Measured Reactive Oxygen Species Production Research Articles

Mechanisms of Siglec-F-induced eosinophil apoptosis: a role for caspases but not for SHP-1, Src kinases, NADPH oxidase or reactive oxygen.

BackgroundSiglec-F and Siglec-8 are functional paralog proapoptotic cell surface receptors expressed on mouse and human eosinophils, respectively. Whereas Siglec-8 mediated death involves caspases and/or reactive oxygen species (ROS) generation and mitochondrial injury, very little is known about Siglec-F-mediated signaling and apoptosis. Therefore the objective of the current experiments was to better define apoptosis pathways mediated by Siglec-F and Siglec-8. Given that Siglec-F-induced apoptosis is much less robust than Siglec-8-induced apoptosis, we hypothesized that mechanisms involved in cell death via these receptors would differ.MethodsConsequences of engagement of Siglec-F on mouse eosinophils were studied by measuring ROS production, and by performing apoptosis assays using eosinophils from normal, hypereosinophilic, NADPH oxidase-deficient, src homology domain-containing protein tyrosine phosphatase (SHP)-1-deficient, and Lyn kinase-deficient mice. Inhibitors of caspase and Src family kinase activity were also used.ResultsEngagement of Siglec-F induced mouse eosinophil apoptosis that was modest in magnitude and dependent on caspase activity. There was no detectable ROS generation, or any role for ROS, NADPH oxidase, SHP-1, or Src family kinases in this apoptotic process.ConclusionsThese data suggest that Siglec-F-mediated apoptosis is different in both magnitude and mechanisms when compared to published data on Siglec-8-mediated human eosinophil apoptosis. One likely implication of this work is that models targeting Siglec-F in vivo in mice may not provide identical mechanistic predictions for consequences of Siglec-8 targeting in vivo in humans.

Extraocular muscles produce more non‐mitochondrial reactive oxygen species

Extraocular muscles (EOM) are constantly active, and have a high mitochondrial content. We previously showed that EOM mitochondria are different (Invest Ophthalmol Vis Sci 50:180–6, 2009). Here, we investigated this hypothesis: EOM mitochondria produce more reactive oxygen species (ROS) than mitochondria from other muscles. First, we measured ROS production in diaphragm muscle bundles and intact EOM, excised from adult male Sprague Dawley rats, and loaded with 2’,7’‐dichlorofluorescein‐diacetate (DCFH‐DA). Next, ROS generation was measured for 60 min with Amplex Red in respiring mitochondria from rat diaphragm, gastrocnemius, and EOM. Intact EOM oxidized more DCFH: 1532 ± 407 vs. 517 ± 77 arbitrary units (AU, mean ± SD), EOM and diaphragm, respectively, n=5 muscles, p=0.04. Amplex Red oxidation was not different in mitochondria from the 3 muscles during state 3 respiration (driven by glutamate/malate), inhibition of complex I with rotenone, or succinate‐driven respiration. EOM mitochondria oxidized less Amplex Red during succinate‐driven respiration combined with complex III inhibition using antimycin: 15570±2135, 13305±1020, 9502±1024 AU, diaphragm, gastrocnemius, and EOM respectively; p=0.0009, n=4 rats). These results show that intact EOM generated ROS faster than diaphragm bundles. However, EOM mitochondria did not produce more ROS than mitochondria from other muscles. Therefore, we reject our initial hypothesis, and propose that the higher rate of ROS production in EOM originates from non‐mitochondrial sources.*Research supported by APS Undergraduate Research Fellowship 2012

Development of an in vitro skin sensitization test based on ROS production in THP-1 cells

Recently, it has been reported that reactive oxygen species (ROS) produced by contact allergens can affect dendritic cell migration and contact hypersensitivity. The aim of the present study was to develop a new in vitro assay that could predict the skin sensitizing potential of chemicals by measuring ROS production in THP-1 (human monocytic leukemia cell line) cells. THP-1 cells were pre-loaded with a ROS sensitive fluorescent dye, 5-(and 6-)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM–H2DCFDA), for 15min, then incubated with test chemicals for 30min. The fluorescence intensity was measured by flow cytometry. For the skin sensitizers, 25 out of 30 induced over a 2-fold ROS production at more than 90% of cell viability. In contrast, increases were only seen in 4 out of 20 non-sensitizers. The overall accuracy for the local lymph node assay (LLNA) was 82% for 50 chemicals tested. A correlation was found between the estimated concentration showing 2-fold ROS production in the ROS assay and the EC3 values (estimated concentration required to induce positive response) of the LLNA. These results indicated that the THP-1 cell-based ROS assay was a rapid and highly sensitive detection system able to predict skin sensitizing potentials and potency of chemicals.

