Effect of antioxidants on chemiluminescence produced by reactive oxygen species

Luminol chemiluminescence was used to evaluate the scavenging of superoxide, hydroxyl and alkoxy radicals by four antioxidants: dipyridamole, diethyldithiocarbamic acid, (+)catechin, and ascorbic acid. Different concentrations of these compounds were compared with well-known oxygen radical scavengers in their capacity to inhibit the chemiluminescence produced in the reaction between luminol and specific oxygen radicals. Hydroxyl radicals were generated using the Fenton reaction and these produced chemiluminescence which was inhibited by diethyldithiocarbamate. Alkoxy radicals were generated using the reaction of tert-butyl hydroperoxide and ferrous ion and produced chemiluminescence which was inhibited equally by all of the compounds tested. For the determination of superoxide scavengers we describe a new, simple, economic, and rapid chemiluminescence method consisting of the reaction between luminol and horseradish peroxidase (HRP). With this method it was found that 40 nmol/l dipyridamole, 0.18 μmol/l ascorbic acid, 0.23 μmol/l (+)catechin, and 3 μmol/l diethyldithiocarbamic acid are equivalent to 3.9 ng/ml superoxide dismutase (specific scavenger of superoxide) in causing the same degree of chemiluminescence inhibition. These results not only indicated that the antioxidative properties of these compounds showed different degrees of effectiveness against a particular radical but also that they may exert their action against more than one radical.     

Scavenging effects of phenolic compounds on reactive oxygen species

Phenolic compounds are widely present in plants and they have received considerable attention due to their antioxidant property. In this article we report the results of a study of the reactivity of 10 selected phenolics (sesamol, three phenolic acids, three flavonols, one flavone, and two flavanones) with superoxide anion radical (O(2) (*)), hydroxyl radical (HO(*)) and singlet oxygen ((1)O(2)). The following generators of reactive oxygen species were used: 18-crown-6/KO(2)/dimethylsulfoxide (DMSO) or hypoxanthine/xanthine oxidase as sources of O(2) (*), the Fenton reaction carried out in a sodium trifluoroacetate (pH 6.15) for HO(*), and a mixture of alkaline aqueous H(2)O(2) and cobalt ions for (1)O(2). We have employed chemiluminescence, electron spin resonance spin trapping, and spectrophotometry techniques to examine an antioxidative property. All tested compounds acted as scavengers of various reactive oxygen species. The reactivity indexes (beta) for the reaction of the phenolic compounds with HO(*) were calculated.     

Chilling-enhanced photooxidation: The production,action and study of reactive oxygen species produced during chilling in the light

