Liver microsomal parameters related to oxidative stress and antioxidant systems in hyperthyroid rats subjected to acute lindane treatment

Liver microsomal functions related to xenobiotic biotransformation and free radical production were studied in control rats and in animals subjected to L-3,3′,5-triiodothyronine (T₃) and/or lindane administration as possible mechanisms contributing to oxidative stress, in relation to the activity of enzymes (superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx), and glucose-6-phosphate dehydrogenase (G-6PDH)) and content of lipid-soluble vitamins (α-tocopherol, β-carotene, and lycopene) affording antioxidant protection. Lindane treatment in euthyroid rats at a dosage of 20 mg/kg did not modify the content of liver microsomal cytochromes P450 and b₅, the activity of NADPH-cytochrome P450 reductase and NADH-cytochrome b₅ reductase, and the production of superoxide radical (O^·-₂), as well as antioxidant systems, except for the reduction in lycopene levels. Hyperthyroidism elicited a calorigenic response and increased specific and molecular activities of NADPH-cytochrome P450 reductase, O^·-₂ generation, and G-6PDH activity, concomitantly with diminution in liver SOD and catalase activities and in α-tocopherol, β-carotene, and lycopene levels. The administration of lindane to hyperthyroid animals led to a further increase in the molecular activity of NADPH-cytochrome P450 reductase and in the O^·-₂ production/SOD activity ratio, and decrease of hepatic α-tocopherol content, in a magnitude exceeding the sum of effects elicited by the separate treatments, as previously reported for reduced glutathione depletion. Collectively, these data support the contention that the increased susceptibility of the liver to the toxic effects of acute lindane treatment in hyperthyroid state is conditioned by potentiation of the hepatic oxidative stress status.     

Exposure to sodium molybdate results in mild oxidative stress in Drosophila melanogaster

Objectives: The study was conducted to assess the redox status of Drosophila flies upon oral intake of insulin-mimetic salt, sodium molybdate (Na₂MoO₄).

Methods: Oxidative stress parameters and activities of antioxidant and associated enzymes were analyzed in two-day-old D. melanogaster insects after exposure of larvae and newly eclosed adults to three molybdate levels (0.025, 0.5, or 10 mM) in the food.

Results: Molybdate increased content of low molecular mass thiols and activities of catalase, superoxide dismutase, glutathione-S-transferase, and glucose-6-phosphate dehydrogenase in males. The activities of these enzymes were not affected in females. Males exposed to molybdate demonstrated lower carbonyl protein levels than the control cohort, whereas females at the same conditions had higher carbonyl protein content and catalase activity than ones in the control cohort. The exposure to 10 mM sodium molybdate decreased the content of protein thiols in adult flies of both sexes. Sodium molybdate did not affect the activities of NADP-dependent malate dehydrogenase and thioredoxin reductase in males or NADP-dependent isocitrate dehydrogenase in either sex at any concentration.

Discussion: Enhanced antioxidant capacity in upon Drosophila flies low molybdate levels in the food suggests that molybdate can be potentially useful for the treatment of certain pathologies associated with oxidative stress.     

Tolerance,arsenic uptake,and oxidative stress in Acacia farnesiana under arsenate-stress

Acacia farnesiana is a shrub widely distributed in soils heavily polluted with arsenic in Mexico. However, the mechanisms by which this species tolerates the phytotoxic effects of arsenic are unknown. This study aimed to investigate the tolerance and bioaccumulation of As by A. farnesiana seedlings exposed to high doses of arsenate (AsV) and the role of peroxidases (POX) and glutathione S-transferases (GST) in alleviating As-stress. For that, long-period tests were performed in vitro under different AsV treatments. A. farnesiana showed a remarkable tolerance to AsV, achieving a half-inhibitory concentration (IC₅₀) of about 2.8 mM. Bioaccumulation reached about 940 and 4380 mg As·kg^?1 of dry weight in shoots and roots, respectively, exposed for 60 days to 0.58 mM AsV. Seedlings exposed to such conditions registered a growth delay during the first 15 days, when the fastest As uptake rate (117 mg kg^?1 day^?1) occurred, coinciding with both the highest rate of lipid peroxidation and the strongest up-regulation of enzyme activities. GST activity showed a strong correlation with the As bioaccumulated, suggesting its role in imparting AsV tolerance. This study demonstrated that besides tolerance to AsV, A. farnesiana bioaccumulates considerable amounts of As, suggesting that it may be useful for phytostabilization purposes.     

