• aldehydes;
  • chromosome territory;
  • Nrf2;
  • oxygenomics;
  • oxyproteomics;
  • reactive species


  1. Top of page
  2. Abstract

Aerobes, including humans, are consistently exposed to oxidative stress by consuming oxygen. The biological significance of oxidative stress via reactive oxygen and nitrogen species consists of two stages: reversible redox regulation and irreversible oxidative molecular damage, which are sometimes intermingled. During the past decade, many signaling cascades associated with oxidative stress have been discovered. An interaction between Keap1 and the Nrf2 transcription factor is among the most fundamental mechanisms of the defense system against oxidative or similar stress. Furthermore, it became apparent that reactive oxygen species are actively produced through enzymes such as xanthine oxidoreductase and nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidases in non-phagocytic cells as well. The role of α-tocopherol solely as an anti-oxidant was also questioned. Now there is a long list of pathological states implicating oxidative stress. At the same time, genome projects on various species have been completed. These efforts convincingly led to a new era of oxidative stress investigation, contributing powerful strategies to select candidate genes or biomolecules. Herein are reviewed recent advances and novel concepts in this field, including oxygenomics. These fruitful results may lead to more accurate and useful pathological diagnosis and more efficient prophylaxis and therapeutic interventions on human diseases.

The longevity of the Japanese people has been continuously prolonged ever since World War II, and averaged 79 years in men and 86 years in women in the world health report from 2006 (, and are world records. The top three causes of death in Japan were cancer, coronary heart disease and cerebral vascular disease in 2005.1 Of note is the fact that all these diseases are closely associated with oxidative stress. Under the current near-optimal environmental conditions, we may have to reconsider the ‘double-edged sword’ aspect of oxygen that may be regulating longevity.

Oxygen is indispensable for aerobes including humans. We utilize absorbed organic compounds via foods in combination with oxygen to produce energy. This process, called oxidative phosphorylation, uses the ability of O2 to be reduced to H2O via 4-electron reduction. These catalytic reactions are efficient but unfortunately not 100% efficient, because certain unexpected bypassed reactions produce reactive oxygen species in mitochondria. It is believed that approximately 2–3% of oxygen taken up will become a toxic intermediate.2 Furthermore, it has recently become clear that cells have mechanisms to positively produce reactive oxygen species either in the cytoplasm or on the plasma membrane via enzymatic reactions with a number of different mechanisms (Table 1). Here, we introduce two enzymes, xanthine oxidoreductase (XOR) and the nicotinamide adenine dinucleotide phosphate, reduced (NADPH) oxidase family. The former works in normal conditions as a xanthine reductase, reducing nicotinamide adenine dinucleotide+ (NAD+) to nicotinamde adenine dinucleotide, reduced (NADH). However, this enzyme, in case of either intramolecular disulfide formation3 or scission of the linker portion14 under oxidative stress, is converted to xanthine oxidase, and has been associated with the pathology of ischemia–reperfusion injury,15 producing either O2 or H2O2. Members of the NADPH oxidase (Nox) family, Nox2 and gp91phox, have long been recognized as the defense mechanisms against foreign materials and all kinds of infection in macrophages and B lymphocytes. However, this recognition is changing quite rapidly due to the presence of this enzyme in many other kinds of cells including endothelial cells.5 Another family protein, Nox1, is expressed abundantly in thecolonic epithelial cells, playing a role in immunity,16 but in vascular smooth muscle cells Nox1 is induced by angiotensin II. Indeed, in Nox1 knockout mice, hypertension is not induced by continuous administration of angiotensin II.4

Table 1.  Enzymes producing reactive oxygen species
  1. NAD, nicotinamide adenine dinucleotide; NADH, nicotinamde adenine dinucleotide, reduced; NADPH, nicotinamide adenine dinucleotide phosphate, reduced.

Xanthine oxidoreductaseUbiquitousHypoxanthine, xanthine and NAD+Xanthine, uric acid and NADHDehydrogenase/Oxidase conversion by disulfide formation or scission (substrates, O2 instead of NAD+ products, O2 instead of NADH)3
NADPH oxidase 1Colon, vascular smooth muscleO2, NADPHO2, NADP+, H+Colon mucosal immunity, hypertension by angiotensin II4
NADPH oxidase 2/gp91phoxPhagocytes, B-cellO2, NADPHO2, NADP+, H+Immunity5
NADPH oxidase 3Inner ear, fetal kidneyO2, NADPHO2, NADP+, H+Otolith formation in mice6
NADPH oxidase 4Kidney, endotheliumO2, NADPHO2, NADP+, H+Not known7
NADPH oxidase 5Spermatozoa, spleen, lymphocytesO2, NADPHO2, NADP+, H+Not known8
Dual oxidase 1Thyroid gland, bronchus, salivary glandO2, NADPHH2O2, NADP+, H+Not known9
Dual oxidase 2Thyroid gland, colonO2, NADPHH2O2, NADP+, H+Thyroid hormone10
Indoleamine 2,3-dioxygenaseUbiquitous but not in liverTryptophan, serotonin, O2O2, cleavage of indole ringMetabolism11
Tryptophan dioxygenaseLiverTryptophan, serotonin, O2O2, cleavage of indole ringMetabolism12
Aldehyde oxidaseLiverAldehydes, O2, NADHO2, NAD+, aldehyde oxidationMetabolism13

Aerobes are indeed cells that have succeeded in developing anti-oxidant defense mechanisms, and thus they are not stressed under ordinary conditions. However, there are many factors that greatly increase oxidative stress. Indeed, oxidative stress is associated with an array of pathological phenomena, including infection, inflammation, ultraviolet and gamma radiation, and overload of transition metals and certain chemical agents as well as ischemia–reperfusion injury.2Table 2 summarizes an updated list of pathological conditions associated with oxidative stress.

Table 2.  Selected reports on pathological conditions associated with oxidative stress
Pathological conditionsReferencesPathological conditionsReferencesPathological conditionsReferences
Acute coronary syndrome17Diabetic vasculopathy36Liver transplantation52
Acute iron poisoning18Environmental pollutants37Lung transplantation53
Acute lung injury with diesel exhaust19Exercise38Methylmercury54
Age-related macular degeneration of retina20Genetic hemochromatosis39Myocarditis55
Aging21,22Glomerulonephritis40Non-alcoholic steatohepatitis56
Alzheimer's disease23Heart failure41Non-erosive reflux disease57
Asthma24Helicobacter pylori infection42Pancreatitis58
Atherosclerosis25Hepatitis C infection43Parkinson's disease59
Atopic dermatitis26Hyperhomocysteinemia44Periodontitis60
Cardiac hypertrophy27Hyperoxic lung injury45Psoriasis61
Chronic obstructive pulmonary disease30Idiopathic pulmonary fibrosis47Reflux esophagitis57
Corneal neovascularization31Inflammatory acne48Renal failure63
Diabetes mellitus32Inflammatory bowel disease49Retinopathy of prematurity64
Diabetic nephropathy33,34Influenza virus pneumonia50Traumatic brain damage65
Diabetic retinopathy35Kawasaki disease51UV radiation66

