, nevertheless, suggest possible misinterpretations of results in previous studies dealing with AH2-dependent, oxygen free radical induced, 'lipofuscin-related fluorescence'. Thus, similar to nonenzymatic glycosylation (Maillard) reactions, AH2 autoxidation as well as reactions between AH2 and AA may result in 'lipofuscin-like material', as judged from their fluorescence spectral patterns.

Aging↗

PubMed1991

Microfluorometric and fluorometric lipofuscin spectral discrepancies: a concentration-dependent metachromatic effect?

The fluorescent spectral patterns of some lipid peroxidation products and their derivatives have been investigated. A significant concentration-dependent fluorescence shift was found. A variety of suggested age pigment fluorophores, 1,4-dihydropyridines, Schiff base and MDA polymers, all demonstrated a potential for spectral shifts. Along with increased concentration, the fluorescence peaks of these fluorophores shifted from blue (400-490 nm) to golden-yellow or orange-red (500-600 nm). The demonstrated metachromasia is supposed to be an inner-filter effect resulting from molecular polymerization or stacking. Thus, the striking differences between lipofuscin fluorescence spectra obtained by different investigators may be explained as due to large differences in lipofuscin concentration during measurement with different techniques. The pigments are either studied in situ by morphologists and recorded by microscopic fluorometry or by biochemists using spectrofluorometers to measure the extracted and dissolved pigments.

Aging↗

PubMed1991

Effects of alloxan and reducing agents on macrophages in culture.

The diabetogenic effect of the quinonoid compound alloxan is not understood in detail although it supposedly involves reactions mediated by alloxan and oxygen radicals. These reactive species may form extra- or intracellularly and cause cell damage through a variety of complex interactions with several macromolecules. The purpose of this study was to elucidate early (less than or equal to 60 min) effects of alloxan and reducing agents (cysteine and ascorbic acid) on cultured macrophages, as assayed by the trypan blue dye exclusion test and the sensitive fluorescein diacetate and propidium iodide (FDA/PI) double staining technique. During the reactions between alloxan and reducing agents, oxygen was consumed as a sign of superoxide anion radical formation. When alloxan alone was added to two different culture media without serum, oxygen was still consumed, indicating formation of oxygen radicals due to the occurrence of reducing substances in cell culture media. This finding demonstrated the necessity of performing further studies in solutions without reducing capacity, e.g. in phosphate-buffered saline. The experiments showed that exposure of normal and malignant macrophages to alloxan and reducing substances resulted in rapidly occurring plasma membrane damage and ensuing cell death. Separate addition of catalase, desferrioxamine or superoxide dismutase resulted in evident, slight and no protection, respectively. The combinations of (i) catalase and desferrioxamine, and (ii) catalase, desferrioxamine and superoxide dismutase, however, inhibited cell damage in a pronounced and complete way, respectively. The results are interpreted as indicating cell damage due to the extracellular formation of hydrogen peroxide and hydroxyl radicals. The latter in close proximity to the cells and acting on the plasma membrane, while the former, after diffusing into the cell, may have several intracellular targets. The FDA/PI technique proved its value as a quantifiable method for the evaluation not only of cell death but also of cell damage with computer-based fluorometry.

Alloxan↗

PubMed1991

Mechanisms of lipofuscinogenesis: effect of the inhibition of lysosomal proteinases and lipases under varying concentrations of ambient oxygen in cultured rat neonatal myocardial cells.

The objective of this study was to analyse the relative roles of oxidative stress and lysosomal lytic enzymes in lipofuscin formation in cultured neonatal rat cardiac myocytes. Myocytes were exposed to E-64 (an inhibitor of lysosomal cathepsins L. D and H), netilmicin (an inhibitor of lysosomal phospholipases A1 and C) and leupeptin (an inhibitor of cytosolic and lysosomal thiolproteinases) under varied conditions of oxidative stress (20% and 40% ambient oxygen) for up 14 days. Lipofuscin was quantified by microspectrofluorometry. The amount of lipofuscin accumulation was enhanced by the lytic enzyme inhibitors as well as by the increase in the ambient oxygen concentration. However, the effects of enzyme inhibitors was less obvious under 40% ambient oxygen than under 20% oxygen. Data are interpreted as suggesting that, under high levels of oxidative stress, intralysosomal peroxidative changes related to lipofuscin formation occur so rapidly that lytic activity assumes a minor role in lipofuscinogenesis whereas, under low oxidative stress, inhibition of lytic activity makes a greater contribution to lipofuscinogenesis by allowing a longer period of time for peroxidative changes. The results further substantiate the hypotheses that (a) lipofuscinogenesis is strongly related to oxidative stress, and (b) lipofuscin forms intralysosomally as a result of processes involving incomplete degradation of heterophagocytosed and or autophagocytosed material.