Arsenic induces oxidative DNA damage in mammalian cells

Although arsenic is a well-established human carcinogen, the underlying carcinogenic mechanism(s) is not known. Using the human-hamster hybrid (A_L) cell mutagenic assay that is sensitive in detecting mutagens that induce predominately multilocus deletions, we showed previously that arsenite is indeed a potent gene and chromosomal mutagen and that oxyradicals may be involved in the mutagenic process. In the present study, the effects of free radical scavenging enzymes on the cytotoxic and mutagenic potential of arsenic were examined using the A_L cells. Concurrent treatment of cells with either superoxide dismutase or catalase reduced both the cytotoxicity and mutagenicity of arsenite by an average of 2–3 fold, respectively. Using immunoperoxidase staining with a monoclonal antibody specific for 8-hydroxy-2-deoxyguanosine (8-OHdG), we demonstrated that arsenic induced oxidative DNA damage in A_L cells. This induction was significantly reduced in the presence of the antioxidant enzymes. Furthermore, reducing the intracellular levels of non-protein sulfhydryls (mainly glutathione) using buthionine S-R-Sulfoximine increased the total mutant yield by more than 3-fold as well as the proportion of mutants with multilocus deletions. Taken together, our data provide clear evidence that reactive oxygen species play an important causal role in the genotoxicity of arsenic in mammalian cells.     

Response of arsenic-induced oxidative stress,DNA damage,and metal imbalance to combined administration of DMSA and monoisoamyl-DMSA during chronic arsenic poisoning in rats

Arsenic and its compounds cause adverse health effects in humans. Current treatment employs administration of thiol chelators, such as meso-2,3-dimercaptosuccinic acid (DMSA) and sodium 2,3-dimercaptopropane 1-sulfonate (DMPS), which facilitate its excretion from the body. However, these chelating agents are compromised by number of limitations due to their lipophobic nature, particularly in case of chronic poisoning. Combination therapy is a new approach to ensure enhanced removal of metal from the body, reduced doses of potentially toxic chelators, and no redistribution of metal from one organ to another, following chronic metal exposure. The present study attempts to investigate dose-related effects of two thiol chelators, DMSA and one of its new analogues, monoisoamyl dimercaptosuccinic acid (MiADMSA), when administered in combination with the aim of achieving normalization of altered biochemical parameters suggestive of oxidative stress and depletion of inorganic arsenic following chronic arsenic exposure. Twenty-five adult male Wistar rats were given 25 ppm arsenic for 10 weeks followed by chelation therapy with the above chelating agents at a dose of 0.3 mmol/kg (orally) when administered individually or 0.15 mmol/kg and 0.3 mmol/kg (once daily for 5 consecutive days), respectively, when administered in combination. Arsenic exposure led to the inhibition of blood δ-aminolevulinic acid dehydratase (ALAD) activity and depletion of glutathione (GSH) level. These changes were accompanied by significant depletion of hemoglobin, RBC and Hct as well as blood superoxide dismutase (SOD) acitivity. There was an increase in hepatic and renal levels of thiobarbituric acid-reactive substances, while GSH:GSSG ratio decreased significantly, accompanied by a significant increase in metallothionein (MT) in hepatocytes. DNA damage based on denaturing polyacrylamide gel electrophoresis revealed significant loss in the integrity of DNA extracted from the liver of arsenic-exposed rats compared to that of normal animals. These changes were accompanied by a significant elevation in blood and soft-tissue arsenic concentration. Co-administration of DMSA and MiADMSA at lower dose (0.15 mmol/kg) was most effective not only in reducing arsenic-induced oxidative stress but also in depleting arsenic from blood and soft tissues compared to other treatments. This combination was also able to repair DNA damage caused following arsenic exposure. We thus recommend combined administration of DMSA and MiADMSA for achieving optimum effects of chelation therapy.     