Chilling-enhanced photooxidation is the light- and oxygen-dependent bleaching of photosynthetic pigments that occurs upon the exposure of chilling-sensitive plants to temperatures below approximately 10 °C. The oxidants responsible for the bleaching are the reactive oxygen species (ROS) singlet oxygen (¹O₂), superoxide anion radical (O ₂ ,hydrogen peroxide (H₂O₂), the hydroxyl radical (OH·), and the monodehydroascorbate radical (MDA) which are generated by a leakage of absorbed light energy from the photosynthetic electron transport chain. Cold temperatures slow the energy-consuming Calvin-Benson Cycle enzymes more than the energy-transducing light reactions, thus causing leakage of energy to oxygen. ROS and MDA are removed, in part, by the action of antioxidant enzymes of the Halliwell/Foyer/Asada Cycle. Chloroplasts also contain high levels of both lipid- and water-soluble antioxidants that act alone or in concert with the HFA Cycle enzymes to scavenge ROS. The ability of chilling-resistant plants to maintain active HFA Cycle enzymes and adequate levels of antioxidants in the cold and light contributes to their ability to resist chilling-enhanced photooxidation. The absence of this ability in chilling-sensitive species makes them susceptible to chilling-enhanced photooxidation. Chloroplasts may reduce the generation of ROS by dissipating the absorbed energy through a number of quenching mechanisms involving zeaxanthin formation, state changes and the increased usage of reducing equivalents by other anabolic pathways found in the stroma. During chilling in the light, ROS produced in chilling-sensitive plants lower the redox potential of the chloroplast stroma to such a degree that reductively-activated regulatory enzymes of the Calvin Cycle, sedohepulose 1,7 bisphosphatase (EC 3.1.3.37) and fructose 1,6 bisphosphatase (EC 3.1.3.11), are oxidatively inhibited. This inhibition is reversible in vitro with a DTT treatment indicating that the enzymes themselves are not permanently damaged. The inhibition of SBPase and FBPase may fully explain the inhibition in whole leaf gas exchange seen upon the rewarming of chilling-sensitive plants chilled in the light. Methods for the study of ROS in chilling-enhanced photooxidation and challenges for the future are discussed.Abbreviations ASP ascorbate-specific peroxidase - -TH reduced -tocopherol - DTT dithiothreitol - FBP fructose 1,6 bisphosphate - FBPase fructose 1,6 bisphosphatase (EC 3.1.3.11) - HFA Cycle the Halliwell/Foyer/Asada Cycle responsible for the enzymatic removal of ROS in the chloroplast stroma - MDA monodehydroascorbate radical - MDAR monodehydroascorbate reductase - ROS reactive oxygen species - SBP sedohepulose 1,7 bisphosphate - SBPase sedohepulose 1,7 bisphosphatase (EC 3.1.3.37) - SOD superoxide dismutase     

Lidocaine: a hydroxyl radical scavenger and singlet oxygen quencher

Lidocaine, a local anaesthetic, has been shown to reduce ventricular arrhythmias associated with myocardial infarction and ischemic myocardial injury and its protective effects has been attributed to its membrane stabilizing properties. Since oxygen radicals are known to be produced during ischemia induced tissue damage, we have investigated the possible antioxidant properties of lidocaine and found that lidocaine does not scavenge 0₂ ^–· radicals at 1 to 20 mM concentrations. However, lidocaine was found to be a potent scavenger of hydroxyl radicals and singlet oxygen. Hydroxyl radicals were produced in a Fenton type reaction and detected as DMPO-OH adducts by electron paramagnetic resonance spectroscopic techniques. Lidocaine inhibited DMPO-OH adduct formation in a dose dependent manner. The amount of lidocaine needed to cause 50% inhibition of that rate was found to be approximately 80 M and at 300 M concentration it virtually eliminated the DMPO-OH adduct formation. The production of OH-dependent TBA reactive products of deoxyribose was also inhibited by lidocaine in a dose dependent manner. Lidocaine was also found to inhibit the ¹O₂-dependent 2,2,6,6-tetramethylpiperidine N-oxyl (TEMPO) formation in a dose dependent manner. ¹O₂ was produced in a photosensitizing system using Rose Bengal or Methylene Blue as photosensitizers and was detected as TEMP-¹O₂ adduct by EPR spectroscopy. The amount of lidocaine required to cause 50% inhibition of TEMP-¹O₂ adduct formation was found to be 500 M. These results suggest that the protective effect of lidocaine on myocardial injury may, in part, be due to its reactive oxygen scavenging properties. These results may also explain the membrane stabilizing actions of lidocaine by scavenging OH · and ¹O₂ that are implicated in membrane lipid peroxidation.     

Effect of low-frequency alternating magnetic fields on the rate of biochemical reactions proceeding with formation of reactive oxygen species

It is (theoretically) shown by an example of the reaction of a radical with an oxygen molecule that the alternating component of a combined weak magnetic field affects the rate constants of chemical reactions. The mechanism of transduction of a weak magnetic perturbation from the primary receptor of the field to experimentally observed biological effects is followed. It is stated that the external magnetic field alters the initial population of energy levels. The magnitude of these changes depends on the field parameters. The exposure to an alternating field with proper parameters can substantially increase the concentration of reactive oxygen species in biological systems. By controlling their concentration by means of weak magnetic field, it is possible to affect the key links of metabolism.     