As for the top cause of death in Japan, many epidemiological studies have demonstrated a close correlation between chronically oxidative conditions and carcinogenesis. For example, chronic tuberculous pleuritis causes a high incidence of malignant lymphoma;67 repeated exposure to asbestos fibers (crocidolite and amosite asbestos fibers are rich in iron68) is often associated with mesothelioma and lung carcinoma;69 chronic Helicobacter pylori infection is associated with a high incidence of gastric adenocarcinoma and lymphoma;42,70 the incidence of colorectal cancer is increased in inflammatory bowel diseases;71,72 a high risk for hepatocellular carcinoma is observed in patients with genetic hemochromatosis;73,74 severe burn by ultraviolet radiation is a risk factor for skin cancer;66,75 and gamma radiation causes a high incidence of leukemia.76 At least in the mentioned situations, and probably also in other more common types of carcinogenesis, oxidative stress appears to play a role.


  1. Top of page
  2. Abstract

Oxidative stress depends on a balance between the load of reactive species and the anti-oxidant systems responsible for them. The latter contains (i) protective mechanisms for the initiation of the free radical reaction; (ii) what are called ‘anti-oxidants’ that efficiently retard the free radical reaction after its initiation; and last, (iii) repair mechanisms for modified or damaged biomolecules. There are two seamless stages in the biological significance of oxidative stress both historically and experimentally (Fig. 1).


Figure 1. Cellular response and chemical modifications of biomolecules under oxidative stress. Different levels of oxidative stress cause different outcomes in cells. Cysteine residues of proteins are recognized as one of the most important targets for oxidative stress. 8-Hydroxy-2′-deoxyguanosine (8-OHdG) is a major oxidative DNA modification. Lipid peroxidation leads to formation of a variety of aldehydes.

Download figure to PowerPoint

The history of free radical chemistry dates back to 113 years ago. Fenton, a British chemist, reported in 1894 that ferrous sulfate and hydrogen peroxide cause the oxidation of tartaric acid, resulting in a beautiful violet color on the addition of caustic alkali.77 This was the basis for the discovery of the Fenton reaction, which produces hydroxyl radicals, presently known as the most reactive endogenous chemical species in living cells. In 1954 Harman proposed a hypothesis of ‘free radicals as a cause of aging’ for the first time.78 A decade was needed to integrate this idea into science, when McCord and Fridovich discovered superoxide dismutase, the first enzyme whose substrate is a free radical, in 1969.79 For the next two decades, studies based on the hypothesis that reactive species are damaging biomolecules were planned, and those efforts led to pioneering works on anti-oxidants, anti-oxidant enzymes, and protein oxidation,80,81 as well as DNA modification.82–84 Thus, the concept of ‘oxidative stress’ in the original sense was established.85

During the 1990s more scientists joined this research area using molecular biology techniques, and low levels of reactive oxygen and nitrogen species were established as signaling molecules in the cell.86,87 Multiple repair enzymes for oxidative DNA damage were also identified;88,89 nitric oxide (NO), a vasodilator, was integrated into free radical and oxidative stress research with reactions of NO and O290 and semiquantitative morphological analysis of oxidative damage was advanced.91 Indeed, reactive species were found to have two completely different characteristics that are seamless and sometimes intermingled, depending on the dose loaded and the situation each living cell or tissue has faced. When the reactions involved are reversible, it is described as redox regulation. In contrast, when the reactions are at the irreversible stage, they are designated oxidative molecular damage (Fig. 1). Aging may be a shift from redox regulation to oxidative damage.92


  1. Top of page
  2. Abstract

It is not surprising that the antagonizing system for oxidative stress is reduction because oxidative stress oxidizes biomolecules. There are basically two major reductant systems, namely glutathione (GSH) and thioredoxin (TRX). A tripeptide, GSH was reported in the 1920s,93 whereas mammalian TRX was reported in 1988.94 Both of the systems constitute an array of associated enzymes and they work together at different levels. Although the intracellular concentration of TRX is 1/1000 that of GSH, TRX is essential for development because absence of this is lethal in TRX knockout mice.

Redox regulation is one of the key mechanisms for adaptation to a variety of stresses, including oxidative stress.95,96 Recently, it was reported from several independent laboratories including the authors' at Kyoto University that an antagonizing protein for TRX (thioredoxin-binding protein-2, TBP-2; also known as vitamin D3 upregulated protein-1, VDUP-1)97 is downregulated in cancers including human adult T-cell leukemia,98,99 human gastric cancer100,101 and iron chelate (ferric nitrilotriacetate)-induced renal cell carcinoma of rats.102 The mechanism of its inactivation was consistently methylation of the promoter region. Furthermore, TBP-2 is expressed at higher levels in non-metastatic melanomas than metastatic melanomas.103 Studies of a TBP-2-null mouse104 provided evidence that loss of TBP-2 results in enhanced sulfhydryl reduction and dysregulated carbohydrate and lipid metabolism, namely hyperinsulinemia, hypoglycemia, hypertriglyceridemia and increased levels of ketone bodies, at least in the liver and pancreatic β-cells.105 This was confirmed by producing TBP-2-deficient mice.106 The loss of TBP-2 appears advantageous to cancer cells because it ultimately results in facilitation of the glycolytic pathway via increasing the TRX activity. This makes sense in consideration of the fact that cancer cells are usually exposed to a lower oxygen environment.107 It is interesting to further note that the vitamin D3 receptor is one of the targets of p53-mediated transcriptional activation.108

One of the most striking findings during this decade is that many of the anti-oxidant enzymatic systems are under the control of a transcription factor, Nrf2.109,110 The cytoplasmic sensor for oxidative stress is now established as Keap1, a partner protein of Nrf2.111 In normal conditions, Nrf2 is bound to Keap1 in the cytosol and continuously degraded via the ubiquitin–proteasome system as soon as Nrf2 is translated. However, when oxidative stress is enhanced, Nrf2 is released from Keap1,112 translocates to the nuclear fraction, and binds to the anti-oxidant-responsive element, initiating the transcription of a variety of anti-oxidant enzymes including the GSH and TRX systems (Fig. 2). The target genes include (i) phase II detoxifying enzymes such as GSH-S-transferase and NAD(P)H:quinone oxidoreductase; (ii) GSH-associated enzymes such as γ-glutamylcysteine synthase and GSH peroxidase; and (iii) anti-oxidant enzymes such as heme oxygenase-1, peroxiredoxin, ferritin and TRX. Of note is the fact that this system can respond immediately to oxidative stress without producing new proteins. The cost of this system is the consumption of energy. Namely, the cells are consistently producing Nrf2 that is instantly decomposed via the ubiquitin–proteasome system.113 This appears to be a waste of energy, but this mechanism is essential for the fastest response against oxidative stress.