Multistationary and Oscillatory Modes of Free Radicals Generation by the Mitochondrial Respiratory Chain Revealed by a Bifurcation Analysis

The mitochondrial electron transport chain transforms energy satisfying cellular demand and generates reactive oxygen species (ROS) that act as metabolic signals or destructive factors. Therefore, knowledge of the possible modes and bifurcations of electron transport that affect ROS signaling provides insight into the interrelationship of mitochondrial respiration with cellular metabolism. Here, a bifurcation analysis of a sequence of the electron transport chain models of increasing complexity was used to analyze the contribution of individual components to the modes of respiratory chain behavior. Our algorithm constructed models as large systems of ordinary differential equations describing the time evolution of the distribution of redox states of the respiratory complexes. The most complete model of the respiratory chain and linked metabolic reactions predicted that condensed mitochondria produce more ROS at low succinate concentration and less ROS at high succinate levels than swelled mitochondria. This prediction was validated by measuring ROS production under various swelling conditions. A numerical bifurcation analysis revealed qualitatively different types of multistationary behavior and sustained oscillations in the parameter space near a region that was previously found to describe the behavior of isolated mitochondria. The oscillations in transmembrane potential and ROS generation, observed in living cells were reproduced in the model that includes interaction of respiratory complexes with the reactions of TCA cycle. Whereas multistationarity is an internal characteristic of the respiratory chain, the functional link of respiration with central metabolism creates oscillations, which can be understood as a means of auto-regulation of cell metabolism.

A High Content Drug Screen Identifies Ursolic Acid as an Inhibitor of Amyloid β Protein Interactions with Its Receptor CD36

A pathological hallmark of Alzheimer disease (AD) is deposition of amyloid β (Aβ) in the brain. Aβ binds to microglia via a receptor complex that includes CD36 leading to production of proinflammatory cytokines and neurotoxic reactive oxygen species and subsequent neurodegeneration. Interruption of Aβ binding to CD36 is a potential therapeutic strategy for AD. To identify pharmacologic inhibitors of Aβ binding to CD36, we developed a 384-well plate assay for binding of fluorescently labeled Aβ to Chinese hamster ovary cells stably expressing human CD36 (CHO-CD36) and screened an Food and Drug Administration-approved compound library. The assay was optimized based on the cells' tolerance to dimethyl sulfoxide, Aβ concentration, time required for Aβ binding, reproducibility, and signal-to-background ratio. Using this assay, we identified four compounds as potential inhibitors of Aβ binding to CD36. These compounds were ursolic acid, ellipticine, zoxazolamine, and homomoschatoline. Of these compounds, only ursolic acid, a naturally occurring pentacyclic triterpenoid, successfully inhibited binding of Aβ to CHO-CD36 cells in a dose-dependent manner. The ursolic acid effect reached a plateau at ~20 μm, with a maximal inhibition of 64%. Ursolic acid also blocked binding of Aβ to microglial cells and subsequent ROS production. Our data indicate that cell-based high-content screening of small molecule libraries for their ability to block binding of Aβ to its receptors is a useful tool to identify novel inhibitors of receptors involved in AD pathogenesis. Our data also suggest that ursolic acid is a potential therapeutic agent for AD via its ability to block Aβ-CD36 interactions.

In Vivo and In Vitro Effects of Metals in Reactive Oxygen Species Production, Protein Carbonylation, and DNA Damage in Land Snails Eobania vermiculata

Heavy metals are known to induce oxidative damage by way of enhancement of intracellular reactive oxygen species (ROS) production, which often preludes the onset of alterations, such as protein carbonylation and DNA damage. In this study, our aim was to examine the early responses of land snails Eobania vermiculata to environmental contaminants by investigating the use of a modified enzyme-linked immunosorbent assay (ELISA) assay for the measurement of protein carbonylation as a new biomarker of terrestrial pollution as well as by measuring ROS production and DNA damage. Land snails were treated with heavy metals-cadmium, lead, or copper-in vivo (15 or 40ppm) for 25days or in vitro (0.5, 5, 50 or 500μM) for 30min in the laboratory, and the previously mentioned biomarkers were determined in digestive gland and haemolymph of the treated animals. A statistically significant increase in ROS production, protein carbonylation, and DNA damage was shown in the snails treated with pollutants compared with the untreated snails. The results indicate the effectiveness of measuring ROS production and DNA damage, as well as using the present ELISA method, as sensitive tools of terrestrial pollution biomonitoring studies. Statistically significant correlations among the previously mentioned techniques further enhance their role as promising biomarkers in terrestrial pollution studies.

