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PubMed1987

Calmodulin and calmodulin binding proteins in amphibian rod outer segments.

The calmodulin (CaM) content of fully intact frog rod outer segments (ROS) has been measured. The molar ratio between rhodopsin and total CaM in ROS is 800:1. This is in good agreement with the data reported for bovine ROS CaM [Kohnken, R. E., Chafouleas, J. G., Eadie, D. M., Means, A. R., & McConnell, D.G. (1981) J. Biol. Chem. 256, 12517-12522]. In the absence of Ca2+, the ROS membrane fraction contains only 4% of total ROS CaM. In contrast, in the presence of Ca2+, 15% of total ROS CaM is found in the membrane fraction. For half-maximal binding of CaM to CaM-depleted ROS membranes, 3 X 10(-7) M Ca2+ is required. This CaM binding is inhibited by trifluoperazine. CaM binding proteins in the ROS membrane fraction are identified by using two different methods: the overlay method and the use of 3,3'-dithiobis(sulfosuccinimidyl propionate) (DTSSP), a bifunctional cross-linking reagent. Ca2+-dependent CaM binding proteins with apparent molecular weights of 240,000, 140,000, 53,000, and 47,000 are detected in the ROS membrane fraction by the overlay method. Anomalous, Ca2+-independent CaM binding to rhodopsin is also detected with this method, and this CaM binding is inhibited by the presence of Ca2+. With the bifunctional cross-linking reagent, DTSSP, three discrete proteins with molecular weights of 240,000, 53,000, and 47,000 are detected in the native ROS membrane fraction. CaM binding to rhodopsin is not detected with this method. Moreover, while the Mr 140,000 band is not detected with DTSSP, a smeared band with a molecular weight between 78,000 and 93,000 is identified (with DTSSP) in the ROS membrane fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed2002

Complex I-mediated reactive oxygen species generation: modulation by cytochrome c and NAD(P)+ oxidation-reduction state.

Several lines of evidence indicate that mitochondrial reactive oxygen species (ROS) generation is the major source of oxidative stress in the cell. It has been shown that ROS production accompanies cytochrome c release in different apoptotic paradigms, but the site(s) of ROS production remain obscure. In the current study, we demonstrate that loss of cytochrome c by mitochondria oxidizing NAD(+)-linked substrates results in a dramatic increase of ROS production and respiratory inhibition. This increased ROS production can be mimicked by rotenone, a complex I inhibitor, as well as other chemical inhibitors of electron flow that act further downstream in the electron transport chain. The effects of cytochrome c depletion from mitoplasts on ROS production and respiration are reversible upon addition of exogenous cytochrome c. Thus in these models of mitochondrial injury, a primary site of ROS generation in both brain and heart mitochondria is proximal to the rotenone inhibitory site, rather than in complex III. ROS production at complex I is critically dependent upon a highly reduced state of the mitochondrial NAD(P)(+) pool and is achieved upon nearly complete inhibition of the respiratory chain. Redox clamp experiments using the acetoacetate/L-beta-hydroxybutyrate couple in the presence of a maximally inhibitory rotenone concentration suggest that the site is approx. 50 mV more electronegative than the NADH/NAD(+) couple. In the absence of inhibitors, this highly reduced state of mitochondria can be induced by reverse electron flow from succinate to NAD(+), accounting for profound ROS production in the presence of succinate. These results lead us to propose a model of thermodynamic control of mitochondrial ROS production which suggests that the ROS-generating site of complex I is the Fe-S centre N-1a.

3-Hydroxybutyric Acid↗

PubMed2001

DeltaPsi(m)-Dependent and -independent production of reactive oxygen species by rat brain mitochondria.

