Intravascular origin of metastasis from the proliferation of endothelium-attached tumor cells: a new model for metastasis.

Metastasis is a frequent complication of cancer, yet the process through which circulating tumor cells form distant colonies is poorly understood. We have been able to observe the steps in early hematogenous metastasis by epifluorescence microscopy of tumor cells expressing green fluorescent protein in subpleural microvessels in intact, perfused mouse and rat lungs. Metastatic tumor cells attached to the endothelia of pulmonary pre-capillary arterioles and capillaries. Extravasation of tumor cells was rare, and it seemed that the transmigrated cells were cleared quickly by the lung, leaving only the endothelium-attached cells as the seeds of secondary tumors. Early colonies were entirely within the blood vessels. Although most models of metastasis include an extravasation step early in the process, here we show that in the lung, metastasis is initiated by attachment of tumor cells to the vascular endothelium and that hematogenous metastasis originates from the proliferation of attached intravascular tumor cells rather than from extravasated ones. Intravascular metastasis formation would make early colonies especially vulnerable to intravascular drugs, and this possibility has potential for the prevention of tumor cell attachment to the endothelium.

Lung metastatic load limitation with hyperbaric oxygen.

Despite some theoretical concern about cancer-enhancing effects ofhyperbaric oxygen (HBO2) therapy, it is frequently administered to cancer patients. We evaluated the growth of murine breast cancer cells in the lung after hyperbaric oxygen treatment in an experimental metastasis assay. Young nu/nu mice were injected intravenously with 3 x 10(3) 4T1-GFP tumor cells per g body weight followed by lung isolation, perfusion, and intact organ epifluorescence microscopy 1 to 37 days after injection. A group of animals (n=32) was exposed once daily for 5 days a week to 45 min of 2.8 ATA hyperbaric oxygen (HBO2) in a research animal HBO2 chamber. Control animals (n=31) were not subjected to HBO2 treatment, but received similar intravenous administration of 3 x 10(3) 4T 1-GFP tumor cells. Single tumor cells and colonies were counted in the subpleural vessels in areas of about 0.5 cm2 of lung surface. HBO2 treatment did not lead to an increase in the number of the large or small colonies in the lungs. Rather, a significant reduction in the number of the large colonies was observed at 1 and 16 to 21-day periods of measurements after hyperbaric treatment. However, most importantly, there was a significant decrease in large colony size in the HBO2 group during all periods of observation. The results indicate that HBO2 is not prometastatic for breast cancer cells; rather it restricts the growth of large tumor cell colonies.

Simulated ischemia in flow-adapted endothelial cells leads to generation of reactive oxygen species and cell signaling.

We have previously shown that increased reactive oxygen species (ROS) generation occurs with ischemia in the oxygenated lung and have hypothesized that mechanotransduction is the initiating event. In the present study, we developed an in vitro model of oxygenated ischemia by interrupting medium flow to flow-adapted bovine pulmonary artery endothelial cells in an artificial capillary system. Cellular oxygenation during the "ischemic" period was maintained by perfusing medium over the abluminal surface of porous capillaries. Cells were assessed for ROS generation, nuclear factor-kappaB (NF-kappaB) and activator protein-1 (AP-1) binding activities, and DNA synthesis using dichlorofluorescein fluorescence by flow cytometry and spectrofluorometry, electrophoretic mobility shift assay of nuclear extracts with NF-kappaB-specific or AP-1-specific (32)P-labeled oligonucleotides, and (3)H-thymidine incorporation into DNA. Cells that were flow adapted for 2 to 7 days with 1 to 2 dyne/cm(2) shear stress exhibited a 1.6- to 1.9-fold increase in ROS generation during 1 hour of simulated ischemia compared with continuously perfused cells. This effect was abolished by diphenyleneiodonium chloride (DPI), indicating a role for a flavoprotein such as NADPH oxidase. The increase in ROS generation with ischemia was similar for cells from low and high passages. With ischemia, flow-adapted cells exhibited increases of 1.7-fold in nuclear NF-kappaB and 1.5-fold in nuclear AP-1; these changes were abolished by pretreatment with N-acetylcysteine or DPI. Ischemia for 24 hours resulted in a 1.8-fold increase of (3)H-thymidine incorporation into DNA and a significant increase of cells entering the cell cycle, as indicated by flow cytometry with propidium iodide. We conclude that flow-adapted endothelial cells generate ROS with ischemia that results in activation of NF-kappaB and AP-1 and an increase of DNA synthesis. This effect is not mediated by hypoxia, implicating a role for mechanotransduction in ischemia-mediated cell signaling.