Role of reactive oxygen species in gender-associated autoimmunity (83.15)

Abstract Gender-bias exists in proteolipid protein (PLP) 139-151-induced experimental autoimmune encephalomyelitis (EAE) in that both male and female SJL mice develop disease but only females show chronic relapses and their underlying mechanisms are unknown. It has been shown that the reactive oxygen species (ROS) play a role in the mediation of autoimmune diseases, but the recent evidence suggests that ROS can suppress autoimmune responses. We hypothesize that T cell-derived ROS are critical to the maintenance of self-tolerance and favor developing resistance to autoimmunity in male mice. To address this hypothesis, we used PLP 139-151-specific T cell receptor (5B6) transgenic (tg) mice and measured ROS production in activated 5B6 T cells by flow cytometry using CM-H2DCFDA as an ROS indicator. Unexpectedly, we found that T cells from female mice produced two fold more ROS when compared to males. We verified these differences by evaluating mRNA expression of oxidative and anti-oxidative defense genes in 5B6 tg T cells and PLP 139-151-specific T cells sorted from immunized wild type (wt) SJL mice by using PLP 139-151 tetramers. TaqMan PCR analysis revealed that interleukin-22 mRNA, a cytokine implicated in ROS metabolism and EAE pathogenesis was increased by three fold in T cells obtained from tg or wt female mice. The data suggests that sex steroids and T cell-derived cytokines have a role in the maintenance of redox homeostasis in autoreactive T cells and T cell tolerance.

Antioxidant, Free Radical–Scavenging, and NF-κB–Inhibitory Activities of Phytosteryl Ferulates: Structure–Activity Studies

Some of the pharmacological properties of phytosteryl ferulates may be linked to their antioxidant potential. In this study, 2,2-diphenyl-1-picrylhydrazyl (DPPH), electron spin resonance (ESR), and thiobarbituric acid–reactive substances (TBARS) assays demonstrated that phytosteryl ferulates such as cycloartenyl ferulate (CAF), 24-methylenecycloartanyl ferulate (24-mCAF), and β-sitosteryl ferulate (β-SF) and ferulic acid (FA) each exerted strong free radical scavenging and antioxidation of lipid membrane, which were comparable to α-tocopherol. However, the sterol moiety alone, such as cycloartenol (CA), had neither activity. Since, the reactive oxygen species (ROS) production in the cell complex defense mechanism cannot be ruled out with the cell free system, we measured ROS production in NIH 3T3 fibroblast cells induced by H2O2. CAF and ethyl ferulate (eFA) greatly decreased the ROS level in this system. CA also significantly inhibited the ROS level, suggesting that CA could inhibit ROS production in living cells. Besides these, CAF, 24-mCAF, β-SF, as well as eFA and CA, all these chemicals significantly inhibited the NF-κB activity as analyzed by measuring translocation of NF-κB p65 in LPS-stimulated RAW 264.7 macrophages. These observations revealed that phytosteryl ferulates are responsible for the antioxidant and anti-inflammatory activity via ROS scavenging and inhibition of ROS production.