Figure 2. Keap1 as a cytoplasmic sensor for oxidative stress. Cysteine residues of Keap1 work as a cytoplasmic sensor for oxidative stress. Without Keap1, Nrf2 is not degraded via proteasome system, but is translocated to nucleus and activates transcription of target downstream genes.

Download figure to PowerPoint

Here we have to point out the interaction between redox signals and classic phosphorylation signals. Regulation of phosphorylation of proteins with protein kinases and phosphatases is one of the most fundamental systems that living cells acquired during the evolution process. Some kinases are activated after exposure to oxidative stress;114 protein kinase C δ is activated via phosphorylation of Tyr512 by another kinase, c-Abl;115 the phosphatidylinositol-3 kinase-Akt(protein kinase B) pathway is also activated;116 apoptosis signal-regulating kinase-1 (ASK1), a mitogen-activated protein kinase kinase kinase, is also activated, further activating the c-jun N-terminal kinase and p38 pathways.117 Cells derived from ASK1 knockout mice are resistant to oxidative stress and septic shock by lipopolysaccharide.118 Interestingly, in the control condition, ASK is bound to TRX, producing an ASK1-signalsome where ASK1 is in the form of a homooligomer and resistant to phosphorylation. Oxidative stress releases ASK1 from the signalsome in parallel with the oxidation of TRX, leading to activation of the p38 pathway.119 It is now known that Toll-like receptor 4 is upstream of this pathway, with ASK1 pathway closely associated in natural immunity.120

Currently there has been great interest in the generation of O2 by the Nox family in association with redox signaling (Table 1). As has been demonstrated in the structural study of Nox, O2 is released to the outside of the cell or intracellular compartments. However, O2 cannot easily move through the membrane. It is assumed that O2 is converted to H2O2 through the dismutation reaction without the enzyme and taken up into the cytosolic compartment. One of the aquaporins (water channels) can take up H2O2, and this mechanism may also be utilized.121 Recently, it was reported that generation of reactive oxygen species through Nox1 is essential for cellular transformation by the ras oncogene.122 Furthermore, the involvement of Nox4 was reported in the signaling pathway of adipocytes after insulin stimulation.123

Even the role of vitamin E (α-tocopherol) as an anti-oxidant was questioned. It can at least suppress the activity of protein kinase C via activating protein phosphatase.124 There is a possibility that α-tocopheryl phosphate is a more efficient biomolecule.125 We recently observed that one of the anti-oxidant mechanisms of vitamin E is via inducing chaperone proteins associated with glycoproteins.126


  1. Top of page
  2. Abstract

A great advance was achieved with the understanding of the two gaseous mediators of oxidation, NO and carbon monoxide (CO). NO is enzymatically produced by nitric synthase from l-arginine, and there are basically two types of enzymes, constitutive and inducible. NO was identified as an endothelium-derived relaxing factor.127 There are three types of modifications by NO: S-nitrosylation (-S-NO) of cysteine residues in proteins, nitration (-NO2) of tyrosine residues in proteins and iron nitrosylation or chelation to heme. Activation of guanylate cyclase by NO is indeed achieved by its chelation to heme. These NO-associated modifications modulate protein interactions.128 For example, the interactions between HIF-1α (a transcription factor modulating responses for hypoxia) and p300129 or glyceraldehyde-3-phosphate dehydrogenase (a glycolytic enzyme) and Siah1130 are enhanced with these modifications, whereas those between N-ethylmaleimide-sensitive factor (NSF) and soluble NSF attachment protein receptor are compromised, relieving endothelial injury.131 Another important point is that in the presence of O2, NO can produce a reactive species, peroxynitrite.90 This is the basis for the reaction of nitration in which 3-nitrotyrosine and 8-nitroguanine are produced. It was also hypothesized that nitrotyrosine-containing peptide with an affinity to SH2 domain of src kinase can activate src enzymatic activity by halting autoinhibition with intramolecular interaction to expose the catalytic domain.132

The majority of CO is produced via heme oxygenase in living cells, leading to the formation of CO, Fe(II) and biliverdin. This enzyme is abundantly present in the liver, brain, spleen, testis and placenta and is induced by oxidative stress. It is now known that CO has anti-inflammatory and cytoprotective activities,133 as well as modulatory actions on vascular tone.134 It is believed that low vascular tonus in the hepatic sinusoid is maintained by the presence of high concentrations of CO. Currently, even H2S has been recognized as a gaseous mediator.135


  1. Top of page
  2. Abstract

Oxygenomics, a new member of the omics family, is a synthetic word combining oxygen and genomics. We have recently proposed this research area136 in the hope that the biased distribution of oxidative DNA damage in the genome is associated with pathology of oxidative stress, especially in carcinogenesis, for the following reasons: the generation of excess reactive oxygen and nitrogen species causes DNA strand breaks, cross-links and modifications,82,84,91,137 leading to alterations in the genome information in spite of robust counteractions by repair enzymes and apoptotic pathways.138 These events may cause persistent activation of oncogenes or inactivation of tumor suppressor genes.139

Most scientists assume that free radical reactions present little specificity based on in vitro studies, in contrast to the extremely selective antigen–antibody interactions. Indeed, the second-order rate constant for the reaction of a hydroxyl radical with the DNA base, guanine is approximately 1.0 × 1010/s mol/L. Thus, one might consider that the genome is damaged at random and that there are no specific target genes or signaling pathways in oxidative stress-associated carcinogenesis. However, it is time to reconsider this assumption. We have been challenging this hypothesis recently, because ferric nitrilotriacetate (Fe-NTA)-induced renal cell carcinomas are homogeneous in histology.91,140 At the early stage of this rat carcinogenesis model, increased amounts of oxidatively modified DNA bases including 8-oxoguanine141 and a major lipid peroxidation product, 4-hydroxy-2-nonenal, and its modified proteins81,142–144 are observed.

In order to clarify whether there is any target tumor suppressor gene, we used a genetic strategy using microsatellite analysis in F1 hybrid rats145 and recently used comparative genomic hybridization (S. Akatsuka and S. Toyokuni, unpubl. data, 2007). This study found that the p15INK4B(p15) and p16INK4A(p16) tumor suppressor genes are two of the major target genes with a high incidence of homozygous deletion as well as methylation of the promoter region. This was the first report that showed the presence of a target gene in an oxidative stress-induced carcinogenesis model.146 The significance of this finding is enormous, especially because p16 is associated not only with the retinoblastoma protein pathway as an inhibitor of cyclin-dependent kinases 4 and 6, but also with the TP53 pathway via p19ARF and MDM2.147 p19ARF is an alternatively spliced transcript from the p16 tumor suppressor gene.148 Indeed, iron-mediated oxidative damage appears to attack one of the most critical loci of the genome. We then showed that allelic loss of p16 occurs as early as 1 week after the start of the animal experiment and is p16 gene-specific.149 Thus, we believe that these results suggest the presence of fragile sites in the genome. Elucidation of this mechanism and principle would change the chemopreventive strategy in particular cases of carcinogenesis.