Apoptosis: an early event in metastatic inefficiency.

Whereas large numbers of cells from a primary tumor may gain access to the circulation, few of them will give rise to metastases. The mechanism of elimination of these tumor cells, often termed "metastatic inefficiency," is poorly understood. In this study, we show that apoptosis in the lungs within 1-2 days of introduction of the cells is an important component of metastatic inefficiency. First, we show that death of transformed, metastatic rat embryo cells occurred via apoptosis in the lungs 24-48 h after injection into the circulation. Second, we show that Bcl-2 overexpression in these cells inhibited apoptosis in culture and also conferred resistance to apoptosis in vivo in the lungs 24-48 h after injection. This inhibition of apoptosis led to significantly more macroscopic metastases. Third, comparison between the extent of apoptosis by a poorly metastatic cell line to that by a highly metastatic cell line 24 h after injection in the lungs revealed more apoptosis by the poorly metastatic cell line. These results indicate that apoptosis, which occurs at 24-48 h after hematogenous dissemination in the lungs is an important determinant of metastatic inefficiency. Although prior work has shown an association between apoptosis in culture and metastasis in vivo, this work shows that apoptosis in vivo corresponds to decreased metastasis in vivo.

A phospholipase A2 inhibitor decreases generation of thiobarbituric acid reactive substance during lung ischemia-reperfusion.

A novel active-site directed specific inhibitor of phospholipase A2 (PLA2), 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33), administered endotracheally co-dispersed in liposomes, significantly reduced the formation of thiobarbituric acid reactive substances (TBARS) in isolated rat lungs subjected to ischemia-reperfusion. Elevated conjugated dienes were unaffected. This contrasts with the effects of the cyclo-/lipoxygenase inhibitor 5,8,11,14-eicosatetraynoic acid (ETYA), which decreased formation of both TBARS and conjugated dienes (CD). The effects of MJ33 plus ETYA were additive for TBARS but results for CD were similar to ETYA alone. A similar dissociation of inhibition of TBARS and CD formation by MJ33 was observed with t-butyl hydroperoxide induced lipid peroxidation of isolated lung microsomes. Assay of lung homogenate with phosphatidylcholine as substrate showed that MJ33 selectively inhibited the Ca(2+)-independent acidic PLA2. MJ33 had no effect on thromboxane B2 release by the isolated lung, indicating the effects of acidic PLA2 inhibition do not involve the arachidonate cascade. MJ33 also partially prevented lung edema and lactate dehydrogenase release associated with ischemia-reperfusion. The observations show that this PLA2 inhibitor can be delivered to oxidant-sensitive lung sites by its co-dispersal in liposomes, and that oxidant-induced lipid peroxidation in this model of lung injury occurs in a complex lipid prior to PLA2 activity.

Peroxynitrite-mediated oxidative protein modifications.

Proteins are targets of reactive species and detection of oxidatively modified proteins is often used as an index of oxidative stress. Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide. Using fatty acid-free bovine serum albumin as a model we examined peroxynitrite-mediated protein modifications. The reaction of protein with peroxynitrite resulted in the oxidation of tryptophan and cysteine, in the nitration of tyrosine, in the formation of dityrosine, in the production of 2,4 dinitrophenylhydrazine-reactive carbonyls and in protein fragmentation. The formation of 3-nitrotyrosine represents a specific peroxynitrite-mediated protein modification that is different from modifications mediated by reactive oxygen species.

Oxidant generation with K(+)-induced depolarization in the isolated perfused lung.

This study evaluated whether cell membrane depolarization can induce oxidant generation in the isolated perfused rat lung as has been demonstrated with bovine pulmonary artery endothelial cells. Depolarization was produced by perfusing the lungs with high [K+] or with glyburide and was evaluated with bis-oxonol lung surface fluorometry. Lung surface bis-oxonol fluorescence increased above baseline (at 5.9 mM K+) by 18.5% with 24 mM K+, 35% with 48 mM K+, and 67% with 96 mM K+, indicating graded membrane depolarization, and by 75% during perfusion with 10 microM glyburide. Oxidant generation was evaluated with hydroethidine lung surface fluorometry, and with assay of tissue thiobarbituric acid reactive substance (TBARS), conjugated dienes, and perfusate H2O2. Depolarization by high K+ or glyburide led to significant increases in generation of tissue oxidants and lipid peroxidation. Bodipy-FL-glyburide microfluorography showed localization of glyburide binding primarily to vascular endothelial cells vascular and airway smooth muscle cells, alveolar type II cells, and to nonciliated cells of the airway epithelium. These results indicate that cellular depolarization is associated with oxidant generation by the lung and suggests a role for K(+)-channels in these events.