Combined administration of N-acetylcysteine and monoisoamyl DMSA on tissue oxidative stress during arsenic chelation therapy

The present study deals with the therapeutic potential of combined administration of N-acetylcysteine (NAC) along with monoisoamyl DMSA (MiADMSA) against chronic arsenic poisoning in guinea pigs. Animal were exposed to 50 ppm arsenic in drinking water for 8 mo and subsequently treated for 5 consecutive days with 100 mg/kg NAC (orally) and MiADMSA (intraperitoneally), individually or in combination (50 mg/kg each). Arsenic exposure produced a significant depletion of blood δ-aminolevulinic acid dehydrate (ALAD) activity, increased the blood zinc protoporphyrin (ZPP) level, and reduced blood and liver glutathione (GSH) levels in guinea pigs. Hepatic oxidized glutathione (GSSG) and thiobarbituric acid reactive substance (TBARS) levels showed a marked increase, whereas hepatic alkaline phosphatase (ALP) activity decreased and acid phosphatase (ACP) activity increased on arsenic exposure. Significant depletion of liver transaminase activities on arsenic exposure suggests organ injury. Administration of MiADMSA, alone and in combination with NAC after arsenic exposure, was able to significantly enhance hepatic GSH and to reduce GSSG and TBARS levels compared to the arsenic control. Biochemical variables indicative of liver injury generally remained insensitive to any of these treatments. The recoveries in parameters indicative of oxidative stress were more marked in guinea pigs treated with combined administration of NAC and MiADMSA than monotherapy. Interestingly, there was a more pronounced depletion of arsenic from blood and tissues after combined treatment with NAC plus MiADMSA than MiADMSA. Blood and tissues copper, zinc, iron, and calcium concentrations showed a significant increase after arsenic exposure, which showed improvement, particularly after combined administration of MiADMSA and NAC. Based on these data, a proposal can be made that greater effectiveness in chelation treatment against chronic arsenic poisoning (i.e., turnover in the oxidative stress and removed of arsenic from the system) could be achieved by combined administration of an antioxidant (preferably having a thiol moiety) with MiADMSA.     

Physiological responses of Populus przewalskii to oxidative burst caused by drought stress

We measured dry matter accumulation and allocation to the roots, leaf gas exchange, chlorophyll fluorescence, antioxidant enzymes, and ABA and polyamine (PA) contents in Populus przewalskii under three different watering regimes (100, 50, and 25% of the field capacity) to investigate the morphological and physiological responses to water deficit in woody plants. The results showed that drought stress retarded P. przewalskii as evident from a decreased biomass accumulation and the reduced increment of shoot height and basal diameter. Drought stress also affected the biomass partition by higher biomass allocated to the root systems for water uptake. The contents of ABA and PAs especially were increased under stressful conditions. Drought stress caused oxidative burst indicated by the accumulation of peroxide (H₂O₂), and fluorimetric detection also confirmed the increased accumulation of H₂O₂. The antioxidant enzymes, including superoxide dimutase, peroxidase, ascorbate peroxidase, and reductase, were activated to bring the reactive oxygen species to their homeostasis; however, oxidative damages to lipids, proteins, and membranes were significantly manifested by the increase in total carbonyl (C=O) and electric conductance (EC).     

Walker-256 tumor growth causes oxidative stress in rat brain

The elevated rate of oxygen consumption and high amount of polyunsaturated fatty acids make the central nervous system vulnerable to oxidative stress. The effect of Walker-256 tumor growth on oxi-reduction indexes in the hypothalamus (HT), cortex (CT), hippocampus (HC) and cerebellum (CB) of male Wistar rats was investigated. The presence of the tumor caused an increase in thiobarbituric acid reactant substances (TBARs) in the HT, CB and HC. Due to tumor growth, the activity of glucose-6-phosphate dehydrogenase increased in the HT and CB, whereas citrate synthase activity was reduced in the HT, CT and CB. Therefore, the potential for generation of reducing power is increased in the cytosol and decreased in the mitochondria of various brain regions of Walker-256 tumor-bearing rats. These changes occurred concomitantly with an unbalance in the brain enzymatic antioxidant system. The tumor decreased the activities of catalase in the HT and CB and of glutathione peroxidase in the HT, CB and HC, and raised the CuZn-superoxide dismutase activity in the HT, CB and HC. These combined findings indicate that Walker-256 tumor growth causes oxidative stress in the brain.     