Studying the localization of oxidative DNA damage in comparison with genome information and chromosomal structure is becoming increasingly important. There are numerous reports published on oxidative DNA damage in vitro using purified DNA or cultured cells. Based on these data, it has been claimed that certain specific sequences such as telomeres150,151 are especially vulnerable to oxidative damage. Currently, however, limited data are available on which part of the genome is susceptible to oxidative damage in vivo at the tissue or organ levels.

This is the post-genomic era, given the completion of genome projects of humans, mice, rats and other species ( The information in public databases is a great advantage for researchers of oxidative stress. We recently developed an immunoprecipitation-based method to make libraries of approximately 1 kb DNA fragments136 that contain one or more 8-oxoguanine152 or acrolein-modified adenine residues153 (Fig. 3). This is a versatile method, allowing the evaluation of relative content of selected oxidative DNA base modifications at any genomic locus when used in combination with polymerase chain reaction. Nuclear genomic DNA is integrated into the chromatin structure in association with histones. Some parts of the chromatin structure are open for transcription, whereas others are closed, constituting heterochromatin. Furthermore, genomic information, a blueprint for a cell, is not continuous information, but is divided into many pieces by the existence of chromosomes, although we cannot routinely observe chromosomal structure at interphase. Recently, the concept of chromosome territory has been established.154,155 This concept indicates that genome information corresponding to each chromosome is located at fixed areas, namely central nuclear or peripheral nuclear, in the nucleus even at the interphase. The localization appears to be different among different kinds of cells.156 This concept has been reviewed in a recent issue of Nature.157 It is possible that the genome areas susceptible to oxidative stress may differ depending on the kind of cells and the situation in which the cells are placed. Such a difference would help to explain the different signaling pathways each type of cancer has acquired, because not a few signaling pathways start with the recognition of DNA damage. We propose to call this novel research area ‘oxygenomics’. Oxygenomics is defined as the study of the localization of oxidative DNA damage in the genome in living cells. Using a DNA immunoprecipitation method, we found that the distribution of modified DNA bases is not random. Rather, the distribution of 8-oxoguanine and acrolein-modifed adenine was contrasting. Interestingly, aldehyde-modified DNA fragments were present in proximity to the nuclear membrane.136 This may be explained by the fact that acrolein, an aldehyde, comes from the cytoplasm or directly from the nuclear membrane.


Figure 3. Concept of oxygenomics. The word ‘oxygenomics’ is a combination of oxygen and genomics. With this terminology we conduct studies to determine the genomic distribution of fragile sites under oxidative stress. DNA immunoprecipitation (DnaIP) is a major strategy. At least three factors (chromosome territory, chromatin status and responsible chemical species) are associated with the pathological processes.

Download figure to PowerPoint


  1. Top of page
  2. Abstract

The functions of proteins are finely regulated with a variety of modifications such as activation by proteolytic enzymes, modulation of activity and interaction by addition of sugars and phosphates, and localization to the membrane by palmitoylation or S-acylation.158 It is quite rare in eukaryotes that translated proteins function as they are, but rather they are subject to a variety of modifications after leaving ribosomes. Thus, post-translational protein modifications are essential for cellular activity.

In addition to these physiological modifications, proteins are subject to a variety of modifications by reactive species in pathological conditions; side chains of amino acids are directly oxidized (Fig. 4a) or modified by aldehydes that are secondary products of lipid peroxidation (Fig. 4b,c). These oxidative modifications may inactivate enzymatic functions and cause degeneration of structural proteins, but in contrast they may start activation of transcription factors or proteolytic systems. Thus, it is now recognized that comprehensive analyses of oxidatively modified proteins by the use of proteomics are important. This may be called oxyproteomics or oxidative stress proteomics. At least three different kinds of modifications are under investigation at present: oxidized cysteine, carbonyls and aldehydes.


Figure 4. Reversible and irreversible modifications at specific amino acid residues in proteins. (a) Reversible alterations at cysteine residues of proteins. (b) Major pathological aldehydes formed during lipid peroxidation. (c) Michael addition reaction. X can be either a cysteine, histidine or lysine residue of amino acids.

Download figure to PowerPoint

Cysteine residues in proteins may work as a catalytic center and contribute to the formation of the stable 3-D structure of proteins by producing disulfide bonds with another cysteine residues within the same molecule. However, because sulfhydryl functions are quite susceptible to oxidative stress, they can be easily oxidized or subject to S-thiolation (Cys-S-S-X; Fig. 4a). This kind of modification occurs relatively early in oxidative stress, so they are early markers for protein modification (Fig. 1). There are two different methods for identification after 2-D gel analysis: one using the radioisotope 35S159 and the other using biotinylated cysteine.160 The latter has the merit of collecting cysteine-labeled proteins with avidin beads.

The most common method currently used for the detection of oxidatively modified proteins is the detection of carbonylated proteins.161 The principle of this method is the detection of dinitrophenylated proteins with monoclonal antibodies after the reaction of the carbonyl function (-C = O) with 2,4-dinitrophenylhydrazine. The merit of this method is its sensitivity, because carbonyl functions are contained not only in carbonylated amino acids but also in aldehydes and glycation products. In a recent work, target proteins were identified from cardiac and skeletal muscle of diabetic rats.162

Aldehydes are the major final products of lipid peroxidation, but are reactive enough to form covalent bonds with the side chains of certain amino acids (Fig. 4c). Recently it has become clear that these aldehydes can be second messengers for neighboring cells, generating hydrogen peroxide. Indeed, acrolein, 2-alkenal, 4-hydroxy-2-alkenal and ketoaldehydes (Fig. 4b) are among the aldehydes that have been associated with oxidative stress and cytotoxicity.81 We previously succeeded in producing monoclonal antibodies against 4-hydroxy-2-nonenal modified proteins.163 One of the major target proteins in the kidney after ferric nitrilotriacetate administration is β-actin.144 A recent report that modification of actin with electrophilic lipids alters its filament structure164 is consistent with our results. These kinds of studies are progressing quite rapidly, as shown in Table 3. These efforts would certainly lead to the discovery of new serum markers for oxidative stress-associated diseases.

Table 3.  Target proteins of 4-hydroxy-2-nonenal modification
ModelChemical administeredPathologyTarget cellsTarget proteinsReferences
  1. ApoE, apolipoprotein E; HNE, 4-hydroxy-2-nonenal; NA, not applied.

