Sestrin2 inhibits uncoupling protein 1 expression through suppressing reactive oxygen species

Uncoupling protein 1 (Ucp1), which is localized in the mitochondrial inner membrane of mammalian brown adipose tissue (BAT), generates heat by uncoupling oxidative phosphorylation. Upon cold exposure or nutritional abundance, sympathetic neurons stimulate BAT to express Ucp1 to induce energy dissipation and thermogenesis. Accordingly, increased Ucp1 expression reduces obesity in mice and is correlated with leanness in humans. Despite this significance, there is currently a limited understanding of how Ucp1 expression is physiologically regulated at the molecular level. Here, we describe the involvement of Sestrin2 and reactive oxygen species (ROS) in regulation of Ucp1 expression. Transgenic overexpression of Sestrin2 in adipose tissues inhibited both basal and cold-induced Ucp1 expression in interscapular BAT, culminating in decreased thermogenesis and increased fat accumulation. Endogenous Sestrin2 is also important for suppressing Ucp1 expression because BAT from Sestrin2^−/− mice exhibited a highly elevated level of Ucp1 expression. The redox-inactive mutant of Sestrin2 was incapable of regulating Ucp1 expression, suggesting that Sestrin2 inhibits Ucp1 expression primarily through reducing ROS accumulation. Consistently, ROS-suppressing antioxidant chemicals, such as butylated hydroxyanisole and N-acetylcysteine, inhibited cold- or cAMP-induced Ucp1 expression as well. p38 MAPK, a signaling mediator required for cAMP-induced Ucp1 expression, was inhibited by either Sestrin2 overexpression or antioxidant treatments. Taken together, these results suggest that Sestrin2 and antioxidants inhibit Ucp1 expression through suppressing ROS-mediated p38 MAPK activation, implying a critical role of ROS in proper BAT metabolism.Although reactive oxygen species (ROS) are normal products of cellular metabolism, excessive accumulation of ROS resulting from nutritional imbalance and/or environmental stresses can provoke oxidative damage of diverse cellular macromolecules, such as DNA, RNA, and proteins (1). Accumulation of ROS has been associated with diverse degenerative diseases, such as cancer, neurodegeneration, and obesity-associated metabolic syndrome (2–4). To minimize detrimental consequences of ROS accumulation, cells are equipped with various antioxidant proteins, including superoxide dismutases, catalases, peroxiredoxins, and sestrins (5–7). Several ROS-scavenging chemicals or dietary supplements, such as butylated hydroxyanisole (BHA), N-acetylcysteine (NAC), and antioxidant vitamins, can assist with eliminating excessive amounts of ROS (8–10) and were once considered to be potential inhibitors of degenerative diseases associated with aging and obesity (11–13). However, most animal and human clinical studies failed to demonstrate the benefits of dietary antioxidants in restoring metabolic homeostasis or in promoting health and lifespan (13, 14).Uncoupling protein 1 (Ucp1) is an anion-carrier protein located in the inner membrane of the mitochondria. By dissipating the proton gradient across the mitochondrial inner membrane, Ucp1 uncouples substrate oxidation from ATP synthesis, ultimately reducing ATP production and generating heat (15). Ucp1-mediated mitochondrial uncoupling also suppresses ROS production during respiration (16). Ucp1 expression is induced upon exposure to cold temperature or nutritional overload, and this induction is important for protection of organisms against cold and obesity (17). Despite the significance of Ucp1 in energy metabolism, it is poorly understood how Ucp1 expression is regulated, other than the fact that cAMP and p38 MAPK signaling pathways (18–20) are necessary for Ucp1 induction upon cold stimuli. It also has not yet been explored whether subcellular ROS can regulate Ucp1 expression and subsequent heat generation.Sestrins are a family of stress-inducible proteins that regulate metabolic homeostasis (21). Sestrins have two independent biological activities largely divided into regulating AMP-activated protein kinase (AMPK)-mammalian target of rapamycin complex 1 (mTORC1) signaling and suppressing ROS accumulation (22). Loss of endogenous sestrins can provoke a variety of metabolic pathologies, including insulin resistance, fat accumulation, mitochondrial dysfunction, and oxidative damage (23, 24). Given the known roles of endogenous sestrins in reducing oxidative stress, fat accumulation, and insulin resistance, we thought that overexpression of sestrins may protect animals from developing obesity or obesity-associated metabolic pathologies. Among the three mammalian sestrins (Sestrin1–3), the metabolism-regulating functions of Sestrin2 have been the most rigorously characterized in metabolic organs, such as the liver and adipose tissue (AT) (23, 25). Thus, to examine the effects of Sestrin2 overexpression, we generated tetracyclin-regulated promoter-Sestrin2 (tet-Sesn2) transgenic mice that can express Sestrin2 in a tissue-specific manner when crossed with tissue-specific tetracycline activator (tTA) strains.Using tet-Sesn2 and AT-specific peroxisome proliferator-activated receptor γ (Pparγ)-tTA (Pparγ-tTA) strains, we generated Pparγ-tTA/tet-Sesn2 mice that express Sestrin2 specifically in AT. Given the ROS- and mTORC1-suppressing functions of Sestrin2 (21), we expected that the Pparγ-tTA/tet-Sesn2 (PG-Sn2) strain would exhibit decreased fat accumulation and improved metabolic homeostasis compared with the control strain. However, we found that Sestrin2 overexpression unexpectedly increased fat accumulation, which is associated with dramatic suppression of Ucp1 expression in brown adipose tissue (BAT). The redox-regulating function of Sestrin2, rather than its mTORC1-inhibiting function, was responsible for its Ucp1 regulation. We also discovered that administration of chemical antioxidants, such as BHA or NAC, could inhibit cAMP (in vitro)- or cold (in vivo)-induced Ucp1 expression. These results reveal a critical role of ROS in basal and cold-induced expression of Ucp1 in BAT.     