Mitochondria are widely believed to be the source of reactive oxygen species (ROS) in a number of neurodegenerative disease states. However, conditions associated with neuronal injury are accompanied by other alterations in mitochondrial physiology, including profound changes in the mitochondrial membrane potential DeltaPsi(m). In this study we have investigated the effects of DeltaPsi(m) on ROS production by rat brain mitochondria using the fluorescent peroxidase substrates scopoletin and Amplex red. The highest rates of mitochondrial ROS generation were observed while mitochondria were respiring on the complex II substrate succinate. Under this condition, the majority of the ROS signal was derived from reverse electron transport to complex I, because it was inhibited by rotenone. This mode of ROS generation is very sensitive to depolarization of DeltaPsi(m), and even the depolarization associated with ATP generation was sufficient to inhibit ROS production. Mitochondria respiring on the complex I substrates, glutamate and malate, produce very little ROS until complex I is inhibited with rotenone, which is also consistent with complex I being the major site of ROS generation. This mode of oxidant production is insensitive to changes in DeltaPsi(m). With both substrates, ubiquinone-derived ROS can be detected, but they represent a more minor component of the overall oxidant signal. These studies demonstrate that rat brain mitochondria can be effective producers of ROS. However, the optimal conditions for ROS generation require either a hyperpolarized membrane potential or a substantial level of complex I inhibition.

Animals↗

PubMed2003

Quantitative relationship between inhibition of respiratory complexes and formation of reactive oxygen species in isolated nerve terminals.

In this study reactive oxygen species (ROS) generated in the respiratory chain were measured and the quantitative relationship between inhibition of the respiratory chain complexes and ROS formation was investigated in isolated nerve terminals. We addressed to what extent complex I, III and IV,respectively, should be inhibited to cause ROS generation. For inhibition of complex I, III and IV, rotenone, antimycin and cyanide were used, respectively, and ROS formation was followed by measuring the activity of aconitase enzyme. ROS formation was not detected until complex III was inhibited by up to 71 +/- 4%, above that threshold inhibition, decrease in aconitase activity indicated an enhanced ROS generation. Similarly, threshold inhibition of complex IV caused an accelerated ROS production. By contrast, inactivation of complex I to a small extent (16 +/- 2%) resulted in a significant increase in ROS formation, and no clear threshold inhibition could be determined. However, the magnitude of ROS generated at complex I when it is completely inhibited is smaller than that observed when complex III or complex IV was fully inactivated. Our findings may add a novel aspect to the pathology of Parkinson's disease, showing that a moderate level of complex I inhibition characteristic in Parkinson's disease leads to significant ROS formation. The amount of ROS generated by complex I inhibition is sufficient to inhibit in situ the activity of endogenous aconitase.

Aconitate Hydratase↗

PubMed2006

[Physiological and physiopathological consequences of mitochondrial reactive oxygen species].

Literature on reactive oxygen species (ROS) effects on cell biology and physiopathology is huge and appears to be controversial. This could be explained by the fact that very few studies take into account the real subcellular source of ROS production, their chemical nature and the intensity of their production. In spite of the importance of the other sites of ROS production in the cell, we decided to focus on mitochondrial ROS. Besides their key role in bioenergetics and ATP synthesis, mitochondria are one of the main sites of ROS generation within the cell. 80 % of intracellular superoxide anion is provided by the mitochondrial respiratory chain. Mitochondrial ROS production is closely associated with activity of the respiratory chain and is modulated by environmental factors which can induce constraints on respiratory chain components. Nutrient availability as well as oxygen pressure can both modulate mitochondrial ROS production. When moderately produced, ROS specifically regulate intracellular signalling pathways by reversible oxidation of proteins such as transcription factors or proteins kinases. In this way, they can trigger cell adaptation to environmental changes as modifications of energetic metabolism or hypoxia. Indeed, we demonstrated that mitochondrial ROS act as key elements in the control of white adipose tissue development by specific up-regulation of the anti-adipogenic transcription factor CHOP-10/GADD153. However, when they are produced at high level and in a chronic manner, mitochondrial ROS can also have deleterious effects by massive and irreversible oxidation of their principals targets i.e. lipids, DNA and proteins. In these conditions, mitochondrial ROS are involved in aging process and in pathological situations as metabolic disease.