Depolarization-associated iron release with abrupt reduction in pulmonary endothelial shear stress in situ.

This study evaluated the roles of endothelial cell membrane potential and reactive oxygen species (ROS) in the increase of tissue free iron during lung ischemia. Oxygenated ischemia was produced in the isolated rat lung by discontinuing perfusion while ventilation with O2 was maintained. We have shown previously that tissue oxygenation is maintained in this model of ischemia and that biochemical changes are the result of an abrupt reduction in endothelial shear stress. With 1 hr oxygenated ischemia, generation of ROS, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 8.0-fold, lung thiobarbituric acid reactive substances (TBARS) increased 3.4-fold, and lung protein carbonyl content increased 2.4-fold. Lung tissue free iron, measured in the lung homogenate with a fluorescent desferrioxamine derivative, increased 4.0-fold during ischemia. Pretreatment of lungs with thapsigargin abolished the increase in free iron with ischemia indicating that this effect is dependent on Ca2+ release from intracellular stores. Perfusion of lungs with high (25 mM) K+ to depolarize the endothelium also led to a significant increase in tissue free iron. Pretreatment of lungs with 35 microM cromakalim, a K+-channel agonist, significantly inhibited both ischemia-induced tissue oxidant injury and the increase in free iron with ischemia or with high K+ perfusion. A similar increase in free iron was observed when lungs were ventilated with either O2 or N2 during the ischemic period or were pre-perfused with an inhibitor of ROS production (diphenyleneiodonium). These results indicate that ROS generation is not required for ischemia-mediated iron release. Thus, ROS generation and iron release with ischemia are independent although both are subsequent to endothelial cell membrane depolarization.

In situ imaging of intracellular calcium with ischemia in lung subpleural microvascular endothelial cells.

We propose that generation of reactive oxygen species (ROS) during ischemia is associated with an increase in intracellular calcium ([Ca2+]i) in pulmonary capillary endothelial cells. We used an isolated rat lung model and epifluorescence microscopy to evaluate [Ca2+]i in subpleural microvascular endothelial cells in situ by ratio imaging of the fluorophores, Calcium Green and Fura Red (CG/FR). Lungs were ventilated continuously under control (continuously perfused) or global ischemia (no perfusion) and thus remained adequately oxygenated even with ischemia. Ischemia for 5 min led to increase in CG/FR, indicating increase in [Ca2+]i in endothelial cells in situ; CG/FR remained elevated during a subsequent 10 min of ischemia. Ca(2+)-free perfusion and gadolinium (100 microM) inhibited the increase in [Ca2+]i, while thapsigargin (250 nM) had no effect. These results indicate that increase in endothelial cell [Ca2+]i with ischemia was due to influx from the extracellular medium. Perfusion with N-acetyl-L-cysteine (20 mM) or diphenyleneiodonium chloride (10 microM) prevented the ischemia-mediated [Ca2+]i increase, suggesting a role for ROS in the Ca2+ changes with ischemia. Membrane depolarization by perfusion with high potassium (K+) or glyburide also resulted in increased [Ca2+]i whereas the K(+)-channel agonist cromakalim, inhibited ischemia-mediated Ca2+ influx. We conclude that increased ROS generation with 'oxygenated' lung ischemia is associated with influx of Ca2+ and an increase in endothelial cell cytosolic calcium concentration.

Mice lacking in gp91 phox subunit of NAD(P)H oxidase showed glomus cell [Ca(2+)](i) and respiratory responses to hypoxia.

The hypothesis that NAD(P)H oxidase may serve as an oxygen sensor was tested using the mice deficient (knock-out) in gp91phox subunit of NAD(P)H oxidase enzyme complex and compared with wild-type (C57BL/6J) strain measuring the ventilatory and glomus cell intracellular calcium ([Ca(2+)](i)) responses of carotid body to hypoxia. The hypoxic ventilatory responses as well as the [Ca(2+)](i) were preserved in the NAD(P)H oxidase knock-out mice. NAD(P)H oxidase, though a major source of oxygen radical production, is not the oxygen sensor in mice carotid body.