醛固酮对脂肪细胞因子表达及活性氧簇生成的影响

目的 探讨醛固酮对3T3-L1脂肪细胞脂肪细胞因子表达、分泌,活性氧簇(ROS)生成水平的影响.方法 诱导分化成熟的3T3-L1脂肪细胞经醛固酮(1μmol/L)孵育4h、24h,实时定量PCR检测脂肪细胞因子脂联素、白细胞介素-6(IL-6)、纤溶酶原激活物抑制剂-1(PAI-1)、单核细胞趋化蛋白-1( MCP-1...     

雌激素与活性氧

大量证据表明活性氧(ROS)对心脑血管疾病的发生和发展有重要影响。它可以直接氧化膜脂质和DNA,造成细胞氧化损伤和异常;同时还作为信使参与细胞各种生理和病理活动,例如调节基因表达和信号转导。近几年,雌激素对心脑血管疾病的保护作用越来越受到人们的重视,它主要参与细胞内的氧化应激反应,但在某些方面也存在争议。     

Isoform-specific regulation of Akt by PDGF-induced reactive oxygen species

Isoform-specific signaling of Akt, a major signaling hub and a prominent therapeutic target, remained poorly defined until recently. Subcellular distribution, tissue-specific expression, substrate specificity, and posttranslational modifications are believed to underlie isoform-specific signaling of Akt. The studies reported here show inhibition of Akt2 activity under physiologically relevant conditions of oxidation created by PDGF-induced reactive oxygen species. Combined MS and functional assays identified Cys124 located in the linker region between the N-terminal pleckstrin homology domain and the catalytic kinase domain as one of the unique regulatory redox sites in Akt2 with functional consequence on PDGF-stimulated glucose uptake. A model is proposed describing the consequence of increased endogenous oxidation induced by extracellular cues such as PDGF on Akt2 activity.     

Generation of reactive oxygen species by the faecal matrix

BACKGROUND: Reactive oxygen species are implicated in the aetiology of a range of human diseases and there is increasing interest in their role in the development of cancer. AIM: To develop a suitable method for the detection of reactive oxygen species produced by the faecal matrix. METHODS: A refined high performance liquid chromatography system for the detection of reactive oxygen species is described. RESULTS: The method allows baseline separation of the products of hydroxyl radical attack on salicylic acid in the hypoxanthine/xanthine oxidase system, namely 2,5-dihydroxybenzoic acid, 2,3-dihydroxybenzoic acid, and catechol. The increased efficiency and precision of the method has allowed a detailed evaluation of the dynamics of reactive oxygen species generation in the faecal matrix. The data show that the faecal matrix is capable of generating reactive oxygen species in abundance. This ability cannot be attributed to the bacteria present, but rather to a soluble component within the matrix. As yet, the nature of this soluble factor is not entirely clear but is likely to be a reducing agent. CONCLUSIONS: The soluble nature of the promoting factor renders it amenable to absorption, and circumstances may exist in which either it comes into contact with either free or chelated iron in the colonocyte, leading to direct attack on cellular DNA, or else it initiates lipid peroxidation processes whereby membrane polyunsaturated fatty acids are attacked by reactive oxygen species propagating chain reactions leading to the generation of promutagenic lesions such as etheno based DNA adducts.     