Isolated respiring heart mitochondria release reactive oxygen species in states 4 and 3

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37°C, the rate of H₂O₂ release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03–0.15?mg?protein/ml), to decrease (0.2–0.5?mg?protein/ml) and to be negligible (over 0.5?mg?protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2?mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1?mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.     

Isolated respiring heart mitochondria release reactive oxygen species in states 4 and 3

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37°C, the rate of H₂O₂ release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03-0.15 mg protein/ml), to decrease (0.2-0.5 mg protein/ml) and to be negligible (over 0.5 mg protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2 mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1 mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.     

Analysis of dichlorodihydrofluorescein and dihydrocalcein as probes for the detection of intracellular reactive oxygen species

Dihydrocalcein (H₂-calcein) is recommended as a superior probe for intracellular radical (ROS) detection as different to dichlorodihydrofluorescein (H₂-DCF), its oxidation product calcein is thought not to leak out of cells. We determined whether H₂-calcein is a useful tool to measure ROS in vascular smooth muscle cells. In vitro, both compounds were oxidized by peroxynitrite, hydroxyl radicals and peroxidase, but not hydrogen peroxide or nitric oxide. The intracellular half-life of calcein was several hours whereas that of DCF was approximately 5 min. Intracellular ROS, as generated by the angiotensin II (Ang II)-activated NADPH oxidase, did not increase the oxidation of H₂-calcein but increased the oxidation of H₂-DCF by approximately 50%. Similar changes were detected using electron spin resonance spectroscopy. Inhibition of the NADPH oxidase using gp91ds-tat prevented the Ang II-induced increase in DCF fluorescence, without affecting cells loaded with H₂-calcein. Diphenylene iodonium (DPI), which inhibits all flavin-dependent enzymes, including those in the respiratory chain, had little effect on the basal but prevented the Ang II-induced oxidation of H₂-DCF. In contrast, DPI inhibited H₂-calcein oxidation in non-stimulated cells by almost 50%. Blockade of respiratory chain complex I inhibited H₂-calcein oxidation, whereas inhibitors of complex III were without effect. Calcein accumulated in the mitochondria, whereas DCF was localized in the cytoplasm. In submitochondrial particles, H₂-calcein, but not H₂-DCF inhibited complex I activity.

These observations indicate that H₂-DCF is an indicator for intracellular ROS, whereas the oxidation of H₂-calcein most likely occurs as a consequence of direct electron transfer to mitochondrial complex I.     

Generator-specific targets of mitochondrial reactive oxygen species

To understand the role of reactive oxygen species (ROS) in oxidative stress and redox signaling it is necessary to link their site of generation to the oxidative modification of specific targets. Here we have studied the selective modification of protein thiols by mitochondrial ROS that have been implicated as deleterious agents in a number of degenerative diseases and in the process of biological aging, but also as important players in cellular signal transduction. We hypothesized that this bipartite role might be based on different generator sites for “signaling” and “damaging” ROS and a directed release into different mitochondrial compartments. Because two main mitochondrial ROS generators, complex I (NADH:ubiquinone oxidoreductase) and complex III (ubiquinol:cytochrome c oxidoreductase; cytochrome bc₁ complex), are known to predominantly release superoxide and the derived hydrogen peroxide (H₂O₂) into the mitochondrial matrix and the intermembrane space, respectively, we investigated whether these ROS generators selectively oxidize specific protein thiols. We used redox fluorescence difference gel electrophoresis analysis to identify redox-sensitive targets in the mitochondrial proteome of intact rat heart mitochondria. We observed that the modified target proteins were distinctly different when complex I or complex III was employed as the source of ROS. These proteins are potential targets involved in mitochondrial redox signaling and may serve as biomarkers to study the generator-dependent dual role of mitochondrial ROS in redox signaling and oxidative stress.     