Animals↗

PubMed1990

Hydrogen peroxide production by liver mitochondria in different species.

The objective of this study was to further examine the relationship between oxygen free radicals and the aging process in animals. The rate of H2O2, a product of superoxide anion radical dismutation, released by liver mitochondria, was measured in the mouse, rat, guinea pig, rabbit, pig and cow as well as in flight muscle mitochondria of Drosophila melanogaster and the housefly, Musca domestica. The rate of H2O2 generation was inversely related to the maximum life span potential (MLSP) of the species. A 42-fold difference in the rate of H2O2 secretion was found among mammals and an up to 300-fold difference was noted between the insects and the mammals. Results of this study indicate that under identical conditions mitochondria from animals with low MLSP release relatively greater amounts of H2O2.

Animals↗

PubMed1990

Relationship between antioxidant defenses and longevity in different mammalian species.

The general objective of this investigation was to examine the relationship between oxygen free radicals and the aging process. Comparisons of antioxidant defenses were made in six different mammalian species, namely, mouse, rat, guinea pig, rabbit, pig and cow, which range from 3.5 to 30 years in their maximum life span potential (MLSP). Activities of superoxide dismutase (SOD), catalase, and glutathione peroxidase, and concentration of glutathione were measured in the liver, the heart, and the brain. SOD and catalase activities were positively correlated whereas glutathione concentration was negatively correlated with MLSP. Glutathione peroxidase activity exhibited a variable pattern: being positively correlated with MLSP in the brain and negatively correlated in the liver and the heart. In each organ, MLSP was correlated with relatively high levels of one or two of the above antioxidants and low levels of the other antioxidants, indicating the possibility of a compensatory balance among various components of the antioxidant system. No obvious pattern of a relationship was detectable between the overall level of antioxidant defenses and MLSP among the mammalian species examined. The implications of this finding concerning the role of oxidative stress in the aging process and the free radical hypothesis of aging are discussed.

Aging↗

PubMed1990

Photooxidative damage to lysosomes of cultured macrophages by acridine orange.

Cultured cells accumulate acridine orange (AO), which is a weak basic dye and a photosensitizer, in lysosomes and other acidic compartments. During exposure to blue light, AO-loaded macrophages show decreasing red granular fluorescence and increasing green diffuse fluorescence. This is hypothesized to represent peroxidative damage to lysosomal membranes resulting in an impaired proton gradient with deprotonation of the AO to its uncharged form and subsequent leakage of the dye. Further damage to the lysosomal membranes will result in release of lytic enzymes from the lysosomal compartment into the cytosol, leading to degeneration and finally cell death. The survival of AO-loaded and light-exposed macrophages is controllable by varying the exposure times to blue light. Inhibition of lysosomal proteases by E-64 results in increased cell survival after AO and blue light-mediated damage, indicating a role of proteolytic enzymes in this type of damage. Morphological analysis shows 'rounding up' with formation of retraction fibrils and pronounced plasma membrane blebbing. The formation of autophagic vacuoles is an early and pronounced event. After protease inhibition, however, all these phenomena are inhibitable to a considerable degree. We have thus directed photooxidative damage selectively to lysosomal membranes and their contents. This technique will allow further detailed studies of the role of lysosomes in degeneration-regeneration processes.

Acridine Orange↗

PubMed1990

Effect of alpha-tocopherol and some metal chelators on lipofuscin accumulation in cultured neonatal rat cardiac myocytes.