Animals↗

PubMed2005

Reactive oxygen species stimulates receptor activator of NF-kappaB ligand expression in osteoblast.

It has been established that reactive oxygen species (ROS) such as H2O2 or superoxide anion is involved in bone loss-related diseases by stimulating osteoclast differentiation and bone resorption and that receptor activator of NF-kappaB ligand (RANKL) is a critical osteoclastogenic factor expressed on stromal/osteoblastic cells. However, the roles of ROS in RANKL expression and signaling mechanisms through which ROS regulates RANKL genes are not known. Here we report that increased intracellular ROS levels by H2O2 or xanthine/xanthine oxidase-generated superoxide anion stimulated RANKL mRNA and protein expression in human osteoblast-like MG63 cell line and primary mouse bone marrow stromal cells and calvarial osteoblasts. Further analysis revealed that ROS promoted phosphorylation of cAMP response element-binding protein (CREB)/ATF2 and its binding to CRE-domain in the murine RANKL promoter region. Moreover, the results of protein kinase A (PKA) inhibitor KT5720 and CREB1 RNA interference transfection clearly showed that PKA-CREB signaling pathway was necessary for ROS stimulation of RANKL in mouse osteoblasts. In human MG63 cells, however, we found that ROS promoted heat shock factor 2 (HSF2) binding to heat shock element in human RANKL promoter region and that HSF2, but not PKA, was required for ROS up-regulation of RANKL as revealed by KT5720 and HSF2 RNA interference transfection. We also found that ROS stimulated phosphorylation of extracellular signal-regulated kinases (ERKs) and that PD98059, the inhibitor for ERKs suppressed ROS-induced RANKL expression either in mouse osteoblasts or in MG63 cells. These results demonstrate that ROS stimulates RANKL expression via ERKs and PKA-CREB pathway in mouse osteoblasts and via ERKs and HSF2 in human MG63 cells.

Animals↗

PubMed2003

Reactive oxygen species and adhesion formation: clinical implications in adhesion prevention.

Postoperative adhesion formation is a major clinical problem. It has been demonstrated that the pneumoperitoneum used during laparoscopy is a cofactor in adhesion formation. Reactive oxygen species (ROS) are produced in a hyperoxic environment and during the ischaemia/reperfusion process. ROS activity is deleterious for cells, which protect themselves by an antioxidant system known as ROS scavengers. ROS activity can increase by up-regulation of ROS themselves or by down-regulation of ROS scavengers. Recent data also point to a role for ROS in adhesion formation since the administration of ROS scavengers decreases adhesion formation in several animal models. ROS activity increases during both laparotomy and laparoscopy. During laparoscopy, the pneumoperitoneum determines ischaemia at the time of insuflation and reperfusion at the time of deflation. During laparotomy, the environment has a 150 mmHg partial pressure of oxygen (pO(2)), which is much higher than the intracellular pO(2) (5-40 mmHg). This can explain the increase in ROS activity. The aim of this debate is to open a discussion about the importance of ROS activity, besides the known players and mechanisms involved, in adhesion formation and in adhesion prevention.

Female↗

PubMed1999

Formation of reactive oxygen species by the contracting diaphragm is PLA(2) dependent.