RatFerric nitrilotriacetateRenal carcinogenesisproximal tubular cellsProteasome, β-actin144,165
P19 neuroglial cultureHNENeurodegenerationNATau166
Human brain sampleNoneLate-onset Alzheimer's diseasePyramidal neuronEpsilon 4 allele of ApoE167
Rat heart mitochondriaHNEHeart failureNAα-ketoglutarate dehydrogenase, pyruvate dehydrogenase168
Human brain sampleNoneAlzheimer's diseaseInferior parietal lobuleGlutamate transporter169
SynaptosomeHNENeurodegenerationNAGlutamate transporter169
Leukemia, colorectal and lung cancer cell linesHNETumor biologyNAIκB kinase170
Human serumNoneType 2 diabetesNAAlbumin171
G93A-SOD1 knockout mouseNoneAmyotropic lateral sclerosisSpinal cordDihydropyrimidinase-related protein-2, heat-shock protein 70172
RatWhite fluorescent lightRetinal degenerationRetinaHeterogenous nuclear ribonucleoprotein A2/B1, glutamine synthetase173
RatHigh fat/ethanol dietChronic alcoholic hepatitisLiverHeat shock protein 90174


  1. Top of page
  2. Abstract

During the past decade not a few scientists came into this research area and it expanded a lot. However, this research area still has room for more expansion. The use of oxygen in life is a fundamental issue. Whether the surrounding environment on Earth gets better or worse, we cannot ignore the issue of oxygen. As the average lifespan is prolonged, the challenging problem for humans is the wearing-out of the blueprint of our system, the genome, and the other fundamental structures of the body. The fact that cancer and atherosclerotic diseases are the leading causes of death in most countries is the basis of this idea. At the same time, there are some emerging areas in science such as non-coding RNA.175,176 This is a rapidly expanding new field. So far, there are almost no reports on the association of oxidative stress and microRNAs. Also, epigenetics would be not less important for the pathological investigation of oxidative stress (Fig. 5).


Figure 5. Less explored research areas in oxidative stress pathology. ncRNA, non-coding RNA. Shadowed items are less explored research areas at present in the fields of oxidative stress pathology.

Download figure to PowerPoint

Recently, a new technique was proposed to visualize free radicals by the use of an antibody against the 5,5-dimethyl-1-pyrroline N-oxide (DMPO)-nitorone spin trap form of molecules.177,178 This is an interesting idea because DMPO has been established as a probe for electron spin resonance. Most researchers call this research area oxidative stress research and we also used this term in the present review. However, we also have to return to the original idea of oxidative stress starting from the load of free radicals, especially from the point of spatial restriction of each reactive species. We hope that the present review article stimulates pathologists to work in the area of oxidative stress. Last, these fruitful results should be utilized to develop more accurate pathological diagnosis and more efficient prophylaxis and therapeutic intervention in the near future.


  1. Top of page
  2. Abstract

This work was supported in part by a MEXT grant (Special Coordination Funds for Promoting Science and Technology), a Grant-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology of Japan, a Grant-in-Aid for Cancer Research from the Ministry of Health, Labour and Welfare of Japan, and a grant of Long-range Research Initiative (LRI) by Japan Chemical Industry Association (JCIA).