An immediate endothelial cell signaling response to lung ischemia.

Abrupt cessation of lung perfusion induces a rapid endothelial response that is not associated with anoxia but reflects loss of normal shear stress. This response includes membrane depolarization, H(2)O(2) generation, and increased intracellular Ca(2+). We evaluated these parameters immediately upon nonhypoxic ischemia using fluorescence videomicroscopy to image in situ endothelial cells in isolated, ventilated rat lungs. Lungs labeled with 4-(2-[6-(dioctylamino)-2-naphthalenyl]ethenyl)1-(3-sulfopropyl)-pyridinium (di-8-ANEPPS; a membrane potential probe), Amplex Red (an extracellular H(2)O(2) probe), or fluo 3-AM (a Ca(2+) indicator) were subjected to control perfusion followed by global ischemia. Endothelial di-8-ANEPPS fluorescence increased significantly within the first second of ischemia and stabilized at 15 s, indicating membrane depolarization by approximately 17 mV; depolarization was blocked by preperfusion with the K(+) channel agonist lemakalim. Increased H(2)O(2), inhibitable by catalase, was detected in the vascular space at 1-2 s after the onset of ischemia. Increased intracellular Ca(2+) was detected 10-15 s after the onset of ischemia; the initial increase was inhibited by preperfusion with thapsigargin. Thus the temporal sequence of the initial response of endothelial cells in situ to loss of shear stress (i.e., ischemia) is as follows: membrane depolarization, H(2)O(2) release, and increased intracellular Ca(2+).

Intracellular generation of reactive oxygen species during nonhypoxic lung ischemia.

Surface fluorometry with 40 microM hydroethidine (HE) as a probe was used to detect oxidant generation in isolated, ventilated rat lungs during lung ischemia. Ethidium fluorescence due to HE oxidation was continuously monitored with 470 nm excitation and 610 nm emission. Fluorescence increased with ischemia in O2-ventilated lungs [0.98 +/- 0.08 arbitrary fluorescence units (AFU)/min vs. 0.58 +/- 0.07 with control perfusion]. HE oxidation during ischemia was prevented by N2 ventilation but was unaltered by preperfusion with superoxide dismutase. Ethidium fluorescence in homogenate prepared from lungs subjected to 1 h of nonhypoxic ischemia was increased (16.8 +/- 1.5 vs. 9.8 +/- 0.4 AFU/mg protein in control) but was unchanged in lungs that had been N2 ventilated. Microfluorographs of HE perfused and fixed lung sections demonstrated marked generalized increases in ethidium fluorescence with ischemia compared with control perfusion. Ischemia resulted in significant increases in tissue thiobarbituric acid reactive substance (176 +/- 13 vs. 44 +/- 3 pmol/mg protein for control) and in lung conjugated dienes (0.90 +/- 0.07 vs. 0.48 +/- 0.06 U/mg protein for control), indicating peroxidation of lung lipids. These results indicate that lung ischemia leads to intracellular oxidant generation that can be continuously monitored by surface fluorometry.

Signaling pathway for nitric oxide generation with simulated ischemia in flow-adapted endothelial cells.

Ischemia in the intact ventilated lung (oxygenated ischemia) leads to endothelial generation of reactive oxygen species (ROS) and nitric oxide (NO). This study investigated the signaling pathway for NO generation with oxygenated ischemia in bovine pulmonary artery endothelial cells (BPAEC) that were flow adapted in vitro. BPAECs were cultured in an artificial capillary system and subjected to abrupt cessation of flow (ischemia) under conditions where cellular oxygenation was maintained. Immunoblotting and dichlorofluorescein/triazolofluorescein fluorescence were used to assess extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation and ROS/NO generation, respectively. ERK1/2 phosphorylation significantly increased during ischemia, whereas total ERK1/2 did not change. ERK1/2 phosphorylation was suppressed by an inhibitor of tyrosine phosphorylation (genestein), cholesterol-binding reagents (filipin or cyclodextrin), or inhibitors of ROS (diphenyleneiodonium, N-acetylcysteine, or catalase), suggesting a role for both membrane cholesterol and ROS in ERK1/2 activation. Ischemia resulted in a 1.8-fold increase in NO generation that was suppressed by inhibitors of ERK1/2 activation (PD-98059 or U-0126). A calmodulin inhibitor (calmidizolium) or removal of Ca2+ from the medium also blocked NO generation, indicating that endothelial NO synthase (eNOS) is the activated isoform. These results indicate ischemia induces NO generation (possibly through a membrane cholesterol-sensitive flow sensor), the ERK1/2 cascade mediates signaling from the sensor to eNOS, and ROS are required for ERK activation.