Mitochondria-derived reactive oxygen species dilate cerebral arteries by activating Ca2+ sparks

Mitochondria regulate intracellular calcium (Ca2+) signals in smooth muscle cells, but mechanisms mediating these effects, and the functional relevance, are poorly understood. Similarly, antihypertensive ATP-sensitive potassium (KATP) channel openers (KCOs) activate plasma membrane KATP channels and depolarize mitochondria in several cell types, but the contribution of each of these mechanisms to vasodilation is unclear. Here, we show that cerebral artery smooth muscle cell mitochondria are most effectively depolarized by diazoxide (-15%, tetramethylrhodamine [TMRM]), less so by levcromakalim, and not depolarized by pinacidil. KCO-induced mitochondrial depolarization increased the generation of mitochondria-derived reactive oxygen species (ROS) that stimulated Ca2+ sparks and large-conductance Ca2+-activated potassium (KCa) channels, leading to transient KCa current activation. KCO-induced mitochondrial depolarization and transient KCa current activation were attenuated by 5-HD and glibenclamide, KATP channel blockers. MnTMPyP, an antioxidant, and Ca2+ spark and KCa channel blockers reduced diazoxide-induced vasodilations by >60%, but did not alter dilations induced by pinacidil, which did not elevate ROS. Data suggest diazoxide drives ROS generation by inducing a small mitochondrial depolarization, because nanomolar CCCP, a protonophore, similarly depolarized mitochondria, elevated ROS, and activated transient KCa currents. In contrast, micromolar CCCP, or rotenone, an electron transport chain blocker, induced a large mitochondrial depolarization (-84%, TMRM), reduced ROS, and inhibited transient KCa currents. In summary, data demonstrate that mitochondria-derived ROS dilate cerebral arteries by activating Ca2+ sparks, that some antihypertensive KCOs dilate by stimulating this pathway, and that small and large mitochondrial depolarizations lead to differential regulation of ROS and Ca2+ sparks.     

Attenuation by reactive oxygen species of glucocorticoid suppression on proopiomelanocortin gene expression in pituitary corticotroph cells

Up-regulation of hypothalamo-pituitary-adrenal axis is maintained during acute inflammation and/or infection, in the face of sustained elevation of plasma glucocorticoid hormone. Inflammatory stress is usually associated with high plasma cytokine levels and increased generation of reactive oxygen species (ROS) as well. In this study, we examined the effect of ROS on the negative feedback regulation of glucocorticoid in hypothalamo-pituitary-adrenal axis using AtT20 corticotroph cells in vitro. When the cells were treated with H2O2, glucocorticoid suppression on the proopiomelanocortin gene promoter activity was attenuated in a dose-dependent manner. H2O2 also inhibited the ligand-stimulated nuclear translocation of glucocorticoid receptor. The released glucocorticoid suppression by H2O2 was not observed when the cells were cotreated with antioxidants. Together, these results suggest that increased ROS generation in the oxidative redox state attenuates the glucocorticoid negative feedback system, at least in part, by interfering with the nuclear translocation of glucocorticoid receptor and eliminating the repression on proopiomelanocortin gene expression.     

氧自由基降低脂肪细胞脂联素表达的机制研究

目的 研究活性氧簇(ROS)对3T3-LI脂肪细胞脂联素表达的调节机制.方法 以实时定量PCR检测分化成熟的3T3-L1脂肪细胞脂联素mRNA表达水平,联合应用多重磷酸化蛋白分析系统与各种蛋白激酶抑制剂,筛查ROS下调脂联索表达的信号通路.结果 作为一种重要的ROS,H_2O_2激活了313-L1脂肪细胞细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基端激酶(JNK)、蛋白激酶B(Akt)、p70 S6 激酶(p70 S6K)及Janus激酶/信号转导和转录激活因子(JAK/STAT)等多种信号转导通路.其中Akt和JAK/STAT抑制剂完全逆转H_2_O2对脂联素表达的下调作用(均P<0.01).结论 ROS可能通过激活Akt、JAK/STAT信号途径下调脂肪细胞脂联素的表达,从而在肥胖及其相关疾病的发生、发展中发挥作用.     