The objective of this study was to test further the hypothesis that oxidative stress is a major causal factor in lipofuscin formation. We have previously shown that cultured cardiac myocytes constitute a suitable model system for the study of factors influencing lipofuscinogenesis. The specific aim of the present study was to elucidate the effects of the chain-breaking free radical scavenger alpha-tocopherol, and the chelators desferrioxamine, EDTA and DTPA on the accumulation of lipofuscin. The effects were examined at different degrees of oxidative stress, obtained by varying the ambient oxygen concentration from 5 to 40%. Lipofuscin was quantified by microspectrofluorometry. Lipofuscin-specific, yellow autofluorescence increased with time in culture, and with enhanced oxidative stress. Increasing concentration of alpha-tocopherol, up to 40 microM, had an inhibitory effect on lipofuscin accumulation that was most pronounced at high oxidative stress. Desferrioxamine and DTPA, both caused a pronounced reduction in lipofuscin formation, while EDTA had no significant effect. The findings are interpreted to support the concept that oxidative stress is a causal factor in lipofuscinogenesis, and that lipofuscin is a product of autophagocytosed, membrane-rich material subjected to free radical-induced, metal-catalyzed peroxidation, fragmentation, and polymerization within the lysosomal vacuome.

Animals↗

PubMed1989

Lipofuscin as an indicator of oxidative stress and aging.

There is a considerable body of evidence indicating that oxidative stress is a causal factor both in lipofuscinogenesis as well as in aging. Studies on the effects of pro-oxidants and antioxidants on lipofuscin accumulation in cultured rat cardiac myocytes and human glial cells indicated that pro-oxidants accelerate while antioxidants retard the rate of lipofuscin accumulation. Lipofuscin was measured by microspectrofluorometry; the reliability of this method was independently validated by comparison with electron microscopical morphometry. In vivo studies on hibernating mammals and on insects indicate that rate of lipofuscin is enhanced while life span is shortened by elevation in metabolite rate. The increase in metabolic rate has been shown to enhance the rate of lipid peroxidation, measured as n-pentane exhalation in vivo. Overall, it seems that there is enough evidence at hand to reasonably infer that lipofuscin can be used as a marker of oxidative stress and aging.

Animals↗

PubMed1989

On the origin of lipofuscin; the iron content of residual bodies, and the relation of these organelles to the lysosomal vacuome. A study on cultured human glial cells.

Cultured human glial cells constitute a suitable model system for the study of lipofuscinogenesis in vitro. These cells, although not post-mitotic, can be kept for several months in stable monolayers due to their display of very pronounced density-dependent inhibition of cell growth. Residual bodies, or lipofuscin pigment granules, accumulate over time in this "pseudo" post-mitotic cell system. I. In early dense cultures, exposed to purified rat liver mitochondriae, it was possible to follow the uptake of mitochondriae and their degradation, which was found to be incomplete and result in the formation of numerous residual bodies containing lipofuscin-type material. It was concluded that incomplete degradation of mitochondriae may be an important origin of lipofuscin. II. Dense, older cultures exposed to electron dense marker particles (colloidal thorium dioxide) accumulated these markers within endosomes, and later in secondary lysosomes of various types, including residual bodies. It was concluded that residual bodies constitute an integral part of the lysosomal vacuome system. III. Phase III glial cells were cultured on formvar-coated gold EM-grids and studied by whole cell transmission electron microscopy using TEM and STEM techniques in combination with energy dispersive X-ray microanalysis. It was found that residual bodies contained iron. This fact was taken as a further indication that lipofuscin has its origin in autophagocytosed mitochondriae and ER-material rich in metallo-enzymes. Due to their high concentration of iron, residual bodies may constitute unstable structures within the cells. Since iron is a well known catalyst of various peroxidative processes, the surrounding lysosomal membrane might be damaged, e.g. by oxidative stress, with risk for leakage of degradative lysosomal enzymes into the cell sap.

Cell Line↗

PubMed1989

Discharge of newly-synthesized dolichol and ubiquinone with lipoproteins to rat liver perfusate and to the bile.