Reactive species in ischemic rat lung injury: contribution of peroxynitrite.

Lung ischemia-reperfusion represents a potentially important mechanism for diverse forms of tissue injury associated with decreased pulmonary flow. Previous studies demonstrated oxidative injury in ischemic-reperfused lungs. The present study was designed to evaluate the contribution of nitric oxide and peroxynitrite in tissue injury. The levels of the stable decomposition products of nitric oxide and peroxynitrite, nitrite plus nitrate, were twofold greater than control during reperfusion after 60 min of ischemia. Inhibition of nitric oxide synthesis by endotracheal insufflation of 5 mM NG-nitro-L-arginine methyl ester, 30 min before the induction of ischemia, decreased the production of lung thiobarbituric acid reactive substances (TBARS) by 67% (P < 0.05, n = 5), TBARS released into the lung perfusate by 55% (P < 0.05, n = 5), lung-conjugated dienes by 61% (P < 0.05, n = 5), and dinitrophenylhydrazine-reactive protein carbonyl levels by 86% (P < 0.05, n = 5). Amino acid analysis of tissue homogenates from lungs exposed to 60 min of ischemia and 60 min of reperfusion revealed a 1.8-fold (P < 0.05, n = 5) increase in nitrotyrosine concentration compared with 2 h continuously perfused lungs. Inhibition of nitric oxide synthesis abolished the increase in nitrotyrosine levels. Furthermore, lungs exposed to 60 min of reperfusion after 60 min of ischemia showed specific binding of an anti-nitrotyrosine antibody. In reperfused tissues, antibody binding was observed throughout the lung. The binding was blocked with excess of nitrotyrosine, and minimal binding was observed in nonperfused blood-free control lungs.(ABSTRACT TRUNCATED AT 250 WORDS)

Anoxia-reoxygenation versus ischemia in isolated rat lungs.

Oxidant generation in anoxia-reoxygenation and ischemia-reperfusion was compared in isolated rat lungs. Anoxia-reoxygenation was produced by N2 ventilation followed by O2 ventilation. After anoxia, lung ATP content was decreased by 59%. Oxygenated ischemia was produced by discontinuing perfusion while ventilation with O2 was maintained. With anoxia-reoxygenation, oxidant generation, evaluated by oxidation of dichlorodihydrofluorescein (H2DCF) to fluorescent dichlorofluorescein, increased 3.6-fold, lung thiobarbituric acid reactive substances (TBARS) increased 342%, conjugated dienes increased 285%, and protein carbonyl content increased 46%. Pretreatment of lungs with 100 microM allopurinol inhibited the reoxygenation-mediated increase in lung fluorescence by 75% and TBARS by 69%. Oxygenated ischemia resulted in an approximately eightfold increase in lung H2DCF oxidation and a fourfold increase in TBARS, but allopurinol had no effect. On the other hand, 100 microM diphenyliodonium (DPI) inhibited the ischemia-mediated increase in lung fluorescence by 69% and lung TBARS by 70%, but it had no effect on the increase with anoxia-reoxygenation. Therefore, both ischemia-reperfusion and anoxia-reoxygenation result in oxidant generation by the lung, but a comparison of results with a xanthine oxidase inhibitor (allopurinol) and a flavoprotein inhibitor (DPI) indicate that the pathways for oxidant generation are distinctly different.

ATP-independent membrane depolarization with ischemia in the oxygen-ventilated isolated rat lung.