Recent work indicates that respiratory muscles generate superoxide radicals during contraction (M. B. Reid, K. E. Haack, K. M. Francik, P. A. Volberg, L. Kabzik, and M. S. West. J. Appl. Physiol. 73: 1797-1804, 1992). The intracellular pathways involved in this process are, however, unknown. The purpose of the present study was to test the hypothesis that contraction-related formation of reactive oxygen species (ROS) by skeletal muscle is linked to activation of the 14-kDa isoform of phospholipase A(2) (PLA(2)). Studies were performed by using an in vitro hemidiaphragm preparation submerged in an organ bath, and formation of ROS in muscles was assessed by using a recently described fluorescent indicator technique. We examined ROS formation in resting and contracting muscle preparations and then determined whether contraction-related ROS generation could be altered by administration of various PLA(2) inhibitors: manoalide and aristolochic acid, both inhibitors of 14-kDa PLA(2); arachidonyltrifluoromethyl ketone (AACOCF(3)), an inhibitor of 85-kDa PLA(2); and haloenol lactone suicide substrate (HELSS), an inhibitor of calcium-independent PLA(2). We found 1) little ROS formation [2.0 +/- 0.8 (SE) ng/mg] in noncontracting control diaphragms, 2) a high level of ROS (20.0 +/- 2.0 ng/mg) in electrically stimulated contracting diaphragms (trains of 20-Hz stimuli for 10 min, train rate 0.25 s(-1)), 3) near-complete suppression of ROS generation in manoalide (3.0 +/- 0.5 ng/mg, P < 0. 001)- and aristolochic acid-treated contracting diaphragms (4.0 +/- 1.0 ng/mg, P < 0.001), and 4) no effect of AACOCF(3) or HELSS on ROS formation in contracting diaphragm. During in vitro studies examining fluorescent measurement of ROS formation in response to a hypoxanthine/xanthine oxidase superoxide-generating solution, manoalide, aristolochic acid, AACOCF(3), and HELSS had no effect on signal intensity. These data indicate that ROS formation by contracting diaphragm muscle can be suppressed by the administration of inhibitors of the 14-kDa isoform of PLA(2) and suggest that this enzyme plays a critical role in modulating ROS formation during muscle contraction.

Animals↗

PubMed2005

Detailed characterization of the lipid composition of detergent-resistant membranes from photoreceptor rod outer segment membranes.

PURPOSE: In recent years, detergent-resistant membranes (DRMs) have been isolated in in vitro models of lipid rafts, from photoreceptor outer segments (ROS), and the localization of a specific complement of photoreceptor proteins has been demonstrated. However, surprisingly little is known about the lipid composition of these important membrane domains. The present study provides the first characterization of phospholipids and fatty acids from ROS-derived DRMs. METHODS: Bovine ROS membranes were incubated with 1% Triton X-100 at 4 degrees C and subjected to density gradient centrifugation to isolate DRMs from the parent membranes. Lipids of ROS and DRMs were separated by two-dimensional, thin-layer chromatography and converted to methyl esters, and fatty acids were analyzed by gas chromatography. Proteins of ROS and DRMs were analyzed by SDS-PAGE and Western blot analysis. RESULTS: The DRMs represented 8% and 3%, respectively, of total ROS lipid and protein. In general, DRMs were enriched in saturated fatty acids when compared with ROS membranes. Relative to ROS, DRMs were enriched in free fatty acids (FFAs) and a specific phosphatidylcholine (PC) fraction that was almost devoid of polyunsaturated fatty acids (PUFAs). DRMs contained less phosphatidylethanolamine (PE) and phosphatidylserine (PS). Ceramide (CM) from ROS contained PUFAs but no saturated fatty acids; the converse was true of CM from DRMs. Docosahexaenoic acid was diminished in DRM PS and was not detected in the FFAs, but was equally abundant in ROS and DRM PE. ROS-derived DRMs were dramatically enriched in caveolin-1, contained significant amounts of transducin-alpha and c-Src, and were relatively devoid of arrestin. CONCLUSIONS: The relatively saturated lipid environment observed in DRMs is likely to promote the localization of signaling proteins modified with saturated fatty acyl chains. Based on the lipid composition of DRMs, the authors conclude that they would not efficiently support phototransduction.

Animals↗

PubMed2026

Angiotensin signaling is essential for stress erythropoiesis but causes retention of dysfunctional mitochondria in RBCs.