  1. Top of page
  2. Abstract
  • 1
    Foundation for Promotion of Cancer Research. Cancer Statistics in Japan. Tokyo: Foundation for Promotion of Cancer Research, 2006.
  • 2
    Halliwell B, Gutteridge JMC. Free Radicals in Biology and Medicine. New York: Oxford University Press, 2007.
  • 3
    Nishino T, Okamoto K, Kawaguchi Y et al. Mechanism of the conversion of xanthine dehydrogenase to xanthine oxidase: Identification of the two cysteine disulfide bonds and crystal structure of a non-convertible rat liver xanthine dehydrogenase mutant. J Biol Chem 2005; 280: 24 888–94.
  • 4
    Matsuno K, Yamada H, Iwata K et al. Nox1 is involved in angiotensin II-mediated hypertension: A study in Nox1-deficient mice. Circulation 2005; 112: 267785.
  • 5
    Lambeth J. NOX enzymes and the biology of reactive oxygen. Nat Rev Immunol 2004; 4: 1819.
  • 6
    Paffenholz R, Bergstrom R, Pasutto F et al. Vestibular defects in head-tilt mice result from mutations in Nox3, encoding an NADPH oxidase. Genes Dev 2004; 18: 48691.
  • 7
    Kuroda J, Nakagawa K, Yamasaki T et al. The superoxide-producing NAD (P) H oxidase Nox4 in the nucleus of human vascular endothelial cells. Genes Cells 2005; 10: 113951.
  • 8
    Banfi B, Tirone F, Durussel I et al. Mechanism of Ca2+ activation of the NADPH oxidase 5 (NOX5). J Biol Chem 2004; 279: 18 583–91.
  • 9
    Daiyasu H, Toh H. Molecular evolution of the myeloperoxidase family. J Mol Evol 2000; 51: 43345.
  • 10
    Moreno J, Bikker H, Kempers M et al. Inactivating mutations in the gene for thyroid oxidase 2 (THOX2) and congenital hypothyroidism. N Engl J Med 2002; 347: 95102.
  • 11
    Sono M. The roles of superoxide anion and methylene blue in the reductive activation of indoleamine 2,3-dioxygenase byascorbic acid or by xanthine oxidase-hypoxanthine. J Biol Chem 1989; 264: 161622.
  • 12
    Brady F, Forman H, Feigelson P. The role of superoxide and hydroperoxide in the reductive activation of tryptophan-2,3-dioxygenase. J Biol Chem 1971; 246: 711924.
  • 13
    Mira L, Maia L, Barreira L, Manso C. Evidence for free radical generation due to NADH oxidation by aldehyde oxidase during ethanol metabolism. Arch Biochem Biophys 1995; 318: 538.
  • 14
    Enroth C, Eger B, Okamoto K, Nishino T, Pai E. Crystal structures of bovine milk xanthine dehydrogenase and xanthine oxidase: Structure-based mechanism of conversion. Proc Natl Acad Sci USA 2000; 97: 10 723–8.
  • 15
    McCord JM. Oxygen-derived free radicals in postischemic tissue injury. N Engl J Med 1985; 312: 15963.
  • 16
    Kawahara T, Kuwano Y, Teshima-Kondo S et al. Role of nicotinamide adenine dinucleotide phosphate oxidase 1 in oxidative burst response to Toll-like receptor 5 signaling in large intestinal epithelial cells. J Immunol 2004; 172: 30518.
  • 17
    Heistad D. Unstable coronary-artery plaques. N Engl J Med 2003; 349: 22857.
  • 18
    Pietrangelo A, Montosi G, Garuti C et al. Iron-induced oxidant stress in nonparenchymal liver cells: Mitochondrial derangement and fibrosis in acutely iron-dosed gerbils and its prevention by silybin. J Bioenerg Biomembr 2002; 34: 6779.
  • 19
    Takano H, Yanagisawa R, Ichinose T et al. Diesel exhaust particles enhance lung injury related to bacterial endotoxin through expression of proinflammatory cytokines, chemokines, and intercellular adhesion molecule-1. Am J Respir Crit Care Med 2002; 165: 132935.
  • 20
    Imamura Y, Noda S, Hashizume K et al. Drusen, choroidal neovascularization, and retinal pigment epithelium dysfunction in SOD1-deficient mice: A model of age-related macular degeneration. Proc Natl Acad Sci USA 2006; 103:11 282–7.
  • 21
    Ishii T, Yasuda K, Akatsuka A, Hino O, Hartman P, Ishii N. A mutation in the SDHC gene of complex II increases oxidative stress, resulting in apoptosis and tumorigenesis. Cancer Res 2005; 65: 2039.
  • 22
    Lee S, Lee R, Fraser A, Kamath R, Ahringer J, Ruvkun G. A systematic RNAi screen identifies a critical role for mitochondria in C. elegans longevity. Nat Genet 2003; 33:4048.
  • 23
    Kawaguchi-Niida M, Shibata N, Morikawa S et al. Crotonaldehyde accumulates in glial cells of Alzheimer's disease brain. Acta Neuropathol (Berl) 2006; 111: 4229.
  • 24
    Yamaya M, Sekizawa K, Ishizuka S, Monma M, Mizuta K, Sasaki H. Increased carbon monoxide in exhaled air of subjects with upper respiratory tract infections. Am J Respir Crit Care Med 1998; 158: 31114.
  • 25
    Takabe W, Kanai Y, Chairoungdua A et al. Lysophosphatidylcholine enhances cytokine production of endothelial cells via induction of 1-type amino acid transporter 1 and cell surface antigen 4F2. Arterioscler Thromb Vasc Biol 2004; 24: 164045.
  • 26
    Antille C, Sorg O, Lubbe J, Saurat J. Decreased oxidative state in non-lesional skin of atopic dermatitis. Dermatology 2002; 204: 6971.
  • 27
    Giordano F. Oxygen, oxidative stress, hypoxia, and heart failure. J Clin Invest 2005; 115: 500508.
  • 28
    Lou M. Redox regulation in the lens. Prog Retin Eye Res 2003; 22: 65782.
  • 29
    Goswami S, Sheets N, Zavadil J et al. Spectrum and range of oxidative stress responses of human lens epithelial cells to H2O2 insult. Invest Ophthalmol Vis Sci 2003; 44: 208493.
  • 30
    Barnes P. Reduced histone deacetylase in COPD: Clinical implications. Chest 2006; 129: 1515.
  • 31
    Rao N, Thaete L, Delmage J, Sevanian A. Superoxide dismutase in ocular structures. Invest Ophthalmol Vis Sci 1985; 26: 177881.
  • 32
    Ihara Y, Toyokuni S, Uchida K et al. Hyperglycemia causes oxidative stress in pancreatic beta-cells of GK rats, a model of type 2 diabetes. Diabetes 1999; 48: 92732.
  • 33
    Asaba K, Tojo A, Onozato M et al. Effects of NADPH oxidase inhibitor in diabetic nephropathy. Kidney Int 2005; 67: 189098.
  • 34
    Inagi R, Yamamoto Y, Nangaku M et al. A severe diabetic nephropathy model with early development of nodule-like lesions induced by megsin overexpression in RAGE/iNOS transgenic mice. Diabetes 2006; 55: 35666.
  • 35
    Chen B, Jiang D, Tang L. Advanced glycation end-products induce apoptosis involving the signaling pathways of oxidative stress in bovine retinal pericytes. Life Sci 2006; 79: 104048.
  • 36
    Brownlee M. The pathobiology of diabetic complications: A unifying mechanism. Diabetes 2005; 54: 161525.
  • 37
    Murata M, Nishimura T, Chen F, Kawanishi S. Oxidative DNA damage induced by hair dye components ortho-phenylenediamines and the enhancement by superoxide dismutase. Mutat Res 2006; 607: 18491.
  • 38
    Aoi W, Naito Y, Takanami Y et al. Oxidative stress and delayed-onset muscle damage after exercise. Free Radic Biol Med 2004; 37: 48087.
  • 39
    Houglum K, Ramm G, Crawford D, Witztum J, Powell L, Chojkier M. Excess iron induces hepatic oxidative stress and transforming growth factor beta1 in genetic hemochromatosis. Hepatology 1997; 26: 60510.