Agents capable of eliminating reactive oxygen species

Reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, hydroxyl radical, and hypochlorous acid have been implicated in the pathogenesis of inflammation and tissue injury in colitis. To determine whether or not anti-ROS agents can decrease the severity of colitis, we evaluated the effects of three known anti-ROS agents: catalase, WR-2721, and Cu(II)₂(3,5-DIPS)₄ on acetic acid-induced colonic inflammation in rats. Histologically, all three compounds significantly decreased the severity of colonic inflammation. The anti-ROS activity of these compounds was also tested using the luminol-enhanced chemiluminescence assay. Catalase, WR-2721, or Cu(II)₂(3,5-DIPS)₄ significantly inhibited luminol-enhanced chemiluminescence produced by inflamed colonic mucosa. These findings suggest that ROS, and in particular superoxide, hydrogen peroxide, and/or one of its secondarily derived species, may play an important role in acetic acid-induced colitis. Further studies are needed to determine the potential effectiveness of these compounds in human colitis.     

衰老对硝酸酯类药物引起活性氮介质和活性氧介质增高的影响

目的:以往研究显示,硝酸酯类药物和衰老都会引发体内活性氧介质(ROS)和活性氮介质(RNS)的增加,本研究旨在探讨年龄是否会影响硝酸酯类药物的这种促进作用。方法:75例不稳定心绞痛患者,分成32例中年组和43例老年两组。所有患者均给予硝酸酯类药物(50μg/min)48h。在试验开始时和用药48小时时,获取血样标本,对血样中的ROS[丙二醛(MDA),髓过氧化物酶(MPO)和还原性谷胱甘肽(GSH)]和RNS(硝基、亚硝基,NOX;过氧亚硝酸阴离子,ONOO-)]的水平进行检测。结果:硝酸酯类药物的使用,引起中年组血浆MDA水平[用药前(1.22±0.37)nmol/m L,用药后(1.61±0.47)nmol/m L,P0.05]增加60%;老年组MDA水平[用药前(2.07±0.77)nmol/m L,用药后(4.05±0.80)nmol/m L,P0.05],增加140%;GSH两组分别减少了9%和48%;硝酸酯类药物使用前,老年组血浆硝基化酪氨酸(398.29±117.0)nmol/L水平为仅为中年组(296.57±120.48)nmol/L的105%,药物使用48h后,老年组血浆硝基化酪氨酸水平(1 182.30±295.01)nmol/L增高到中年组(610.82±217.36)nmol/L,增高210%。结论:在硝酸酯类药物的使用过程中,除了药物本身增加机体内ROS和RNS,年龄增加能够促进硝酸酯类药物的这种作用。     

Bidirectional regulation of Ca2+ sparks by mitochondria-derived reactive oxygen species in cardiac myocytes

AIMS: The cardiac ryanodine receptor (RyR) Ca(2+) release channel homotetramer harbours approximately 21 potentially redox-sensitive cysteine residues on each subunit and may act as a sensor for reactive oxygen species (ROS), linking ROS homeostasis to the regulation of Ca(2+) signalling. In cardiac myocytes, arrayed RyRs or Ca(2+) release units are packed in the close proximity of mitochondria, the primary source of intracellular ROS production. The present study investigated whether and how mitochondria-derived ROS regulate Ca(2+) spark activity in intact cardiac myocytes. METHODS AND RESULTS: Bidirectional manipulation of mitochondrial ROS production in intact rat cardiac myocytes was achieved by photostimulation and pharmacological means. Simultaneous measurement of intracellular ROS and Ca(2+) signals was performed using confocal microscopy in conjunction with the indicators 5-(-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (for ROS) and rhod-2 (for Ca(2+)). Photoactivated or antimycin A (AA, 5 microg/mL)-induced mitochondrial ROS production elicited a transient increase in Ca(2+) spark activity, followed by gradual spark suppression. Intriguingly, photoactivated mitochondrial ROS oscillations subsequent to the initial peaks mirrored phasic depressions of the spark activity, suggesting a switch of ROS modulation from spark-activating to spark-suppressing. Partial deletion of Ca(2+) stores in the sarcoplasmic reticulum contributed in part to the gradual, but not the phasic, spark depression. H(2)O(2) at 200 microM elicited a bidirectional effect on sparks and produced sustained spark activation at 50 microM. Lowering basal mitochondrial ROS production, scavenging baseline ROS, and applying the sulphydryl-reducing agent dithiothreitol diminished the incidence of spontaneous Ca(2+) sparks and abolished the Ca(2+) spark responses to mitochondrial ROS. CONCLUSION: Mitochondrial ROS exert bidirectional regulation of Ca(2+) sparks in a dose- and time (history)-dependent manner, and basal ROS constitute a hitherto unappreciated determinant for the production of spontaneous Ca(2+) sparks. As such, ROS signalling may play an important role in Ca(2+) homeostasis as well as Ca(2+) dysregulation in oxidative stress-related diseases.     

