Rate Of Reactive Oxygen Species Formation Research Articles

The study of the protective effect of mitochondrial uncouplers during acute toxicity of the fungicide difenoconazole in different organs of mice.

Pesticides represent a serious problem for agricultural workers due to their neurotoxic effects. The aim of this study was to evaluate the ability of pharmacological oxidative phosphorylation uncouplers to reduce the effect of the difenoconazole fungicide on mitochondrial DNA (mtDNA) of various organs in mice. Injections of difenoconazole caused cognitive deficits in mice, and the protonophore 2,4-dinitrophenol (2,4-DNP) and Azur I (AzI), a demethylated metabolite of methylene blue (MB), prevented the deterioration of cognitive abilities in mice induced by difenoconazole. Difenoconazole increased the rate of reactive oxygen species (ROS) production, likely through inhibition of complex I of the mitochondrial respiratory chain. After intraperitoneal administration of difenoconazole lungs, testes and midbrain were most sensitive to the accumulation of mtDNA damage. In contrast, the cerebral cortex and hippocampus were not tolerant to the effects of difenoconazole. The protonophore 2,4-DNP reduced the rate of ROS formation and significantly reduced the amount of mtDNA damage caused by difenoconazole in the midbrain, and partially, in the lungs and testes. MB, an alternative electron carrier capable of bypassing inhibited complex I, had no effect on the effect of difenoconazole on mtDNA, while its metabolite AzI, a demethylated metabolite of MB, was able to protect the mtDNA of the midbrain and testes. Thus, mitochondria-targeted therapy is a promising approach to reduce pesticide toxicity for agricultural workers.

Effects of Different Bone Substitutes on Reactive Oxygen Species Release in Leukocytes in Vitro: A Pilot Study.

To evaluate the formation of reactive oxygen species in human leukocytes promoted by bone substitutes that are different in origin and morphology used for jawbone tissue regeneration. This preclinical prospective randomized crossover study involved 10 subjects, from whom venous blood samples were taken. Leukocytes were separated and standardized. Sixty experimental samples consisted of leukocytes incubated with allogeneic, xenogeneic, or alloplastic bone substitutes at different bone weights (12.5 and 25 mg). The control samples consisted only of incubated leukocytes. Reactive oxygen species were quantitatively determined with the fluorimetric method. Statistical analysis was carried out using SPSS 23 software. The highest average reactive oxygen species values were obtained in the allogeneic bone substitute group (P < .05), while the xenogeneic bone substitute group and control group presented equal reactive oxygen species formation rates (P > .05). A proportional difference (P < .05) of reactive oxygen species emission was obtained between different masses of bone substitute in the samples. Allogeneic and alloplastic bone substitutes affect leukocytes and promote reactive oxygen species emission. Xenogeneic bone substitute presents no leukocyte stimulation and maintains anti-inflammatory conditions. Larger bone substitute mass provokes greater oxidative stress.

Molecular-level effects on cell membrane models to explain the phototoxicity of gold shell-isolated nanoparticles to cancer cells

Metallic nanoparticles are promising agents for photothermal cancer therapy (PTT) owing to their photostability and efficient light-to-heat conversion, but their possible aggregation remains an issue. In this paper, we report on the photoinduced heating of gold shell-isolated nanoparticles (AuSHINs) in in vitro experiments to kill human oropharyngeal (HEp-2) and breast (BT-474 and MCF-7) carcinoma cells, with cell viability reducing below 50 % with 2.2 × 1012 AuSHINs/mL and 6 h of incubation. This toxicity to cancer cells is significantly higher than in previous works with gold nanoparticles. Considering the AuSHINs dimensions we hypothesize that cell uptake is not straightforward, and the mechanism of action involves accumulation on phospholipid membranes as the PTT target for photoinduced heating and subsequent generation of reactive oxygen species (ROS). Using Langmuir monolayers as simplified membrane models, we confirmed that AuSHINs have a larger effect on 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS), believed to represent cancer cell membranes, than on 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) taken as representative of healthy eukaryotic cells. In particular, data from polarization-modulated infrared reflection absorption spectroscopy (PM-IRRAS) revealed an increased conformational order of DOPS tails due to the stronger adsorption of AuSHINs. Furthermore, light irradiation reduced the stability of AuSHINs containing DOPC and DOPS monolayers owing to oxidative reactions triggered by ROS upon photoinduced heating. Compared to DOPC, DOPS lost nearly twice as much material to the subphase, which is consistent with a higher rate of ROS formation in the vicinity of the DOPS monolayer.