Copper increases the damage to DNA and proteins caused by reactive oxygen species

Copper [Cu(II)] is an ubiquitous transition and trace element in living organisms. It increases reactive oxygen species (ROS) and free-radical generation that might damage biomolecules like DNA, proteins, and lipids. Furthermore, ability of Cu(II) greatly increases in the presence of oxidants. ROS, like hydroxyl (·OH) and superoxide (·O₂) radicals, alter both the structure of the DNA double helix and the nitrogen bases, resulting in mutations like the AT→GC and GC→AT transitions. Proteins, on the other hand, suffer irreversible oxidations and loss in their biological role. Thus, the aim of this investigation is to characterize, in vitro, the structural effects caused by ROS and Cu(II) on bacteriophage λ DNA or proteins using either hydrogen peroxide (H₂O₂) or ascorbic acid with or without Cu(II). Exposure of DNA to ROS-generating mixtures results in electrophoretic (DNA breaks), spectrophotometric (band broadening, hypochromic, hyperchromic, and bathochromic effects), and calorimetric (denaturation temperature [T _d], denaturation enthalpy [ΔH], and heat capacity [C _p] values) changes. As for proteins, ROS increased their thermal stability. However, the extent of the observed changes in DNA and proteins were distinct, depending on the efficiency of the systems assayed to generate ROS. The resulting effects were most evident when Cu(II) was present. In summary, these results show that the ROS, ·O₂ and ·OH radicals, generated by the Cu(II) systems assayed deeply altered the chemical structure of both DNA and proteins. The physiological relevance of these structural effects should be further investigated.     

Scavenging of reactive oxygen species by novel indolin-2-one and indoline-2-thione derivatives

The antioxidant behavior of a series of new synthesized substituted indoline-2-ones and indolin-2-thiones was investigated in this study using an oxygen radical absorbance capacity assay (ORAC(ROO*-) and 2,2'-azobis(2-amidino-propane) dihydrochloride (AAPH) as the radical generator; system generating superoxide anion radical, O2*- (18-crown-6/KO(2)/DMSO), and the Fenton-like reaction [Co(II) + H(2)O(2) --> Co(III) + HO(*) + HO(-)]. Measurements were done using fluorescence, chemiluminescence methods, and a deoxyribose assay based on the spectrophotometry method, respectively. The results obtained indicated that the examined indoline derivatives had effective activities as radical scavengers and may be considered as an effective source for combating oxidative damage.     

Light-dependent generation of reactive oxygen species in cell culture media

Cell culture media (RPMI 1640, Dulbecco’s Minimal Essential Medium and yeast extract-peptone-glucose medium) were found to oxidize dichlorodihydrofluorescein diacetate and dihydrorhodamine 123, and to generate spin adduct of 5,5′-dimethyl-1-pyrroline N-oxide, which indicates formation of reactive oxygen species (ROS). The production of ROS was light dependent. The main component of the media responsible for the generation of ROS was riboflavin, but tryptophan, tyrosine, pyridoxine, and folic acid enhanced the effect of riboflavin. These observations point to exposure of cells to ROS under in vitro culture conditions.     

Caveolin-1 is a negative regulator of NADPH oxidase-derived reactive oxygen species