Generation of reactive oxygen species by lethal attacks from competing microbes

Whether antibiotics induce the production of reactive oxygen species (ROS) that contribute to cell death is an important yet controversial topic. Here, we report that lethal attacks from bacterial and viral species also result in ROS production in target cells. Using soxS as an ROS reporter, we found soxS was highly induced in Escherichia coli exposed to various forms of attacks mediated by the type VI secretion system (T6SS), P1vir phage, and polymyxin B. Using a fluorescence ROS probe, we found enhanced ROS levels correlate with induced soxS in E. coli expressing a toxic T6SS antibacterial effector and in E. coli treated with P1vir phage or polymyxin B. We conclude that both contact-dependent and contact-independent interactions with aggressive competing bacterial species and viruses can induce production of ROS in E. coli target cells.Microbial species exist predominantly in complex communities in the natural environment and animal hosts. To survive in a multispecies environment, bacteria have developed various strategies to compete with other species. For example, some bacteria can exert long-range inhibitory effects by secreting diffusible molecules, such as antibiotics, bacteriocins, and H₂O₂ (1), whereas others require direct cell-to-cell contact to kill nearby organisms (2, 3). One such contact-dependent inhibitory system is the type VI secretion system (T6SS), a protein translocating nanomachine expressed by many Gram-negative bacterial pathogens that can kill both bacterial and eukaryotic cells (3–5). Structurally analogous to an inverted bacteriophage tail, the T6SS delivers effectors into target cells by using a contractile sheath to propel an inner tube out of the producer cell and into nearby target cells. The inner tube (composed of Hcp protein) is thought to carry toxic effector proteins within its lumen or on its tip, which is decorated with VgrG and PAAR proteins (4, 6, 7). Given that some cells can detect T6SS attack but not suffer any measurable loss in viability (8, 9), it would seem that cell killing is likely due to the toxicity of effectors rather than membrane disruptions caused by insertion of the spear-like VgrG/PAAR/Hcp tube complex. T6SS-dependent effectors can attack a number of essential cellular targets, including the cell wall (10, 11), membranes (11, 12), and nucleic acids (13), and thus can mimic the actions of antibiotics and bacteriocins. As a model prey or target organism, Escherichia coli can be killed by the T6SS activities of a number of bacteria including Vibrio cholerae (14), Pseudomonas aeruginosa (10, 15), and Acinetobacter baylyi ADP1 (7).Collins and coworkers (16–18) have reported that antibiotic treatment of E. coli elicits the production of reactive oxygen species (ROS) resulting from a series of events involving perturbation of the central metabolic pathway, NADPH depletion, and the Fenton reaction. ROS can cause lethal damage to DNA, lipid, and proteins (19, 20) and thus can contribute to cell death in combination with the deleterious effects of antibiotics on their primary targets. The idea that antibiotics kill bacterial cells, in part, through the action of ROS has been supported by a number of follow-up studies (18, 21–23) but has also been challenged by others as a result of observations contradictory to a model where ROS is the sole mediator of antibiotic lethality (24–26). These observations include the fact that antibiotics kill under anaerobic conditions, oxidation of the hydroxyphenyl fluorescein fluorescence dye used to measure ROS levels is nonspecific, and the extracellular level of H₂O₂ is not elevated by antibiotic treatment (24, 26). To address these concerns, Dwyer et al. (27) used a panel of ROS-detection fluorescence dyes, a defined growth medium under stringent anaerobic conditions, and an in vivo H₂O₂ enzymatic assay to study the effects of antibiotics on cells. The results further support that antibiotics induce ROS generation, which contributes to the efficacy of antibiotics in addition to their primary lethal actions (18, 27, 28).     