Healthy Aging: Antioxidants, Uncouplers and/or Telomerase?

The free radical theory of aging was proposed in 1956. Although it does not fully describe the mechanisms of aging, it is generally accepted that reactive oxygen species (ROS) are one of the pathogenetic factors in aging and, in particular, in the development of pathologies associated with aging. The main source of ROS in the cell is mitochondria. Antioxidants directed to mitochondria have a positive effect, but have low efficiency. The problem is that increased amounts of antioxidants disrupt normal cellular redox reactions, and a low amount of antioxidants is not able to seriously affect the processes. Protection against ROS may be more effective if the rate of ROS formation is reduced. There is a natural mitochondrial uncoupling process that significantly reduces ROS production. The weak uncoupler dinitrophenol (DNP) prolongs the life span of mice, reduces traumatic brain damage, and inhibits the development of a number of neurodegenerative diseases. Unfortunately, DNP has a number of disadvantages that hinder its practical use. Uncoupling of oxidative phosphorylation by free fatty acids is a natural mechanism, the activation of which can be used in medicine. The third (after antioxidants and uncouplers), but so far little studied, method of reducing ROS is telomerase, which, under conditions of oxidative stress, is transported into the mitochondria and improves cell survival by reducing ROS production.

The effect of trematode infection on the markers of oxidative stress in the offspring of the freshwater snail Lymnaea stagnalis.

Most invertebrate species exhibit immunological responses that can inactivate and eliminate penetrating parasites. Such immune responses in particular involve the formation of potentially toxic reactive oxygen species (ROS). We explored the immune capabilities of the first-generation (F1) offspring of naturally infected freshwater snails, Lymnaea stagnalis, in response to infection by trematode cercariae under laboratory conditions. The rates of ROS formation and peroxidase activity in the hemolymph of the F1 offspring of L. stagnalis parents infected by an asexual stage of trematodes were significantly higher than in F1 offspring of uninfected parents. Compared to offspring from uninfected parents, the growth rate of F1 snails from infected parents was higher, but survival was lower. After infection of F1 snails by trematode cercariae of Echinoparyphium aconiatum under laboratory conditions, the rate of ROS formation and peroxidase activity in the hemolymph of F1 offspring of uninfected parents increased compared to control snails. This pattern persisted throughout the entire 3-week observation period. In contrast, the rate of ROS formation in the hemolymph of F1 snails from infected parents after experimental infection by E. aconiatum cercariae did not differ from controls, and peroxidase activity even decreased. Thus, trematode parthenitae infection of parents could alter the immune response of their offspring.

Voltage gated proton channels modulate mitochondrial reactive oxygen species production by complex I in renal medullary thick ascending limb