Changes in the expression and function of caveolin-1 (Cav-1) have been proposed as a pathogenic mechanism underlying many cardiovascular diseases. Cav-1 binds to and regulates the activity of numerous signaling proteins via interactions with its scaffolding domain. In endothelial cells, Cav-1 has been shown to reduce reactive oxygen species (ROS) production, but whether Cav-1 regulates the activity of NADPH oxidases (Noxes), a major source of cellular ROS, has not yet been shown. Herein, we show that Cav-1 is primarily expressed in the endothelium and adventitia of pulmonary arteries (PAs) and that Cav-1 expression is reduced in isolated PAs from multiple models of pulmonary artery hypertension (PH). Reduced Cav-1 expression correlates with increased ROS production in the adventitia of hypertensive PA. In vitro experiments revealed a significant ability of Cav-1 and its scaffolding domain to inhibit Nox1–5 activity and it was also found that Cav-1 binds to Nox5 and Nox2 but not Nox4. In addition to posttranslational actions, in primary cells, Cav-1 represses the mRNA and protein expression of Nox2 and Nox4 through inhibition of the NF-κB pathway. Last, in a mouse hypoxia model, the genetic ablation of Cav-1 increased the expression of Nox2 and Nox4 and exacerbated PH. Together, these results suggest that Cav-1 is a negative regulator of Nox function via two distinct mechanisms, acutely through direct binding and chronically through alteration of expression levels. Accordingly, the loss of Cav-1 expression in cardiovascular diseases such as PH may account for the increased Nox activity and greater production of ROS.     

Primary mechanisms of photoactivation of molecular oxygen. History of development and the modern status of research

This review starts from a brief historical account devoted to the principles of the Bach-Engler peroxidation theory and experiments and ideas which led A. N. Bach to its creation. Then, the discovery of photodynamic action is described, which was shown to result from pigment photosensitized activation of molecular oxygen. The dramatic history of mechanistic studies of oxygen photoactivation is reviewed starting from the Bach-Engler peroxidation theory to the hypothesis of moloxide, discovery of singlet oxygen and free radicals and, then, to modern views on the primary photoactivation processes. The origin of widely used division of photodynamic processes into type I and type II and the relation of these processes to the nature of the primary photochemical reactions of photosensitizers are discussed. New definitions of these reactions are proposed on the basis of the mechanisms of oxygen photoactivation. Photographs of the scientists who greatly contributed to the development of this field of research are presented. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 10, pp. 1311–1329.     

活性氧、自由基与植物的衰老

介绍近 1 0年来有关活性氧、自由基的产生 ,对植物的伤害及植物对活性氧、自由基清除的研究进展。     

In vitro scavenging activity for reactive oxygen species by N-substituted indole-2-carboxylic acid esters.

The hydroxyl radical (HO*)- and superoxide anion radical (O* (2))-scavenging activity, as well as the singlet oxygen ((1)O(2))-quenching property of N-substituted indole-2-carboxylic acid esters (INDs) were investigated by deoxyribose degradation assay, a chemiluminescence method and the electron spin resonance (ESR) spin-trapping technique. This novel group of compounds was developed as a search for cyclooxygenase-2 (COX-2)-selective enzyme inhibitors. The results obtained demonstrated that of the 16 compounds examined, five inhibited light emission from the superoxide anion radical (O* (2))-DMSO system by at least 60% at a concentration of 1 mmol/L, nine prevented the degradation of deoxyribose induced by the Fenton reaction system (range 3-78%) or scavenged hydroxyl radicals (HO*) directly (range 8-93%) and 14 showed the (1)O(2)-quenching effect (range 10-74%). These results indicate that majority of the indole esters tested possess the ability to scavenge O(-) (2) and HO radicals and to quench (1)O(2) directly, and consequently may be considered effective antioxidative agents.     

Imaging reactive oxygen species in asthma

Inflammatory processes in asthma are characterized by an infiltration of inflammatory cells including mononuclear phagocytes. It has been observed that mononuclear phagocytes, alveolar macrophages and blood monocytes, release higher quantities of reactive oxygen species in asthmatic patients than in healthy subjects. Chemiluminescence assays were developed to measure the superoxide anion and the other reactive oxygen species. The chemiluminescence response was first analysed with a luminometer, which made it possible to study cells in suspension before and after PMA-stimulation. Secondly a video-imaging camera was used in experiments on adherent cells before and after stimulation with PMA and/or specific stimulus IgE/anti-IgE. Both techniques showed that human alveolar macrophages, blood monocytes, PMN and lymphocytes were spontaneously primed in vivo and were more easily stimulated in asthma. Analysis of adherent cells in vitro may provide give information on the physiological condition of adherent cells in vivo.     