We previously reported that excessive angiotensin-II→AT receptor-1 (AT→ATR1) signaling results in sickle cell anemia-associated (SCA-associated) nephropathy. Herein, we showed that hyperangiotensinemia in SCA results from high erythroid cell-generated reactive oxygen species (ROS), which oxidized angiotensinogen (ATGN) and favored its rapid conversion to AT. Increased AT→ATR1 signaling in SCA erythroid cells generated ROS and created a positive feedback loop of ROS→oxidized ATGN→AT→ATR1→ROS, perpetuating the hyperangiotensinemia. ATR1 blocker, losartan, reduced erythrocyte ROS, oxidized ATGN, and AT levels. The ROS→AT→ATR1→ROS loop was driven by sickle erythropoiesis, as it was reproduced when WT mice were transplanted with SCA hematopoiesis. Using SCA and WT mice with germline- and erythroid-specific ATR1 deficiency, we found that stress erythropoiesis, but not steady-state erythropoiesis, was critically dependent on erythroid AT→ATR1 signaling, which acted in harmony with increased erythropoietin signaling. Furthermore, instead of the canonical AT→ATR1→NADPH-oxidase→ROS signaling in steady-state erythropoiesis, AT→ATR1 signaling in stress erythroid cells increased mitochondrial mass and dysfunctional mitochondria, which thereby increased ROS. SCA mice with erythroid-specific ATR1 deficiency had decreased RBC accumulation of dysfunctional mitochondria and decreased ROS, which reduced SCA-associated nephropathy. Overall, we demonstrate that AT→ATR1 signaling was essential for stress erythropoiesis but led to increased dysfunctional mitochondria retention in mature RBCs, which generated ROS and perpetuated hyperangiotensinemia, resulting in end-organ damage.

Animals↗

PubMed2001

Signalling apoptosis: a radical approach.

Reactive oxygen species (ROS) are frequently associated with cytotoxicity, often being described as damaging, harmful or toxic. It is generally assumed that, under pathological circumstances, ROS elicit wide-spread and random acts of oxidation. This passive attack of cellular components by ROS, in conditions where oxidative stress is the initiating stimulus for apoptosis, is assumed to simply trigger cell death as a result of cumulative oxidative damage. However, accumulating evidence now suggests that ROS may act as signalling molecules for the initiation and execution of the apoptotic death programme in many, if not all, current models of apoptotic cell death. Signalling by ROS would not appear to be random, as previously assumed, but targeted at specific metabolic and signal transduction cellular components. There is also evidence that the enzymatic generation of ROS may not simply be an unwanted by-product of the primary reaction catalysed, but that ROS may be used as signalling molecules to regulate cellular processes including apoptosis. This view of ROS as signalling molecules (as opposed to toxic metabolites) has been further bolstered by the findings that cellular antioxidants such as glutathione and thioredoxin not only serve to regulate ROS levels but also act as reversible redox modifiers of enzyme function. This review will attempt to delineate the involvement of ROS in apoptosis in light of these recent discoveries and provide evidence for a crucial role for ROS in the initiation and execution of the death process.

Animals↗

PubMed2005

Redox signaling in colonial hydroids: many pathways for peroxide.

Studies of mitochondrial redox signaling predict that the colonial hydroids Eirene viridula and Podocoryna carnea should respond to manipulations of reactive oxygen species (ROS). Both species encrust surfaces with feeding polyps connected by networks of stolons; P. carnea is more 'sheet-like' with closely spaced polyps and short stolons, while E. viridula is more 'runner-like' with widely spaced polyps and long stolons. Treatment with the chemical antioxidant vitamin C diminishes ROS in mitochondrion-rich epitheliomuscular cells (EMCs) and produces phenotypic effects (sheet-like growth) similar to uncouplers of oxidative phosphorylation. In peripheral stolon tips, treatment with vitamin C triggers a dramatic increase of ROS that is followed by tissue death and stolon regression. The enzymatic anti-oxidant catalase is probably not taken up by the colony but, rather, converts hydrogen peroxide in the medium to water and oxygen. Exogenous catalase does not affect ROS in mitochondrion-rich EMCs, but does increase the amounts of ROS emitted from peripheral stolons, resulting in rapid, runner-like growth. Treatment with exogenous hydrogen peroxide increases ROS levels in stolon tips and results in somewhat faster colony growth. Finally, untreated colonies of E. viridula exhibit higher levels of ROS in stolon tips than untreated colonies of P. carnea. ROS may participate in a number of putative signaling pathways: (1) high levels of ROS may trigger cell and tissue death in peripheral stolon tips; (2) more moderate levels of ROS in stolon tips may trigger outward growth, inhibit branching and, possibly, mediate the redox signaling of mitochondrion-rich EMCs; and (3) ROS may have an extra-colony function, perhaps in suppressing the growth of bacteria.