Hv1 is a voltage-gated proton channel highly expressed in immune cells where, it acts to maintain NAD(P)H oxidase activity during the respiratory burst. We have recently reported that Hv1 is expressed in cells of the medullary thick ascending limb (mTAL) of the kidney and is critical to augment reactive oxygen species (ROS) production by this segment. While Hv1 is associated with NOX2 mediated ROS production in immune cells, the source of the Hv1 dependent ROS in mTAL remains unknown. In the current study, the rate of ROS formation was quantified in freshly isolated mTAL using dihydroethidium and ethidium fluorescence. Hv1 dependent ROS production was stimulated by increasing bath osmolality and ammonium chloride (NH4Cl) loading. Loss of either p67phox or NOX4 did not abolish the formation of ROS in mTAL. Hv1 was localized to mitochondria within mTAL, and the mitochondrial superoxide scavenger mitoTEMPOL reduced ROS formation. Rotenone significantly increased ROS formation and decreased mitochondrial membrane potential in mTAL from wild-type rats, while treatment with this inhibitor decreased ROS formation and increased mitochondrial membrane potential in mTAL from Hv1−/− mutant rats. These data indicate that NADPH oxidase is not the primary source of Hv1 dependent ROS production in mTAL. Rather Hv1 localizes to the mitochondria in mTAL and modulates the formation of ROS by complex I. These data provide a potential explanation for the effects of Hv1 on ROS production in cells independent of its contribution to maintenance of cell membrane potential and intracellular pH.

Towards predicting intracellular radiofrequency radiation effects.

Recent experiments have reported an effect of weak radiofrequency magnetic fields in the MHz-range on the concentrations of reactive oxygen species (ROS) in living cells. Since the energy that could possibly be deposited by the radiation is orders of magnitude smaller than the energy of molecular thermal motion, it was suggested that the effect was caused by the interaction of RF magnetic fields with transient radical pairs within the cells, affecting the ROS formation rates through the radical pair mechanism. It is, however, at present not entirely clear how to predict RF magnetic field effects at certain field frequency and intensity in nanoscale biomolecular systems. We suggest a possible recipe for interpreting the radiofrequency effects in cells by presenting a general workflow for calculation of the reactive perturbations inside a cell as a function of RF magnetic field strength and frequency. To justify the workflow, we discuss the effects of radiofrequency magnetic fields on generic spin systems to particularly illustrate how the reactive radicals could be affected by specific parameters of the experiment. We finally argue that the suggested workflow can be used to predict effects of radiofrequency magnetic fields on radical pairs in biological cells, which is specially important for wireless recharging technologies where one has to know of any harmful effects that exposure to such radiation might cause.

Mechanistic Approach for Thioridazine-Induced Hepatotoxicity and Potential Benefits of Melatonin and/or Coenzyme Q10 on Freshly Isolated Rat Hepatocytes.

Thioridazine (TZ) is used mainly in the treatment of schizophrenia. However, hepatotoxicity as a life-threatening adverse effect is associated with its clinical use. In this context, we examined the cytotoxic mechanisms of TZ on freshly isolated rat hepatocytes to better understanding of the pathogenesis of TZ-induced hepatotoxicity. Hepatocytes were prepared by the method of collagenase enzyme perfusion via the portal vein. The level of parameters such as cell death, reactive oxygen species (ROS) formation, lipid peroxidation (LPO), mitochondrial membrane potential (MMP), lysosomal membrane integrity and cellular glutathione (GSH) content in TZ-treated and non-treated hepatocytes were determined and the mentioned markers were assessed in the presence of Coenzyme Q10 and/or melatonin. Results showed that TZ caused an increase in ROS formation as well as induction of LPO and GSH depletion. Moreover, mitochondria and lysosomes seem to be targets of TZ-induced toxicity. The administration of Coenzyme Q10 and/or melatonin efficiently decreased the rate of ROS formation, LPO and improved cell viability, MMP, GSH level and lysosome membrane integrity. This study proposes the possible protective role of Coenzyme Q10 and/or melatonin against TZ-induced cellular injury probably through their radical scavenging properties and their effects on mitochondria and lysosomes.