Compensatory growth and oxidative stress in a damselfly

Physiological costs of compensatory growth are poorly understood, yet may be the key components in explaining why growth rates are typically submaximal. Here we tested the hypothesized direct costs of compensatory growth in terms of oxidative stress. We assessed oxidative stress in a study where we generated compensatory growth in body mass by exposing larvae of the damselfly Lestes viridis to a transient starvation period followed by ad libitum food. Compensatory growth in the larval stage was associated with higher oxidative stress (as measured by induction of superoxide dismutase and catalase) in the adult stage. Our results challenge two traditional views of life-history theory. First, they indicate that age and mass at metamorphosis not necessarily completely translate larval stress into adult fitness and that the observed physiological cost may explain hidden carry-over effects. Second, they support the notion that costs of compensatory growth may be associated with free-radical-mediated trade-offs and not necessarily with resource-mediated trade-offs.     

Recovery of bean plants from boron-induced oxidative damage by zinc supply

The effects of zinc on growth, boron uptake, lipid peroxidation, membrane permeability (MP), lypoxygenase (LOX) activity, proline and H₂O₂ accumulation, and the activities of major antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX)) in bean plants were investigated under greenhouse conditions. Treatments consisted of control, 20 mg/kg B, and 20 mg/kg B plus 20 mg/kg Zn. When the plants were grown with 20 mg/kg Zn, B toxicity was less severe. Zinc supplied to soil counteracted the deleterious effects of B on root and shoot growth. Excess B significantly increased and Zn treatment reduced B concentrations in shoot and root tissues. Applied Zn increased the Zn concentration in the roots and shoots. While the concentrations of H₂O₂ and proline were increased by B toxicity, their concentrations were decreased by Zn supply. Boron toxicity increased the MP, malondialdehyde content, and LOX activity in excised bean leaves. Applied Zn significantly ameliorated the membrane deterioration. Compared with control plants, the activity of SOD was increased while that of CAT was decreased and APX remained unchanged in B-stressed plants. However, application of Zn decreased the SOD and increased the CAT and APX activities under toxic B conditions. It is concluded that Zn supply alleviates B toxicity by preventing oxidative membrane damage. Published in Russian in Fiziologiya Rastenii, 2009, Vol. 56, No. 4, pp. 555–562. This text was submitted by the authors in English.     

The protective effect of melatonin against cypermethrin-induced oxidative stress damage in Spodoptera litura (Lepidoptera: Noctuidae)

Antioxidant enzymes form the first-line defense against free radicals damage in organisms. Their regulation depends mainly on the oxidant and antioxidant status of the cell, given that oxidants are their principal modulators. Therefore, the aim of the present study was to investigate the effect of melatonin on synthetic pyrethroid insecticide-induced antioxidative enzymes activity in Spodoptera litura larvae. In addition, activities of enzymatic antioxidants viz. superoxide dismutase (SOD), glutathione S-transferase (GST), catalase (CAT), glutathione reductase (GR), α, β-esterase, and acetylcholine esterase (AChE) were assessed. There was no significant change in GST levels in the melatonin-treated groups. Melatonin modulates cypermethrin-induced changes in the activities of esterase and AChE, whereas SOD, CAT, and GR activity was significantly increased in melatonin-treated samples when compared to control. In conclusion, the results of the current study revealed that SP toxicity activated oxidant systems in all antioxidant systems in some tissues of insects. Melatonin administration led to a marked increase in antioxidant activity and inhibited GST and AChE in most of the tissues studied.     

Inorganic arsenic compounds cause oxidative damage to DNA and protein by inducing ROS and RNS generation in human keratinocytes

Arsenic is a naturally occurring element that is present in food, soil, and water. Inorganic arsenic can accumulate in human skin and is associated with increased risk of skin cancer. Oxidative stress due to arsenic exposure is proposed as one potential mode of carcinogenic action. The purpose of this study is to investigate the specific reactive oxygen and nitrogen species that are responsible for the arsenic-induced oxidative damage to DNA and protein. Our results demonstrated that exposure of human keratinocytes to trivalent arsenite caused the generation of 8-hydroxyl-2′-deoxyguanine (8-OHdG) and 3-nitrotyrosine (3-NT) in a concentration- and time-dependent manner. Pentavalent arsenate had similar effects, but to a significantly less extent. The observed oxidative damage can be suppressed by pre-treating cells with specific antioxidants. Furthermore, we found that pre-treating cells with Nω-nitro-l-arginine methyl ester (l-NAME), an inhibitor of nitric oxide synthase (NOS), or with 5,10,15,20-tetrakis (N-methyl-4′-pyridyl) porphinato iron (III) chloride (FeTMPyP), a decomposition catalyst of peroxynitrite, suppressed the generation of both 8-OHdG and 3-NT, which indicated that peroxynitrite, a product of the reaction of nitric oxide and superoxide, played an important role in arsenic-induced oxidative damage to both DNA and protein. These findings highlight the involvement of peroxynitrite in the molecular mechanism underlying arsenic-induced human skin carcinogenesis.     