Analysis of Variance↗

PubMed2006

Adiponectin suppression of high-glucose-induced reactive oxygen species in vascular endothelial cells: evidence for involvement of a cAMP signaling pathway.

Adiponectin is an abundant adipocyte-derived plasma protein with antiatherosclerotic effects. Vascular signal transduction by adiponectin is poorly understood and may involve 5'-AMP-activated protein kinase (AMPK), cAMP signaling, and other pathways. Hyperglycemia sharply increases the production of reactive oxygen species (ROS), which play a key role in endothelial dysfunction in diabetes. Because the recombinant globular domain of human adiponectin (gAd) reduces the generation of endothelial ROS induced by oxidized LDL, we sought to determine whether adiponectin could also suppress ROS production induced by high glucose in cultured human umbilical vein endothelial cells. Incubation in 25 mmol/l glucose for 16 h increased ROS production 3.8-fold (P<0.05), using a luminol assay. Treatment with gAd for 16 h suppressed glucose-induced ROS in a dose-dependent manner up to 81% at 300 nmol/l (P<0.05). The AMPK activator 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR; 1 mmol/l, 16 h) only partially decreased glucose-induced ROS by 22% (P<0.05). Cell pretreatment with AMPK inhibitors, however, failed to block the effect of gAd to suppress glucose-induced ROS, suggesting that the action of gAd was independent of AMPK. Interestingly, activation of cAMP signaling by treatment with forskolin (2 micromol/l) or dibutyryl-cAMP (0.5 mmol/l) reduced glucose-induced ROS generation by 43 and 67%, respectively (both P<0.05). Incubation with the cAMP-dependent protein kinase (PKA) inhibitor H-89 (1 micromol/l) fully abrogated the effect of gAd, but not that of AICAR, on ROS induced by glucose. gAd also increased cellular cAMP content by 70% in an AMPK-independent manner. Full-length adiponectin purified from a eukaryotic expression system also suppressed ROS induced by high glucose or by treatment of endothelial cells with oxidized LDL. Thus, adiponectin suppresses excess ROS production under high-glucose conditions via a cAMP/PKA-dependent pathway, an effect that has implications for vascular protection in diabetes.

AMP-Activated Protein Kinases↗

PubMed1980

Inhibition of DNA and RNA synthesis in lymphocyte cultures by rod outer segments and its counteraction by vitamin E and other antioxidants.

Bovine rod outer segments (ROS) specifically stimulate DNA synthesis in lymphocyte cultures from guinea pigs preimmunized with ROS. At doses above 10 microgram/ml protein, however, preparations of ROS inhibit DNA and RNA synthesis by the sensitized lymphocytes or by normal lymphocytes reacting to mitogens. The inhibtion of RNA synthesis becomes apparent after 24 hr of incubation, whereas little effect was detected after 6 hr. Of the lymphocytes tested, those from mouse and guinea pig spleen were the most susceptible to the effects of ROS, whereas were completely refractory to tested doses of ROS. Bovine retina homogenates were moderately inhibitory, whereas the corresponding cytosol fractions had no detectable effect. Intact ROS were less inhibitory than the disrupted ones, but only minimal amounts of inhibitory activity leaked from disrupted ROS during incubation. Two ROS components, retinoids and docosahexaenoic acid, were also inhibitory to the lymphocytes. The effects of ROS, retinoids, or docosahexaenoic acid were counteracted by antioxidants, with alpha-tocopherol (vitamin E) providing the best protection against ROS. These data thus show lymphocyte cultures to be useful for studying the damaging potential of ROS components and the essential role of antioxidants in protecting the retinal tissues.