The effect of entomopathogenic fungi Metarhizium robertsii of different virulence on the generation of reactive oxygen species in Galleria mellonella larvae

The toxicity of reactive oxygen species (ROS) plays a significant role in the immune response of insects. Little is known about the effect of the virulence of entomopathogenic fungi on the generation of ROS in a host. The aim of the study was to reveal whether the different levels of virulence cause the different ROS production in insects. The half-lethal dosages of two Metarhizium robertsii strains, of high and low virulence, which have shown a similar survival to Galleria mellonella larvae after treatment, were used in the study. The rates of ROS generation were determined in the cuticle, hemocytes and cell-free hemolymph of G. mellonella larvae 1, 3, 5 days after fungi treatment. We have shown that the level of ROS production in the cuticle and hemolymph of G. mellonella larvae depends on the virulence of the M. robertsii strains. The influence of both fungal strains on the rate of ROS formation in hemocytes was the same throughout the observation period. The host’s defense mechanism was activated in the cuticle under the treatment of both low and high virulent fungi strains. In the hemolymph, the activation of the immune response occurred only after treatment with low virulent strain.

Relation Between Mitochondrial Membrane Potential and ROS Formation.

Mitochondria are considered the main source of reactive oxygen species (ROS) in the cell. For this reason they have been recognized as a source of various pathological conditions as well as aging. Chronic increase in the rate of ROS production is responsible for the accumulation of ROS-associated damages in DNA, proteins, and lipids and may result in progressive cell dysfunctions and, in a consequence, apoptosis, increasing the overall probability of an organism's pathological conditions. The superoxide anion is the main undesired by-product of mitochondrial oxidative phosphorylation. Its production is triggered by a leak of electrons from the mitochondrial respiratory chain and the reaction of these electrons with O2. Superoxide dismutase (MnSOD, SOD2) from the mitochondrial matrix, as well as superoxide dismutase (Cu/ZnSOD, SOD1) present in small amounts in the mitochondrial intramembrane space, converts superoxide anion to hydrogen peroxide, which can be then converted by catalase to harmless H2O.In the chapter we describe a relation between mitochondrial membrane potential and the rate of ROS formation. We present different methods applicable for isolated mitochondria or intact cells. We also present experiments demonstrating that a magnitude and a direction (increase or decrease) of a change in mitochondrial ROS production depend on the metabolic state of this organelle.

Modulation of mitochondrial dysfunction-related oxidative stress in fibroblasts of patients with Leigh syndrome by inhibition of prooxidative p66Shc pathway

The mitochondrial respiratory chain, and in particular, complex I, is a major source of reactive oxygen species (ROS) in cells. Elevated levels of ROS are associated with an imbalance between the rate of ROS formation and the capacity of the antioxidant defense system. Increased ROS production may lead to oxidation of DNA, lipids and proteins and thus can affect fundamental cellular processes. The aim of this study was to investigate the magnitude of intracellular oxidative stress in fibroblasts of patients with Leigh syndrome with defined mutations in complex I. Moreover, we hypothesized that activation of the p66Shc protein (phosphorylation of p66Shc at Ser36 by PKCβ), being part of the oxidative stress response pathway, is partially responsible for the increased ROS production in cells with dysfunctional complex I. Characterization of bioenergetic parameters and ROS production showed that the cellular model of Leigh syndrome is described by increased intracellular oxidative stress and oxidative damage to DNA and proteins, which correlate with increased p66Shc phosphorylation at Ser36. Treatment of patients' fibroblasts with hispidin (an inhibitor of the protein kinase PKCβ), in addition to decreasing ROS production and intracellular oxidative stress, resulted in restoration of complex I activity.