Protoporphyrin IX and oxidative stress

The short- and long-term pro-oxidant effect of protoporphyrin IX (PROTO) administration to mice was studied in liver. A peak of liver porphyrin accumulation was found 2 h after the injection of PROTO (3.5 mg/kg, i.p.); then the amount of porphyrins diminished due to biliar excretion. After several doses of PROTO (1 dose every 24 h up to 5 doses) a sustained enhancement of liver porphyrins was observed. The activity of δ-amino-levulinic acid synthetase was induced 70–90% over the control values 4 h after the first injection of PROTO and stayed at these high levels throughout the period of the assay. Administration of PROTO induced rapid liver damage, involving lipid peroxidation. Hepatic GSH content was increased 2 h after the first injection of PROTO, but then decreased below the control values which were maintained after several doses of porphyrin. After a single dose of PROTO, Cu-Zn superoxide dismutase (SOD) was rapidly induced, suggesting that superoxide radicals had been generated. Increased levels of hydrogen peroxide coming from the reaction catalyzed by SOD and lipid peroxides as a consequence of membrane peroxidation, induced the activity of catalase and glutathione peroxidase (GPx), while decreased GSH levels induced glutathione reductase (GRed) activity. However after 5 doses of PROTO, the activity of SOD was reduced reaching control values. GPx and catalase activities slowly went down, while GRed continued increasing as long as the levels of GSH were kept very low. TBARS values, although lower than those observed after a single dose of PROTO, remained above control values; Glutathione S-transferase activity was instead greatly diminished, indicating sustained liver damage.

Our findings would indicate that accumulation of PROTO in liver induces oxidative stress, leading to rapid increase in the activity of the antioxidant enzymes to avoid or revert liver damage. However, constant accumulation of porphyrins provokes a liver damage so severe that the antioxidant system is compromised.     

Nickel exposure and plasma levels of biomarkers for assessing oxidative stress in nickel electroplating workers

Context: The mechanism of nickel-induced pathogenesis remains elusive.

Objective: To examine effects of nickel exposure on plasma oxidative and anti-oxidative biomarkers.

Materials and methods: Biomarker data were collected from 154 workers with various levels of nickel exposure and from 73 controls. Correlations between nickel exposure and oxidative and anti-oxidative biomarkers were determined using linear regression models.

Results: Workers with a exposure to high nickel levels had significantly lower levels of anti-oxidants (glutathione and catalase) than those with a lower exposure to nickel; however, only glutathione showed an independent association after multivariable adjustment.

Discussion and conclusion: Exposure to high levels of nickel may reduce serum anti-oxidative capacity.     

Manipulation of oxidative stress responses as a strategy to generate stress-tolerant crops. From damage to signaling to tolerance

Plants exposed to hostile environmental conditions such as drought or extreme temperatures usually undergo oxidative stress, which has long been assumed to significantly contribute to the damage suffered by the organism. Reactive oxygen species (ROS) overproduced under stress conditions were proposed to destroy membrane lipids and to inactivate proteins and photosystems, ultimately leading to cell death. Accordingly, considerable effort has been devoted, over the years, to improve stress tolerance by strengthening antioxidant and dissipative mechanisms. Although the notion that ROS cause indiscriminate damage in vivo has been progressively replaced by the alternate concept that they act as signaling molecules directing critical plant developmental and environmental responses including cell death, the induction of genes encoding antioxidant activities is commonplace under many environmental stresses, suggesting that their manipulation still offers promise. The features and consequences of ROS effects depend on the balance between various interacting pathways including ROS synthesis and scavenging, energy dissipation, conjugative reactions, and eventually reductive repair. They represent many possibilities for genetic manipulation. We report, herein, a comprehensive survey of transgenic plants in which components of the ROS-associated pathways were overexpressed, and of the stress phenotypes displayed by the corresponding transformants. Genetic engineering of different stages of ROS metabolism such as synthesis, scavenging, and reductive repair revealed a strong correlation between down-regulation of ROS levels and increased stress tolerance in plants grown under controlled conditions. Field assays are scarce, and are eagerly required to assess the possible application of this strategy to agriculture.     