We hypothesize that lung ischemic injury is related to cessation of flow leading to endothelial cell membrane depolarization and activation of oxidant-generating systems. Cell membrane potential was assessed in isolated, oxygen ventilated, Krebs-Ringer bicarbonate buffer-dextran-perfused rat lungs by lung surface fluorescence after infusion of bis-oxonol or 5,5',6,6'-tetrachloro-1, 1',3,3'-tetraethylbenzimidazolyl-carbocyanine iodide (JC-1), voltage-sensitive dyes. Surface fluorometry showed increased bis-oxonol fluorescence (34.7 +/- 3.3% above baseline) and decreased JC-1 fluorescence (24.5 +/- 4.5% below baseline) with ischemia, compatible with membrane depolarization. Fluorescence change was initiated within 1-2 min of the onset of ischemia and was rapidly reversible with reperfusion. Fluorescence changes varied with perfusion flow rate; maximal increase occurred with the transition from 1.8 ml/min to zero flow. Elevation of static intravascular pressure resulted in only a minor increase of bis-oxonol fluorescence. In situ subpleural fluorescence microscopy showed that endothelial cells are the major site of the increased bis-oxonol fluorescence signal with ischemia. These results indicate that endothelial cell membrane depolarization represents an early event with lung ischemia. Since the adenosine triphosphate content of lung was unchanged with ischemia in the O2-ventilated lungs, we postulate that membrane depolarization results from elimination of shear stress, possibly via inactivation of flow-sensitive K+-channels.

Endothelial cell oxidant generation during K(+)-induced membrane depolarization.

We tested the hypothesis that membrane depolarization may initiate oxidant generation in the endothelial cell. Depolarization was produced in bovine pulmonary arterial endothelial cells (BPAEC) in monolayer culture with varying external K+, or with glyburide (10 microM), tetraethylammonium (TEA, 10 mM), gramicidin (1 microM), or nigericin (2 microM). Evaluation of bisoxonol fluorescence of BPAEC indicated concentration-dependent depolarization by high K+ (2% change in fluorescence/mV change in membrane potential in the 5.9-48 mM range of K+) and essentially complete depolarization with glyburide. Generation of oxidants was assessed with o-phenylenediamine dihydrochloride (o-PD) oxidation in the presence of horseradish peroxidase (HRP). There was a time-dependent increase in o-PD oxidation with 24 mM K+, nigericin, and gramicidin over 2 hours compared with control. In 1 hour o-PD oxidation increased 2.8-fold for 24 mM and 3.7-fold for 48 mM K+ compared with control. Catalase reduced 24 mM K(+)-induced o-PD oxidation by 50%, while Cu/Zn-superoxide dismutase (SOD) abolished the increase. Oxidation of o-PD was reduced by 57% in the absence of HRP in the system. With K+ channel blockade, o-PD oxidation increased 3.8-fold with glyburide and 4.6-fold with TEA compared with control. These data indicate formation of H2O2 and possibly other oxidants with depolarization and suggest involvement of K(+)-channels in this process.

Inhibition of lung tissue oxidation during ischemia/reperfusion by 2-mercaptopropionylglycine.

The effect of 2-mercaptopropionylglycine (MPG), a potent free radical scavenger, on ischemia/reperfusion-induced tissue oxidation in isolated perfused rat lung was investigated. The isolated lung, continuously ventilated with 95% oxygen, was subjected to 1 h global ischemia followed by 1 h reperfusion with or without the presence of an antioxidant. In ischemic/reperfused lungs, there was a significant increase in protein oxidation (carbonyl formation) and lipid peroxidation (thiobarbituric acid reactive substances) to 10.7 nmol/mg protein and 176 pmol/mg protein, respectively, at the end of reperfusion. MPG administered at 6 mg/kg body wt intravenously to the rats prior to isolation of lung reduced protein oxidation by 65% and lipid peroxidation by 40%. An additional effect was noted when MPG was also added to the perfusate (0.275 mg/ml) during reperfusion. Pretreatment with dimethylthiourea (DMTU) or addition of desferal to the perfusate also significantly reduced the protein oxidation of lung ischemia/reperfusion. The addition of DMTU or desferal with MPG showed no additive effect. However, eicosatetraynoic acid (100 microM), a cyclooxygenase and lipoxygenase inhibitor, added with MPG reduced ischemia/reperfusion-induced lipid peroxidation by 80%, which was significantly greater than the protective effect exhibited by MPG alone. The oxidative stress on the lung tissue components was also demonstrated by a decrease in the sulfhydryl content of "end ischemic" lungs; MPG pretreatment maintained the sulfhydryl level at the control level in ischemic lungs. The results indicate that MPG at relatively low and non-toxic concentrations can markedly inhibit the oxidation of tissue sulfhydryls, soluble protein, and lipids associated with ischemia/reperfusion injury of the lung.