The Use of Dichlorofluorescein as a Probe for Monitoring the Effects of Calcium on ROS Production in Mitochondria

Dichlorofluorescein (DCF) is widely applied for monitoring reactive oxygen species (ROS) production in cells and isolated mitochondria. Our purpose was to compare different approaches to the use of DCF for monitoring ROS formation accompanying Ca2+ transport in isolated mitochondria. Ca2+ effects on ROS production was studied in rat brain and liver mitochondria using two modes of DCF application: the first was preloading of stock mitochondrial suspension with DCF precursor dichlorofluorescein diacetate (DCFDA), and the other was the addition of the aliquots of DCFDA directly to the incubation medium. Calcium increased ROS production in brain and liver mitochondria both under steady-state and non-equilibrium conditions caused by the gradual release of cytochrome c and the gating of the electron flow. Using DCF-preloaded mitochondria we have shown that the rate of ROS formation under steady-state conditions was linearly dependent on the rate of Ca2+-cycling and Ca2+-stimulated respiration. Transition to non-equilibrium state resulted in the non-linear rise in ROS formation, which too exhibited dependence on Ca2+ concentration. While in DCF-preloaded mitochondria DCF fluorescence closely followed the time course of Ca2+ transport, in unloaded mitochondria probe loading interfered with the detection of ROS formation because of continuing change of DCF concentration into matrix space. Based on the experiments, we came to the conclusion that DCF preloaded in mitochondria could serve a suitable probe for monitoring the effects of Ca2+ transport on ROS production.

Inhibitory Effect of Meclofenamate Sodium on Proteasome Activity in the Cardiac Cells and Reversal of its Effect by Antioxidants

Objective: Prolonged use of nonsteroidal anti-inflammatory drugs (NSAIDs) is reported to increase the occurrences of cardiovascular diseases (CVD). Significant proteasome inhibition is also associated with increased incidences of CVD. Hence, the effect of the NSAIDs meclofenamate sodium (MS) and naproxen sodium (NAP) on the proteasome system was investigated in H9c2 rat cardiac cells. Methods: 26S proteasome activity assays, cell viability assays, reactive oxygen species (ROS) assays, and MV151 fluorescent labelling of proteasome active sites were utilized. Results: Decreased 26S β5 chymotrypsin-like proteasome activity (30%) and β1 caspase-like activity (20%) was seen in H9c2 cells treated with 100µM MS for 24h compared to the cells treated with 500 µM NAP or vehicle. Rat heart lysates showed significant decreases in β5 activity in the presence of 50-500 µM MS. Purified proteasomes also showed decreased β5 activity in the presence of 100 µM MS. MV-151 labeling showed that this decrease in activity was not due to direct interaction between MS and the proteasome active sites. Both NSAIDs caused increased ROS levels in H9c2 cells but the rate of ROS formation was significantly higher in cells treated with 100 µM MS than 500 µM NAP after 2hrs. MS (100 µM) but not NAP (500 µM) significantly decreased cell viability. Pretreatment of the cells with antioxidants, 200 µM ascorbic acid and 100 µM tempol, completely reversed the effect of MS on the proteasome and cell viability. Conclusion Our results suggest for the first time that NSAIDs may have an implication in CVD through the direct inhibition of the proteasome system. Interestingly, this inhibitory effect of NSAIDs could be reversed by using antioxidants.

Effect of aging on formation of reactive oxygen species by mitochondria of rat heart

Mitochondrial electron transport chain is thought to be a major source of reactive oxygen species (ROS) during aging. However, this view is supported mainly by accumulation of mitochondrial oxidative damage with age and the exact sites of ROS formation remains unknown. In the present study, we measured rate of ROS formation using 2',7'-dichlorofluorescein (DCF) probe in cardiac mitochondria from adult (6-month-old), old (15-month-old) and senescent (26-month-old) rats. In mitochondria oxidizing complex II substrate, succinate, the rate of ROS formation progressively increased with age. In the presence of complex I inhibitor rotenone or complex III inhibitor antimycin A, the rate ROS formation significantly decreased, but even the combination of inhibitors could not fully prevent generation of ROS. Age-dependent increase of ROS formation was accompanied by a loss of thiol groups, tryptophan degradation and increased lipid peroxidation. These data suggest that in addition to complex I and complex II other mitochondrial sites can contribute to accelerated ROS generation and oxidative damage during aging.