Animals↗

PubMed1996

Retinal light damage in rats with altered levels of rod outer segment docosahexaenoate.

PURPOSE: To compare retinal light damage in rats with either normal or reduced levels of rod outer segment (ROS) docosahexaenoic acid. METHODS: Weanling male albino rats were maintained in a weak cyclic light environment and fed either a nonpurified control diet or a purified diet deficient in the linolenic acid precursor of docosahexaenoic acid (DHA). Half the rats on the deficient diet were given linseed oil, containing more than 50 mol% linolenic acid, once a week to maintain ROS DHA at near normal levels. Diets and linseed oil supplementation were continued for 7 to 12 weeks. To replenish DHA in their ROS, some 10-week-old rats on the deficient diet were given linseed oil three times a week for up to 3 additional weeks. Groups of animals were killed at various times for ROS fatty acid determinations or were exposed to intense green light using intermittent or hyperthermic light treatments. The extent of retinal light damage was determined biochemically by rhodopsin or photoreceptor cell DNA measurements 2 weeks after exposure and morphologically by light and electron microscopy at various times after light treatment. RESULTS: Rats maintained for 7 to 12 weeks on the linolenic acid-deficient diet had significantly lower levels of DHA and significantly higher levels of n-6 docosapentaenoic acid (22:5n-6) in their ROS than deficient-diet animals supplemented once a week with linseed oil or those fed the nonpurified control diet. As determined by rhodopsin levels and photoreceptor cell DNA measurements, deficient diet rats exhibited protection against retinal damage from either intermittent or hyperthermic light exposure. However, the unsaturated fatty acid content of ROS from all three dietary groups was the same and greater than 60 mol%. In 10 week-old deficient-diet rats given linseed oil three times a week, ROS DHA was unchanged for the first 10 days, whereas 22:5n-6 levels declined by 50%. After 3 weeks of treatment with linseed oil, ROS DHA and 22:5n-6 were nearly the same as in rats supplemented with linseed oil from weaning. The time course of susceptibility to retinal light damage, however, was different. Hyperthermic light damage in rats given linseed oil for only 2 days was the same as for rats always fed the deficient diet. Six days after the start of linseed oil treatment, retinal light damage was the same as in rats given the linseed oil supplement from weaning. Morphologic alterations in ROS of linseed oil-supplemented rats immediately after intermittent light exposure were more extensive than in either the deficient-diet animals or those fed the control diet. The deficient-diet rats also exhibited better preservation of photoreceptor cell nuclei and structure 2 weeks after exposure. CONCLUSIONS: Rats fed a diet deficient in the linolenic acid precursor of DHA are protected against experimental retinal light damage. The relationship between retinal light damage and ROS lipids does not depend on the total unsaturated fatty acid content of ROS; the damage appears to be related to the relative levels of DHA and 22:5n-6.

Animals↗

PubMed2004

Analysis of mitochondrial generation and release of reactive oxygen species.

BACKGROUND: Reactive oxygen species (ROS) are mainly produced in mitochondria and are important contributors to many forms of cell death. ROS also function as second messengers within the cell and may constitute a signaling pathway from mitochondria to the cytoplasm and nucleus. The aim of the present study was to develop a protocol to detect changes in intra- and extramitochondrial releases of ROS, which could be used to analyze the role of mitochondria in cell signaling and cell death. METHODS: Fluorescence-based assays were used to measure (a) total production of ROS, (b) intramitochondrial ROS, (c) extramitochondrial hydrogen peroxide, and (d) superoxide outside inverted (inside-out) submitochondrial particles. ROS generation in the samples was increased or decreased by the addition of different substrates, enzymes, and inhibitors of the electron transport chain. RESULTS: The individual assays used were sensitive to increased (e.g., after addition of antimycin A; increased signal) and decreased (ROS scavenging; decreased signal) levels of ROS. In combination, the assays provided information about mitochondrial ROS generation and release dynamics from small samples of isolated mitochondria. CONCLUSIONS: The combination of fluorescent techniques described is a useful tool to study the role of ROS in cell death and in cellular redox signaling.