HEAT AND CADMIUM: A DAMAGING COCKTAIL

Permanently glued to the rocks of their estuary home, eastern oysters(Crassostrea virginica) can cope with most of the extremes of their environment: daily and seasonal variations in temperature, from 5°C to 35°C, plus the coming and going of the tides. Add environmental stressors like pollution into the mix, however, and the oysters suffer from oxidative stress, where their cells' mitochondria produce more molecule-damaging reactive oxygen species (ROS) than the cells can clean up. It is important to understand how temperature and pollution interact with each other to affect the survival of marine invertebrates, such as oysters, given the predicted rise in global temperatures, explains Inna Sokolova of the University of North Carolina, Charlotte. Sokolova and her colleagues show that as the temperature rises, oysters suffer more from oxidative stress when they are exposed to cadmium, a toxic heavy metal (p. 46).Cadmium is similar in structure to zinc and calcium and it messes up many cellular processes, such as calcium dependent cell signalling, by blocking enzyme active sites. The metal also increases ROS production, and therefore oxidative stress, in mitochondria. Directly measuring ROS production in a cell is tricky, so to measure the level of oxidative stress the team focussed on a Krebs cycle enzyme called aconitase. This enzyme has an iron atom in the middle, which is a bit loose. `If the iron becomes oxidised by ROS, it falls off, and the enzyme doesn't work properly', says Sokolova. So if a cell is producing larger quantities of ROS, the activity of the aconitase enzyme goes down.To investigate the effects of cadmium and temperature on the aconitase enzyme, the team carefully isolated mitochondria from oyster gill tissue and transferred them to culture for experiments. They exposed the mitochondria to cadmium and higher temperatures and measured aconitase enzyme activity. At 20°C, cadmium slightly reduced aconitase activity, but mitochondria could mop up most of the excess ROS before they caused too much damage. However,when they exposed mitochondria to cadmium and raised the temperature to 30°C, the team found that aconitase activity decreased by up to 52%. This was probably because cadmium induces the mitochondria to produce more ROS at higher temperatures, which inhibits aconitase.Having shown that cadmium increases oxidative stress at higher temperatures, the team wondered if a group of mitochondrial membrane proteins called uncoupling proteins, which are known to diminish the effects of ROS,would reduce oxidative stress and the effects of ROS on aconitase. They suggested that stimulating uncoupling protein activity with fatty acids would reduce oxidative stress caused by cadmium and high temperature, while inhibiting uncoupling protein activity with ATP would increase it. They found no evidence that uncoupling proteins help protect aconitase from ROS, and Sokolova explains that the team also found a result that they weren't expecting: ATP directly inhibits the aconitase enzyme. This suggests ATP might play an important role in regulating the activity of the Krebs cycle, and scientists will need to look at the phenomenon more closely to see if it is unique to oysters or not.While the team's unexpected result will give researchers plenty of food for thought, their study also suggests that rising global temperatures could have a serious effect on oyster survival: `moderately polluted areas could become graveyards', says Sokolova. In the future, researchers might have to adjust their criteria for water quality, to take into account the effects of rising temperatures.

Capillary electrophoresis monitors enhancement in subcellular reactive oxygen species production upon treatment with doxorubicin.

This study investigated the role of doxorubicin (DOX) accumulation in reactive oxygen species (ROS) production detected in individually electrophoresed organelles, including mitochondria, acidic organelles, and peroxisomes. While bulk measurements of ROS production in cells and organelles are not capable of discriminating between the effects of preparative procedures on measured ROS production, capillary electrophoresis with dual laser-induced detection of individual organelles demonstrated a difference in the measured ROS production as a result of various preparative procedures. Using this technique, the three different types of detected organelles (i) produce ROS and do not have detectable levels of DOX, (ii) contain detectable DOX but do not produce ROS, or (iii) produce ROS and accumulate DOX. The third type displays two subpopulations of organelles, one of which demonstrated a direct relationship between DOX uptake and subsequent ROS production, corresponding most likely to mitochondria, and a second one with low DOX uptake but large variation in ROS production, corresponding most likely to acidic organelles.

Measurement of myocardial free radical production during exercise using EPR spectroscopy

Exercise is associated with an increase in oxygen flux through the mitochondrial electron transport chain that has recently been demonstrated to increase the production of reactive oxygen species (ROS) in skeletal muscle. This study examined whether exercise also causes free radical production in the heart. We measured ROS production in seven chronically instrumented dogs during rest and treadmill exercise (6.4 km/h at 10 degrees grade; and heart rate, 204 +/- 3 beats/min) using electron paramagnetic resonance spectroscopy in conjunction with the spin trap alpha-phenyl-tert-butylnitrone (PBN) (0.14 mol/l) in blood collected from the aorta and coronary sinus (CS). To improve signal detection, the free radical adducts were deoxygenated over a nitrogen stream for 15 min and extracted with toluene. The hyperfine splitting constants of the radicals were alpha(N) = 13.7 G and alpha(H) = 1.0 G, consistent with an alkoxyl or carbon-centered radical. Resting aortic and CS PBN adduct concentrations were 6.7 and 6.3 x 10(8) arbitrary units (P = not significant). Both aortic and CS adduct concentrations increased during exercise, but there was no significant difference between the aortic and CS concentrations. Thus, in contrast to skeletal muscle, submaximal treadmill exercise did not result in detectable free radical production by the heart.