Gas Flow Effect on E. coli and B. subtilis Bacteria Inactivation in Water Using a Pulsed Dielectric Barrier Discharge

This paper presents an experimental study of the inactivation in water of two representative classes of bacteria, Gram-negative Escherichia coli and Gram-positive Bacillus subtilis, using pulsed dielectric barrier discharges (PDBDs) in a coaxial arrangement. To this purpose, an adjustable plasma source supplies 25-kV/500-Hz pulses, 30 μs long, at atmospheric pressure, with total energy consumption estimated at about 100 mJ/pulse. The inactivation effect of a PDBD on these types of microorganisms has been previously studied in dependence on an oxygen gas flow mode (null, continuous, and modulate). The results have shown a significant bacterial reduction rate from 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">8</sup> to 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> cells/mL with E. coli and from 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">7</sup> to 10 <sup xmlns:mml="http://www.w3.org/1998/Math/MathML" xmlns:xlink="http://www.w3.org/1999/xlink">3</sup> cells/mL with B. subtilis. The inactivation effectiveness is substantially similar in both kinds of bacteria, although some data suggest a greater susceptibility of the Gram-negative E. coli to plasma exposure. Plasma diagnostics was carried out using optical emission spectroscopy whereby the OH radical and reactive oxygen species formation rates in solution were found and the level of ozone produced by the discharge was monitored. Finally, a kinetics model was developed to characterize the chemical species taking place in nonthermal plasma inactivation processes in water.

Bacterial Growth Response to Photoactive Quinones

Quinones are known producers of reactive oxygen species (ROS) that may be toxic in natural aquatic environments. In this study, the effects of parent quinones and their photodegradation products on bacterial growth were determined, and photochemical ROS formation rates were measured. Using (3)H-leucine incorporation to measure growth of the bacterium Pseudomonas aeruginosa and natural seawater bacterioplankton, growth inhibition was observed when samples were exposed to dichlone, chloranil and sodium anthraquinone-2-sulfonate (AQ2S). For seawater, compared with other quinones tested, dichlone showed the greatest toxicity in the dark, and AQ2S toxicity was greatest during simultaneous exposure to sunlight. Photodegraded chloranil and dichlone showed decreased toxicity compared with nonirradiated samples. For P. aeruginosa, AQ2S and its photodegradation products showed the greatest toxicity during simultaneous exposure to sunlight. Chloranil photodegradation products showed reduced toxicity compared with the parent compound during simultaneous exposure to sunlight. Dichlone was the only compound to show any toxicity to P. aeruginosa in the dark, and its photodegradation products were more toxic than the parent compound. Based on the results of dark and light controlled experiments measuring bacterial growth and estimated ROS production rates, ROS alone does not account for relative differences in toxicity between these quinones.

Rapid suppression of multidrug resistance of leukemic cells by oxidative srtess

The effect of a short-time (1 h) oxidative stress on multidrug resistance (MDR) of murine leukemic P388VR cells has been investigated. We studied the production of reactive oxygen species (ROS) in cells depending on the composition of medium and the concentration of cells and hydrogen peroxide, as well as the effect of hydrogen peroxide on MDR of cells. MDR was determined from the transport of calcein acetoxymethyl ester out of the cells and from a change in cell sensitivity to vincristine. The amount of ROS arising in cells was determined using 2′,7′-dichlorodihydrofluorescein diacetate (DCFH2-DA). It was shown that the rate of ROS formation in cells decreases after the addition of serum to the medium and with an increase of the cell number. By the action of hydrogen peroxide, the amount of ROS increases directly with its concentration. Oxidative stress generated by 30–300 μM hydrogen peroxide decreases the MDR of the cells. The effect of hydrogen peroxide increases with the treatment duration and concentration of hydrogen peroxide. MDR determined by the criterion of the efflux of calcein ester from cells is completely suppressed after 1-h exposure to 300 μM hydrogen peroxide. At a concentration of hydrogen peroxide of 60 μM and treatment duration of 1 h, the sensitivity of P388VR cells to vincristine increases to reach the sensitivity of the wild-type P388 cells. Rapid (about 1 h) suppression of MDR is caused by inhibition of the activity of transport proteins. MDR decrease induced by oxidative stress can be used in therapy of tumors resistant to anticancer drugs.