Uptake by rice seedlings and in-plant degradation of atrazine as influenced by the oxidative stress induced by added arsenic or phosphate deficiency

The uptake and degradation of atrazine (ATR) by rice seedlings (Oryza sativa L.) was investigated with and without arsenate and phosphate nutrient in the cultured solution over a period of 48 h. The hydrogen peroxide (H₂O₂) contents in plants under different treatments were measured to evaluate the oxidative stress of the plant cell and its influence on the plant uptake and degradation of ATR. Results indicated that the ATR levels and main degradation products, deethylatrazine (DEA) and deisopropylatrazine (DIA), in plants varied significantly in different treatments. Added arsenate in solution increased the level of DEA and the ratios of DEA to the total (ATR, DEA, and DIA) in roots, while it either increased or decreased the H₂O₂ content in roots. Added arsenate increased the ratios of degradation products to the total in shoots, which corresponded to the 110%–285% increase of the H₂O₂ content. In phosphate-deficient systems, the H₂O₂ contents in shoots increased significantly, especially when exposed to a low level of ATR while the ratios of DIA and DEA to the total in shoots increased. The oxidative stress in rice seedlings induced by arsenic coexisting with ATR and by phosphate deficiency affected the plant uptake and degradation of ATR.     

Oxidative stress induced by lead, cadmium and arsenic mixtures: 30-day, 90-day, and 180-day drinking water studies in rats: An overview

Humans are frequently exposed to combinations of lead (Pb), cadmium (Cd) and Arsenic (As) but there is a paucity of actual data on the molecular effects of these agents at low dose levels. The present factorial design studies were undertaken in rats to examine the effects of these agents at LOEL dose levels on a number of molecular parameters of oxidative stress in hematopoietic and renal organ systems following oral exposure in drinking water at 30, 90 and 180 day time points. Results of these studies demonstrated dynamic, time-dependent alterations in both molecular targets and inducible oxidative stress protective systems in target cell populations. In general, cellular protective systems, which protected against oxidative damage at the 90 day time point, appeared to be finite such that molecular manifestations of oxidative stress became statistically significant at the 180 day time point for several of the combination exposure groups. These data demonstrate the importance of duration of exposure in assessing the toxic potential of Pb, Cd and As mixtures at low dose levels.     

Oxidative stress in silicosis: Evidence for the enhanced clearance of free radicals from whole lungs

We hypothesized that reactive oxygen species (ROS) may be involved in the pathogenesis of silicosis. To investigate ROS' dependent pathophysiological processes during silicosis we studied the kinetic clearance of instilled stable nitroxide radicals (TEMPO). Antioxidant enzymes' superoxide dismutase (SOD) and glutathione peroxidase (GPx), and lipid peroxidation were also studied in whole lungs of rats exposed to crystalline silica (quartz) and sham exposed controls. Low frequency L-band electron spin resonance spectroscopy was used to measure the clearance of TEMPO in whole-rat lungs directly. The clearance of TEMPO followed first order kinetics showing significant differences in the rate for clearance between the diseased and sham exposed control lungs. Comparison of TEMPO clearance rates in the sham exposed controls and silicotic rats showed an oxidative stress in the rats exposed to quartz. Studies on the antioxidant enzymes SOD and GPx in the lungs of silicotic and sham exposed animals supported the oxidative stress and accelerated clearance of TEMPO by up regulated levels of enzymes in quartz exposed animals. Increased lipid peroxidation potential in the silicotics also supported a role for enhanced generation of ROS in the pathogenesis of silica-induced lung injury. These in vivo experiments directly demonstrate, for the first time, that silicotic lungs are in a state of oxidative stress and that increased generation of ROS is associated with enhanced levels of oxidative enzymes and lipid peroxidation. This technique offers great promise for the elucidation of ROS induced lung injury and development of therapeutic strategies for the prevention of damage.