Localizing extracellular signal–regulated kinase (ERK) in pharmacological preconditioning's trigger pathway

Acetylcholine (ACh) and opioid receptor agonists trigger the preconditioned phenotype through sequential activation of the epidermal growth factor (EGF) receptor, phosphatidylinositol 3-kinase (PI3-K), Akt, and nitric oxide synthase (NOS), and opening of mitochondrial (mito) K(ATP) channels with the generation of reactive oxygen species (ROS). Although extracellular signal-regulated kinase (ERK) has recently been reported to be part of this pathway, its location has not been determined. To address this issue, we administered a 5-min pulse of ACh (550 microM) prior to 30 min of ischemia in isolated rabbit hearts. It reduced infarction from 30.4 +/- 2.2% of the risk zone in control hearts to 12.3 +/- 2.8% and co-administration of the MEK, and, therefore, downstream ERK inhibitor U0126 abolished protection (29.1 +/- 4.6% infarction) con.rming ERK's involvement. MitoK(ATP) opening was monitored in adult rabbit cardiomyocytes by measuring ROS production with MitoTracker Red. ROS production was increased by each of three G protein-coupled agonists: ACh (250 microM), bradykinin (BK) (500 nM), and the delta-opioid agonist DADLE (20 nM). Co-incubation with the MEK inhibitors U0126 (500 nM) or PD 98059 (10 microM) blocked the increased ROS production seen with all three agonists. Direct activation of its receptor by EGF increased ROS production and PD 98059 blocked that increase, thus placing ERK downstream of the EGF receptor. Desferoxamine (DFO) which opens mitoK(ATP) through direct activation of NOS also increased ROS. PD 98059 could not block DFO-induced ROS production, placing ERK upstream of NOS. In isolated hearts, ACh caused phosphorylation of both Akt and ERK. U0126 blocked phosphorylation of ERK but not of Akt. The PI3-K inhibitor wortmannin blocked both. Together these data indicate that ERK is located between Akt and NOS.

NAD(P)H oxidase plays a crucial role in PDGF-induced proliferation of hepatic stellate cells

The proliferation of hepatic stellate cells (HSCs) is a critical step in hepatic fibrogenesis. Platelet-derived growth factor (PDGF) is the most potent mitogen for HSCs. We investigated the role of nonphagocytic NAD(P)H oxidase-derived reactive oxygen species (ROS) in PDGF-induced HSC proliferation. The human HSC line, LI-90 cells, murine primary-cultured HSCs, and PDGF-BB were used in this study. We examined the mechanism of PDGF-BB-induced HSC proliferation in relation to the role of a ROS scavenger and diphenylene iodonium, an inhibitor of NAD(P)H oxidase. We also measured ROS production with the aid of chemiluminescence. We showed that PDGF-BB induced proliferation of HSCs through the intracellular production of ROS. We also demonstrated that HSCs expressed key components of nonphagocytic NAD(P)H oxidase (p22phox, gp91phox, p47phox, and p67phox) at both the messenger RNA and protein levels. Diphenylene iodonium suppressed PDGF-BB-induced ROS production and HSC proliferation. Coincubation of H2O2 and PDGF-BB restored the proliferation of HSCs that was inhibited by diphenylene iodonium pretreatment. Phosphorylation of the mitogen-activated protein kinase (MAPK) family constitutes a signal transduction pathway of cell proliferation. Our data demonstrate that NAD(P)H oxidase-derived ROS induce HSC proliferation mainly through the phosphorylation of p38 MAPK. Moreover, an in vivo hepatic fibrosis model also supported the critical role of NAD(P)H oxidase in the activation and proliferation of HSCs. In conclusion, NAD(P)H oxidase is expressed in HSCs and produces ROS via activation of NAD(P)H oxidase in response to PDGF-BB. ROS further induce HSC proliferation through the phosphorylation of p38 MAPK.

Lysophosphatidylcholine enhances superoxide anions production via endothelial NADH/NADPH oxidase.