Copper-induced oxidative stress in rainbow trout gill cells

Copper is known to pose a serious threat to aquatic organisms. However, the mechanisms of its toxicity still remain unclear. Cu is known to exert its toxicity partly due to the formation of reactive oxygen species (ROS). The purpose of this work was therefore to link the exposure to copper at pH 6 and 7 to cellular formation of ROS and effects like cell viability and genotoxicity using the rainbow trout gill cell line RTgill-W1. To relate effects to bioavailable copper, free Cu 2+ concentrations in the medium were calculated using the programm ChemEQL 3.0. 2′,7′-Dichlorodihydrofluorescein-diacetate (H 2DCF-DA) was used as cell-permeant indicator of ROS formation. Cell viability was assessed using the fluorogenic probe 5-carboxyfluorescein diacetate acetoxymethyl ester (CFDA-AM). DNA strand breaks were assessed using the comet assay, and lipid peroxidation was investigated using the thiobarbituric acid-reactive substances assay (TBARS). Copper treatment resulted in a dose-dependent elevation in cytotoxicity and formation of cellular ROS. Cell viability was significantly reduced at total copper (Cu T) concentrations of 5 μM (corresponding to a free Cu 2+ of 0.11 μM at pH 7) and higher, resulting in an EC 50 of Cu T = 29.2 μM (Cu 2+ = 0.63 μM, pH 7). Neither an impairment concerning the viability of control cells due to growth at pH 6 was observed nor significant differences for cytotoxicity in cells exposed to the same nominal Cu T concentrations at pH 6 compared to pH 7. Cellular ROS concentrations increased significantly and decreased with loss of cell viability. After normalizing ROS formation to cell viability, ROS induction up to 25–35-fold compared to the control was detected, but mainly for rather high concentrations (Cu T ≥ 100 μM; Cu 2+ ≥ 2.2 μM, pH 7). ROS formation rates were slightly higher when cells were exposed to Cu at pH 6 compared to pH 7, correlating with the higher free Cu 2+ concentrations. A significant induction of DNA strand breaks was noted at Cu T of 1 and 2.5 μM with greater effects at pH 6 due to higher free Cu 2+ concentrations than at pH 7. No effects on lipid peroxidation were observed. These results lead to the hypothesis that copper-induced loss in viability and genotoxicity in trout gill cells are partially triggered by the generation of ROS and related to the free Cu 2+.

The single subunit NADH dehydrogenase reduces generation of reactive oxygen species from complex I

Using rat dopaminergic and human neuroblastoma cell lines transduced with the NDI1 gene encoding the internal NADH dehydrogenase (Ndi1) from Saccharomyces cerevisiae, we investigated reactive oxygen species (ROS) generation caused by complex I inhibition. Incubation of non-transduced cells with rotenone elicited oxidative damage to mitochondrial DNA as well as lipid peroxidation. In contrast, oxidative stress was significantly decreased when the cells were transduced with NDI1. Furthermore, mitochondria from the NDI1-transduced cells showed a suppressed rate of ROS formation by the complex I inhibitors. We conclude that the Ndi1 enzyme is able to suppress ROS overproduction from defective complex I.

Point:Counterpoint: Hypoxic pulmonary vasoconstriction is/is not mediated by increased production of reactive oxygen species

This series of debates was initiated for the Journal of Applied Physiology because we believe an important means of searching for truth is through debate where contradictory viewpoints are put forward. This dialectic process whereby a thesis is advanced, then opposed by an antithesis, with a