Mitochondrial metabolism of reactive oxygen species

Oxidative stress is considered a major contributor to etiology of both normal senescence and severe pathologies with serious public health implications. Mitochondria generate reactive oxygen species (ROS) that are thought to augment intracellular oxidative stress. Mitochondria possess at least nine known sites that are capable of generating superoxide anion, a progenitor ROS. Mitochondria also possess numerous ROS defense systems that are much less studied. Studies of the last three decades shed light on many important mechanistic details of mitochondrial ROS production, but the bigger picture remains obscure. This review summarizes the current knowledge about major components involved in mitochondrial ROS metabolism and factors that regulate ROS generation and removal. An integrative, systemic approach is applied to analysis of mitochondrial ROS metabolism, which is now dissected into mitochondrial ROS production, mitochondrial ROS removal, and mitochondrial ROS emission. It is suggested that mitochondria augment intracellular oxidative stress due primarily to failure of their ROS removal systems, whereas the role of mitochondrial ROS emission is yet to be determined and a net increase in mitochondrial ROS production in situ remains to be demonstrated.Translated from Biokhimiya, Vol. 70, No. 2, 2005, pp. 246–264.Original Russian Text Copyright © 2005 by Andreyev, Kushnareva, Starkov.This revised version was published online in April 2005 with corrections to the post codes.     

Defence against reactive oxygen species

As part of the European Commission Concerted Action on Functional Food which was managed by the International Life Sciences Institute (Europe) a series of Theme Papers was produced which examined the ‘state of the art’ with respect to the subject matter and made recommendations for research. This paper is a summary of the paper concerned with Defence Against Reactive Oxygen species. Having reviewed the scientific literature the authors concluded that certain stringent criteria, which they identified, would need to be satisfied in order to be able to conclude that free radical events are involved in certain human diseases, and that antioxidants are capable of modulating these events and thus reducing the risk of disease. Although there is some evidence that would lead to this conclusion the authors demonstrated that there is at present insufficient evidence available on which to base a firm conclusion that antioxidants are capable of reducing risk of disease, and very little evidence that addresses the important question as to how much of the nutrients concerned are required in the diet to achieve the objective of reducing risk. Research priorities address the need in particular for the development and validation of cellular markers of oxidative damage which are required before there can be new human studies that address the question. There is also a need for more information as to the pharmacokinetics of uptake from diet, distribution and cellular concentration of the antioxidants.     

A reporter system for the individual detection of hydrogen peroxide and singlet oxygen: its use for the assay of reactive oxygen species produced in vivo

A reporter system for the assay of reactive oxygen species (ROS) was developed in Chlamydomonas reinhardtii, a plant model organism well suited for the application of inhibitors and generators of various types of ROS. This system employs various HSP70A promoter segments fused to a Renilla reniformis luciferase gene as a reporter. Transformants with the complete HSP70A promoter were inducible by both hydrogen peroxide and singlet oxygen. Constructs that lacked upstream heat-shock elements (HSEs) were inducible by hydrogen peroxide, indicating that this induction does not require such HSEs. Rather, downstream elements located between positions -81 to -149 with respect to the translation start site appear to be involved. In contrast, upstream sequences are essential for the response to singlet oxygen. Thus, activation by singlet oxygen appears to require promoter elements that are different from those used by hydrogen peroxide. ROS generated endogenously by treatment of the alga with metronidazole, protoporphyrin IX, dinoterb or high light intensities were detected by this reporter system, and distinguished as production of hydrogen peroxide (metronidazole) and singlet oxygen (protoporphyrin IX, dinoterb, high light). This system thus makes it possible to test whether, under varying environmental conditions including the application of abiotic stress, hydrogen peroxide or singlet oxygen or both are produced.