  • 40
    Kondo S, Shimizu M, Urushihara M et al. Addition of the antioxidant probucol to angiotensin II type I receptor antagonist arrests progressive mesangioproliferative glomerulonephritis in the rat. J Am Soc Nephrol 2006; 17: 78394.
  • 41
    Ide T, Tsutsui H, Hayashidani S et al. Mitochondrial DNA damage and dysfunction associated with oxidative stress in failing hearts after myocardial infarction. Circ Res 2001; 88: 52935.
  • 42
    Naito Y, Yoshikawa T. Carcinogenesis and chemoprevention in gastric cancer associated with helicobacter pylori infection: Role of oxidants and antioxidants. J Clin Biochem Nutr 2005; 36: 3749.
  • 43
    Furutani T, Hino K, Okuda M et al. Hepatic iron overload induces hepatocellular carcinoma in transgenic mice expressing the hepatitis C virus polyprotein. Gastroenterology 2006; 130: 208798.
  • 44
    Loscalzo J. Homocysteine trials: Clear outcomes for complex reasons. N Engl J Med 2006; 354: 162932.
  • 45
    Christofidou-Solomidou M, Muzykantov V. Antioxidant strategies in respiratory medicine. Treat Respir Med 2006; 5: 4778.
  • 46
    Venditti P, Di Meo S. Thyroid hormone-induced oxidative stress. Cell Mol Life Sci 2006; 63: 41434.
  • 47
    Hoshino T, Nakamura H, Okamoto M et al. Redox-active protein thioredoxin prevents proinflammatory cytokine- or bleomycin-induced lung injury. Am J Respir Crit Care Med 2003; 168: 107583.
  • 48
    Miyachi Y, Yoshioka A, Imamura S, Niwa Y. Effect of antibiotics on the generation of reactive oxygen species. J Invest Dermatol 1986; 86: 44953.
  • 49
    Koutroubakis I, Malliaraki N, Dimoulios P, Karmiris K, Castanas E, Kouroumalis E. Decreased total and corrected antioxidant capacity in patients with inflammatory bowel disease. Dig Dis Sci 2004; 49: 14337.
  • 50
    Nakamura H, Tamura S, Watanabe I, Iwasaki T, Yodoi J. Enhanced resistance of thioredoxin-transgenic mice against influenza virus-induced pneumonia. Immunol Lett 2002; 82: 16570.
  • 51
    Deng Y, Xiang H, Chang Q, Li C. Evaluation by high-resolution ultrasonography of endothelial function in brachial artery after Kawasaki disease and the effects of intravenous administration of vitamin C. Circ J 2002; 66: 90812.
  • 52
    Kobayashi T, Sato Y, Yamamoto S et al. Augmentation of heme oxygenase-1 expression in the graft immediately after implantation in adult living-donor liver transplantation. Transplantation 2005; 79: 97780.
  • 53
    Kawashima M, Bando T, Nakamura T et al. Cytoprotective effects of nitroglycerin in ischemia-reperfusion-induced lung injury. Am J Respir Crit Care Med 2000; 161: 93543.
  • 54
    Usuki F, Yasutake A, Umehara F et al. In vivo protection of a water-soluble derivative of vitamin E, Trolox, against methylmercury-intoxication in the rat. Neurosci Lett 2001; 304: 199203.
  • 55
    Nimata M, Okabe T, Hattori M, Yuan Z, Shioji K, Kishimoto C. MCI-186 (edaravone), a novel free radical scavenger, protects against acute autoimmune myocarditis in rats. Am J Physiol Heart Circ Physiol 2005; 289: H251418.
  • 56
    Ikejima K, Okumura K, Lang T et al. The role of leptin in progression of non-alcoholic fatty liver disease. Hepatol Res 2005; 33: 1514.
  • 57
    Rantanen T, Rasanen J, Sihvo E, Ahotupa M, Farkkila M, Salo J. The impact of antireflux surgery on oxidative stress of esophageal mucosa caused by gastroesophageal reflux disease: 4-yr follow-up study. Am J Gastroenterol 2006; 101: 2228.
    Direct Link:
  • 58
    Ohashi S, Nishio A, Nakamura H et al. Clinical significance of serum thioredoxin 1 levels in patients with acute pancreatitis. Pancreas 2006; 32: 26470.
  • 59
    Tabner B, Turnbull S, El-Agnaf O, Allsop D. Formation of hydrogen peroxide and hydroxyl radicals from A(beta) and alpha-synuclein as a possible mechanism of cell death in Alzheimer's disease and Parkinson's disease. Free Radic Biol Med 2002; 32: 107683.
  • 60
    Komatsu T, Lee M, Miyagi A et al. Reactive oxygen species generation in gingival fibroblasts of Down syndrome patients detected by electron spin resonance spectroscopy. Redox Rep 2006; 11: 717.
  • 61
    Peus D, Beyerle A, Rittner H et al. Anti-psoriatic drug anthralin activates JNK via lipid peroxidation: Mononuclear cells are more sensitive than keratinocytes. J Invest Dermatol 2000; 114: 68892.
  • 62
    Anzai K, Ueno M, Yoshida A et al. Comparison of stable nitroxide, 3-substituted 2,2,5,5-tetramethylpyrrolidine-N-oxyls, with respect to protection from radiation, prevention of DNA damage, and distribution in mice. Free Radic Biol Med 2006; 40: 117078.
  • 63
    Morimoto H, Nakao K, Fukuoka K et al. Long-term use of vitamin E-coated polysulfone membrane reduces oxidative stress markers in haemodialysis patients. Nephrol Dial Transplant 2005; 20: 277582.
  • 64
    Cervantes-Munguia R, Espinosa-Lopez L, Gomez-Contreras P, Hernandez-Flores G, Dominguez-Rodriguez J, Bravo-Cuellar A. [Retinopathy of prematurity and oxidative stress]. An Pediatr (Barc) 2006; 64: 12631.
  • 65
    Wang H, Olivero W, Lanzino G et al. Rapid and selective cerebral hypothermia achieved using a cooling helmet. J Neurosurg 2004; 100: 2727.
  • 66
    Nishigori C, Hattori Y, Toyokuni S. Role of reactive oxygen species in skin carcinogenesis. Antioxid Redox Signal 2004; 6: 56170.
  • 67
    Iuchi K, Ichimiya A, Akashi A et al. Non-Hodgkin's lymphoma of the pleural cavity developing from long-standing pyothorax. Cancer 1987; 60: 17715.
  • 68
    Gilmour P, Brown D, Beswick P, MacNee W, Rahman I, Donaldson K. Free radical activity of industrial fibers: Role of iron in oxidative stress and activation of transcription factors. Environ Health Perspect 1997; 105: 131317.
  • 69
    Hodgson J, Darnton A. The quantitative risks of mesothelioma and lung cancer in relation to asbestos exposure. Ann Occup Hyg 2000; 44: 565601.
  • 70
    Uemura N, Okamoto S, Yamamoto S et al. Helicobacter pylori infection and the development of gastric cancer. N Engl J Med 2001; 345: 7849.
  • 71
    Collins R, Feldman M, Fordtran J. Colon cancer, dysplasia, and surveillance in patients with ulcerative colitis. A critical review. N Engl J Med 1987; 316: 16548.
  • 72
    Eaden J, Abrams K, Mayberry J. The risk of colorectal cancer in ulcerative colitis: A meta-analysis. Gut 2001; 48: 52635.
  • 73
    Toyokuni S. Iron-induced carcinogenesis: The role of redox regulation. Free Radic Biol Med 1996; 20: 55366.
  • 74
    Elmberg M, Hultcrantz R, Ekbom A et al. Cancer risk in patients with hereditary hemochromatosis and in their first-degree relatives. Gastroenterology 2003; 125: 173341.
  • 75
    Grodstein F, Speizer F, Hunter D. A prospective study of incident squamous cell carcinoma of the skin in the nurses' health study. J Natl Cancer Inst 1995; 87: 10616.