氧自由基对脂肪细胞中脂联素表达的下调作用

目的研究脂肪细胞中氧自由基（reactive oxygen species,ROS）对脂联素（adiponectin,AD）表达的调节,并探讨其机制。方法培养3T3-L1细胞,并诱导其分化为脂肪细胞,以MTT比色法检测细胞的活性。采用Hydroethidi-neTM（HE）及H2-DCFDA测定细胞内ROS的产生。分别以定量PCR、多重免疫分析及夹心ELISA法,检测AD mR-NA和其蛋白的表达水平。结果低氧条件诱导脂肪细胞中ROS产生。作为一种重要的ROS,H2O2可剂量依赖性地抑制3T3-L1脂肪细胞产生AD,并降低AD基因的转录激活因子增强子结合蛋白α（CCAAT/enhancer binding pro-tein alpha,C/EBPα）及过氧化物酶体增殖激活受体γ（peroxisome proliferator-activated receptor gamma,PPARγ）的表达。结论低氧产生ROS,而ROS导致脂肪细胞中AD的表达抑制,其重要机制之一是ROS可下调C/EBPα和PPARγ,从而减弱AD基因的转录激活。     

Reaction mechanism of melatonin oxidation by reactive oxygen species in vitro

Melatonin (N-acetyl-5-hydroxytryptamine) is a pineal hormone widely known for its antioxidant properties, both in vivo and by direct capture of free radicals in vitro. Although some metabolites and oxidation products of melatonin have been identified, the molecular mechanism by which melatonin exerts its antioxidant properties has not been totally unravelled. This study investigated the reaction mechanism of oxidation of melatonin by radio-induced reactive oxygen species, generated by gamma radiolysis of water for aqueous solutions of melatonin (from 20 to 200 μm), in the presence or absence of molecular oxygen. The hydroxyl radical was found to be the unique species able to initiate the oxidation process, leading to three main products, e.g. N(1)-acetyl-N(2)-formyl-5-methoxykynurenin (AFMK), N(1)-acetyl-5-methoxykynurenin (AMK) and hydroxymelatonin (HO-MLT). The generation of AFMK and HO-MLT strongly depended on the presence of molecular oxygen in solution: AFMK was the major product in aerated solutions (84%), whereas HO-MLT was favoured in the absence of oxygen (86%). Concentrations of AMK remained quite low, and AMK was proposed to result from a chemical hydrolysis of AFMK in solution. A K-value of 1.1 × 10(-4) was calculated for this equilibrium. Both hydrogen peroxide and superoxide dismutase had no effect on the radio-induced oxidation of melatonin, in good accordance for the second case with the poor reactivity of the superoxide anion towards melatonin. Finally, a reaction mechanism was proposed for the oxidation of melatonin in vitro.     

Hemoglobin potentiates the production of reactive oxygen species by alveolar macrophages

The objectives of this investigation were (1) to determine the effects of hemoglobin on the production of reactive oxygen species by activated rat alveolar macrophages, (2) to determine a possible mechanism for these effects, and (3) to determine which part of the hemoglobin molecule is responsible for these effects. Production of reactive oxygen species by phorbol myristate acetate (PMA)-stimulated cells was assessed by measuring luminol-enhanced chemiluminescence (CL). Hemoglobin enhances PMA-stimulated CL in a dose-dependent manner. The effect is maximal at 0.5-1.0 microM hemoglobin where PMA-induced CL is increased by approximately 20-fold. Superoxide anion release from PMA-stimulated cells is not affected by hemoglobin. However, the hemoglobin-induced enhancement of PMA-stimulated CL is inhibited by superoxide dismutase, catalase, dimethylthiourea, or deferoxamine. These results suggest that hydroxyl radical may be formed from hydrogen peroxide which is derived from superoxide anion. Measurements of electron spin resonance spectra following spin trapping of radicals verify that hydroxyl radicals are produced by the cells in the presence of PMA and hemoglobin. The hemoglobin effects appear to require iron in a protoporphyrin complex, because hemin stimulates PMA-induced CL, whereas neither ferrous nor ferric iron has any effect. These findings taken together suggest that hemoglobin can act as a biological Fenton reagent to enhance the production of reactive oxygen species from alveolar macrophages and potentially contribute to lung damage during leakage of blood into the alveolar spaces.