Animals↗

PubMedDate not supplied

Utility of the nitroblue tetrazolium reduction test for assessment of reactive oxygen species production by seminal leukocytes and spermatozoa.

The purpose of this study was to evaluate the ability of spermatozoa and leukocytes in semen to produce reactive oxygen species (ROS) by using nitroblue tetrazolium (NBT) staining and to examine the association between NBT staining and levels of ROS as measured by chemiluminescence. Twenty-one infertility patients (leukocytospermia; n = 8; nonleukocytospermia, n = 13) and 9 healthy donors were included. Standard semen analysis and density gradient centrifugation were performed to test NBT staining, ROS, and total antioxidant capacity. A ROS-total antioxidant capacity (ROS-TAC) score was calculated by using principal component analysis. In the leukocytospermic group, after separation on a density gradient, the percentage of NBT-positive staining was significantly higher in sperm suspensions contaminated with leukocytes (median [25th, 75th percentiles]; 70% [61%, 79%]) compared to the nonleukocytospermic group (14.5% [9%, 25.5%]; P =.03) and donors (7% [3%, 11%]; P =.02), respectively. A strong positive correlation was seen between levels of ROS in whole ejaculates and NBT-positive staining in leukocytes (r = 0.59; P <.0006) and in leukocyte fractions (r = 0.72; P <.0001) after density gradient separation. Similarly, ROS was positively correlated with excessive cytoplasmic retention in spermatozoa from whole ejaculates and abnormal spermatozoa after separation on density gradients (r = 0.72; P <.0001). The ROS-TAC score was inversely correlated with NBT staining in leukocytes in whole ejaculates (r = -0.960, P <.0007) and in both leukocyte fractions (r = -0.39; P <.04) and spermatozoa with cytoplasmic retention (r = -0.38; P <.04). Our results indicate that the NBT reduction test can be used to assess the contribution of seminal leukocytes and defective spermatozoa towards ROS generation in semen. Levels of ROS assessed by chemiluminescence assay are strongly correlated with the results of NBT staining.

Antioxidants↗

PubMed2003

Mitochondrial and microsomal derived reactive oxygen species mediate apoptosis induced by transforming growth factor-beta1 in immortalized rat hepatocytes.

Transforming growth factor-beta1 (TGFbeta1) is a multifunctional cytokine that is over expressed during liver hepatocytes injury and regeneration. SV40-transformed CWSV-1 rat hepatocytes that are p53-defective undergo apoptosis in response to choline deficiency (CD) or TGFbeta1, which mediates CD-apoptosis. Reactive oxygen species (ROS) are essential mediators of apoptosis. We have shown that apoptosis induced by TGFbeta1 is accompanied by ROS generation and the ROS-trapping agent N-acetylcysteine (NAC) inhibits TGFbeta1-induced apoptosis. While persistent induction of ROS contributes to this form of apoptosis, the source of ROS generated downstream of TGFbeta1 is not clear. The mitochondria and the endoplasmic reticulum both harbor potent electron transfer chains that might be the source of ROS essential for completion of TGFbeta1-apoptosis. Here we show that CWSV-1 cells treated with cyclosporine A, which prevents opening of mitochondrial membrane pores required for ROS generation, inhibits TGFbeta1-induced apoptosis. A similar effect was obtained by treating these cells with rotenone, an inhibitor of complex 1 of the mitochondrial electron transfer chain. However, we demonstrate that TGFbeta1 induces cytochrome P450 1A1 and that metyrapone, a potent inhibitor of cytochrome P450 1A1, inhibits TGFbeta1-induced apoptosis. Therefore, our studies indicate that concurrent with promoting generation of ROS from mitochondria, TGFbeta1 also promotes generation of ROS from the cytochrome P450 electron transfer chain. Since inhibition of either of these two sources of ROS interferes with apoptosis, it is reasonable to conclude that the combined involvement of both pathways is essential for completion of TGFbeta1-induced apoptosis.

Animals↗