An effective system for perfusing rat liver using complete tissue culture medium and washed calf erythrocytes as oxygen carriers was devised. Infusion of taurocholate and glucose proved necessary to maintain stable metabolic activity and bile secretion during a 6-hr period. Perfusate pO2, pCO2 and pH values were monitored continuously and found to be stable. Electron microscopic examination revealed the maintenance of normal hepatic structure, even after 6 hr. Normal rates of protein and urea synthesis, no leakage of cytoplasmic enzymes, and continuous bile acid production demonstrated the functional integrity of this system. Using [3H]mevalonic acid as precursor, dolichol, dolichyl phosphate, ubiquinone and cholesterol were found to be continuously synthesized in this perfused liver system. All these lipids appeared in the perfusate, indicating discharge through the ER-Golgi system. The lipoproteins of the perfusate were isolated by ultracentrifugation and characterized with respect to size distribution and lipid composition. Dolichol was found in VLDL, LDL and HDL fractions, with the highest concentration present in the latter. In rat and human blood plasma this lipid was mainly associated with HDL. The ubiquinone in the perfusate was primarily associated with the VLDL fraction, while in rat plasma it was found more evenly distributed among all the three lipoprotein fractions studied. Dolichol, ubiquinone and cholesterol were also discharged to the bile, whereas dolichyl phosphate was not. Thus, newly-synthesized dolichol and ubiquinone are transported out of the hepatocyte to the blood and to the bile.

Animals↗

PubMed1989

Acridine orange-mediated photodamage of microsomal- and lysosomal fractions.

Irradiation of microsomes with visible light in the presence of externally-added acridine orange results in O2 uptake, malondialdehyde accumulation, and inactivation of the microsomal drug-metabolizing system. The latter effect is reflected by a decrease in NADPH-cytochrome P450- and NADH-cytochrome b5 reductase activities and cytochromes P450 and b5 content by 88-, 85-, 60-, and 34%, respectively, after 5-min irradiation. Anoxia prevented inactivation of both reductases by 70-90%, whereas it prevented completely cytochrome b5 destruction. The presence of reducing equivalents, at the expense of NADPH and NADH, exert a partial protection (40-54% residual activities) against photosensitization damage on both reductase activities, whereas it almost fully protected cytochrome b5. Photosensitization of lipid peroxidation, as well as inactivation of the microsomal drug-metabolizing system, appears to involve both a type I and type II process. Products of lipid peroxidation might also play a role in enzyme inactivation and cytochrome destruction, as suggested by kinetic and time course studies and the redox state of microsomes. The uptake of acridine orange by isolated lysosomes is linearly dependent on the concentration of added dye and the distribution between extra- and intralysosomal acridine orange is strongly dependent on the amount of lysosomes. Irradiation of acridine orange-loaded lysosomes (light intensity at the sample position approximately 320 mW/cm2) produces an impairment of the membrane which leads to a rapid release of enzyme (N-acetyl-beta-glucosaminidase activity) into the medium, accompanied by a loss of activity in the lysosome-containing pellet and a partial photodamage of the enzyme. Concomitantly, thiobarbituric acid-reactive material accumulation increases in the reaction mixture with increasing irradiation time. When light intensity at the position was reduced to approximately 3.6 mW/cm2, photodamage of lysosomes was of a lesser magnitude, allowing the demonstration of a lag phase, which decreased with irradiation time, probably reflecting the so-called first-stage activation of lysosomes, preceding the release of lysosomal enzymes.

Acridine Orange↗

PubMed1989

Evaluation of lysosomal stability in living cultured macrophages by cytofluorometry. Effect of silver lactate and hypotonic conditions.

The ability of living mouse peritoneal macrophages to retain the lysosomotropic photosensitizer acridine orange (AO) within their secondary lysosomes was studied with a novel cytofluorometric method. During exposure to blue light, cellular AO fluorescence turned from a red granular pattern to that of diffuse green. The resulting change in total fluorescence intensity versus time - a primary decline due to red fluorescence bleaching and a secondary recovery due to the spectral shift - was interpreted as the result of leakage of AO from the lysosomal vacuome. The hypothesis that this time course should be affected by changes in lysosomal membrane stability was tested by labilizing the lysosomes by exposure of cultured macrophages to either hypotonic medium or silver lactate. In hypotonic medium, the ability to retain AO decreased continuously. Exposure to low concentrations of silver lactate (10 microM) also decreased AO retention time. We suggest that this method could be used, within appropriate experimental conditions, to evaluate lysosomal membrane stability in living cells.

Acridine Orange↗

PubMed1989

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction.

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetylglucosaminidase↗