Reactive oxygen species (ROS) including superoxide anions (O2(-)) play a key role in atherogenesis, and endothelial cells have the ability to generate ROS. To investigate the enzymatic sources of ROS and the effects of lysophosphatidylcholine (LPC), an atherogenic lipid, we measured ROS production in cultured bovine aortic endothelial cells (BAECs) by the lucigenin-enhanced chemiluminescence (CL) method and electron spin resonance (ESR). BAEC homogenates had the enzymatic activity of NADH/NADPH oxidase. BAECs cultured on microcarrier beads generated O2(-) under basal conditions. The inhibition of NADH/ NADPH oxidase by diphenylene iodonium (DPI) significantly attenuated O2(-) production, whereas no inhibitors of other oxidases suppressed it. Although LPC enhanced O2(-) production approximately 3.1-fold, its action was suppressed by DPI. Tyrosine kinase inhibitors significantly attenuated LPC-induced O2(-) production. ESR with DMPO demonstrated that LPC increased the formation of the DMPO-hydroxyl adduct in dose- and time-dependent manners. These data suggest that the basal production of O2(-) in endothelial cells is mainly mediated by the NADH/NADPH oxidase system and that LPC activates this oxidase to enhance O2(-) production through a tyrosine kinase-dependent pathway. The enhancement of ROS production by LPC is probably involved in its atherogenic property.

An anionic impurity in preparations of cytochrome e interferes with assays of cationic catalysts of the dismutation of the superoxide anion radical

Reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide (H2O2), have a diverse array of physiological and pathological effects within living cells depending on the extent, timing, and location of their production. For measuring ROS production in cells, the ESR spin trapping technique using cyclic nitrones distinguishes itself from other methods by its specificity for superoxide and hydroxyl radical. However, several drawbacks, such as the low spin trapping rate and the spontaneous and cell-enhanced decomposition of the spin adducts to ESR-silent products, limit the application of this method to biological systems. Recently, new cyclic nitrones bearing a triphenylphosphonium (Mito-DIPPMPO) or a permethylated β-cyclodextrin moiety (CD-DIPPMPO) have been synthesized and their spin adducts demonstrated increased stability in buffer. In this study, a comparison of the spin trapping efficiency of these new compounds with commonly used cyclic nitrone spin traps, i.e., 5,5-dimethyl-1-pyrroline N-oxide (DMPO), and analogs BMPO, DEPMPO, and DIPPMPO, was performed on RAW 264.7 macrophages stimulated with phorbol 12-myristate 13-acetate. Our results show that Mito-DIPPMPO and CD-DIPPMPO enable a higher detection of superoxide adduct, with a low (if any) amount of hydroxyl adduct. CD-DIPPMPO, especially, appears to be a superior spin trap for extracellular superoxide detection in living macrophages, allowing measurement of superoxide production in unstimulated cells for the first time. The main rationale put forward for this extreme sensitivity is that the extracellular localization of the spin trap prevents the reduction of the spin adducts by ascorbic acid and glutathione within cells.

Reactive Oxygen Species Produced by Liver Mitochondria of Rats in Sepsis

Reactive oxygen species (ROS) can be generated in experimental shock states through several different mechanisms. We measured ROS production in metabolically active liver mitochondria from rats rendered septic by cecal ligation and puncture. By polarography, the State 4 and State 3 respiration rates of liver mitochondria isolated from septic animals were no different from control organelles. During oxidation of succinate, however, nonenzymatic hydroxylation of salicylic acid to 2,3-dihydroxybenzoic acid by mitochondria from septic rats was increased, indicating generation of hydroxyl radical (OH.). Inhibition of electron transport at Complex I with rotenone had no effect on this pattern of OH. production, but rotenone and cyanide abolished the differences in OH. formation between control and septic liver mitochondria. Measurements of H2O2 release suggested that septic mitochondria will increase rates of H2O2 production in the presence of succinate. Additional investigations revealed no difference in the release of iron between septic and control mitochondria. When referenced to respiration rate, both OH. and H2O2 production were greater in septic liver mitochondria. The reproducible effect of sepsis on generation of reactive oxygen species by liver mitochondria utilizing FAD-linked but not NAD-linked substrates suggests that enhanced mitochondrial oxidative stress in sepsis is related to alterations in the activity of Complex II of the electron transport chain.