  • 76
    Preston D, Kusumi S, Tomonaga M et al. Cancer incidence in atomic bomb survivors. Part III. Leukemia, lymphoma and multiple myeloma, 1950–1987. Radiat Res 1994; 137: S6897.
  • 77
    Fenton HJH. Oxidation of tartaric acid in presence of iron. J Chem Soc 1894; 65: 899910.
  • 78
    Harman D. Free radical theory of aging: Role of free radicals in the origination and evolution of life, aging, and disease process. In: JohnsonJEJr, Walford R, Harman D, MiquelsJ, eds. Free Radicals, Aging, and Degenerative Diseases. New York: Liss, 1986; 349.
  • 79
    McCord JM, Fridovich I. Superoxide dismutase: An enzymatic function for erythrocuprein (hemocuprein). J Biol Chem 1969; 244: 604955.
  • 80
    Stadtman ER. Protein oxidation and aging. Science 1992; 257: 122024.
  • 81
    Uchida K. Protein-bound 4-hydroxy-2-nonenal as a marker of oxidative stress. J Clin Biochem Nutr 2005; 36: 110.
  • 82
    Steenken S. Purine bases, nucleosides, and nucleotides: Aqueous solution redox chemistry and transformation reactions of their radical reactions and e and ·OH adducts. Chem Rev 1989; 89: 50320.
  • 83
    Kasai H. Analysis of a form of oxidative DNA damage, 8-hydroxy-2′-deoxyguanosine, as a marker of cellular oxidative stress during carcinogenesis. Mutat Res 1997; 387: 14763.
  • 84
    Dizdaroglu M. Chemical determination of free radical-induced damage to DNA. Free Radic Biol Med 1991; 10: 22542.
  • 85
    Cadenas E, Sies H. Oxidative stress: Excited oxygen species and enzyme activity. Adv Enzyme Regul 1985; 23: 21737.
  • 86
    Toyokuni S, Okamoto K, Yodoi J, Hiai H. Persistent oxidative stress in cancer. FEBS Lett 1995; 358: 13.
  • 87
    Suzuki Y, Forman H, Sevanian A. Oxidants as stimulators of signal transduction. Free Radic Biol Med 1997; 22: 26985.
  • 88
    Mitra S, Boldogh I, Izumi T, Hazra T. Complexities of the DNA base excision repair pathway for repair of oxidative DNA damage. Environ Mol Mutagen 2001; 38: 18090.
  • 89
    Nakabeppu Y, Tsuchimoto D, Furuichi M, Sakumi K. The defense mechanisms in mammalian cells against oxidative damage in nucleic acids and their involvement in the suppression of mutagenesis and cell death. Free Radic Res 2004; 38: 4239.
  • 90
    Beckman J, Koppenol W. Nitric oxide, superoxide, and peroxynitrite: The good, the bad, and ugly. Am J Physiol 1996; 271: C142437.
  • 91
    Toyokuni S. Reactive oxygen species-induced molecular damage and its application in pathology. Pathol Int 1999; 49: 91102.
  • 92
    Humphries K, Szweda P, Szweda L. Aging: A shift from redox regulation to oxidative damage. Free Radic Res 2006; 40: 123943.
  • 93
    Hopkins FG, Dixon M. On glutathione. II. A thermostable oxidation-reduction system. J Biol Chem 1922; 54: 52763.
  • 94
    Tagaya Y, Okada M, Sugie K et al. IL-2 receptor (p55)/Tac-inducing factor. Purification and characterization of adult T cell leukemia-derived factor. J Immunol 1988; 140: 261420.
  • 95
    Sen C, Packer L. Antioxidant and redox regulation of gene transcription. FASEB J 1996; 10: 70920.
  • 96
    Nakamura H, Nakamura K, Yodoi J. Redox regulation of cellular activation. Annu Rev Immunol 1997; 15: 35169.
  • 97
    Nishiyama A, Matsui M, Iwata S et al. Identification of thioredoxin-binding protein-2/vitamin D3 up-regulated protein 1 as a negative regulator of thioredoxin function and expression. J Biol Chem 1999; 274: 21 645–50.
  • 98
    Nishinaka Y, Nishiyama A, Masutani H et al. Loss of thioredoxin-binding protein-2/vitamin D3 up-regulated protein 1 in human T-cell leukemia virus type I-dependent T-cell transformation: Implications for adult T-cell leukemia leukemogenesis. Cancer Res 2004; 64: 128792.
  • 99
    Ahsan M, Masutani H, Yamaguchi Y et al. Loss of interleukin-2-dependency in HTLV-I-infected T cells on gene silencing of thioredoxin-binding protein-2. Oncogene 2005; 25: 218191.
  • 100
    Ikarashi M, Takahashi Y, Ishii Y, Nagata T, Asai S, Ishikawa K. Vitamin D3 up-regulated protein 1 (VDUP1) expression in gastrointestinal cancer and its relation to stage of disease. Anticancer Res 2002; 22: 40458.
  • 101
    Han S, Jeon J, Ju H et al. VDUP1 upregulated by TGF-β1 and 1,25-dihydorxyvitamin D3 inhibits tumor cell growth by blocking cell-cycle progression. Oncogene 2003; 22: 403546.
  • 102
    Dutta KKN, Ishinaka Y, Masutani H et al. Thioredoxin-binding protein-2 is a target gene in oxidative stress-induced renal carcinogenesis. Lab Invest 2005; 85: 798807.
  • 103
    Goldberg S, Miele M, Hatta N et al. Melanoma metastasis suppression by chromosome 6: Evidence for a pathway regulated by CRSP3 and TXNIP. Cancer Res 2003; 63: 43240.
  • 104
    Bodnar J, Chatterjee A, Castellani L et al. Positional cloning of the combined hyperlipidemia gene Hyplip1. Nat Genet 2002; 30: 11016.
  • 105
    Hui T, Sheth S, Diffley J et al. Mice lacking thioredoxin interacting protein provide evidence linking cellular redox state to appropriate response to nutritional signals. J Biol Chem 2004; 279: 24 387–93.
  • 106
    Oka S, Liu W, Masutani H et al. Impaired fatty acid utilization in thioredoxin binding protein-2 (TBP-2) -deficient mice: A unique animal model of Reye syndrome. FASEB J 2005; 147: 73343.
  • 107
    Hockel M, Vaupel P. Tumor hypoxia: Definitions and current clinical, biologic, and molecular aspects. J Natl Cancer Inst 2001; 93: 26676.
  • 108
    Maruyama R, Aoki F, Toyota M et al. Comparative genome analysis identifies the vitamin D receptor gene as a direct target of p53-mediated transcriptional activation. Cancer Res 2006; 66: 457483.
  • 109
    Itoh K, Chiba T, Takahashi S et al. An Nrf2/small Maf heterodimer mediates the induction of phase II detoxifying enzyme genes through antioxidant response elements. Biochem Biophys Res Commun 1997; 236: 31322.
  • 110
    Ishii T, Itoh K, Takahashi S et al. Transcription factor Nrf2 coordinately regulates a group of oxidative stress-inducible genes in macrophages. J Biol Chem 2000; 275: 16 023–9.
  • 111
    Itoh K, Wakabayashi N, Katoh Y et al. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 1999; 13: 7686.
  • 112
    Wakabayashi N, Dinkova-Kostova A, Holtzclaw W et al. Protection against electrophile and oxidant stress by induction of the phase 2 response: Fate of cysteines of the Keap1 sensor modified by inducers. Proc Natl Acad Sci USA 2004; 101: 204045.
  • 113
    Kobayashi A, Kang M, Okawa H et al. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2. Mol Cell Biol 2004; 24: 713039.
  • 114
    Steinberg S. Distinctive activation mechanisms and functions for protein kinase Cdelta. Biochem J 2004; 384: 44959.
  • 115
    Sun X, Wu F, Datta R, Kharbanda S, Kufe D. Interaction between protein kinase C delta and the c-Abl tyrosine kinase in the cellular response to oxidative stress. J Biol Chem 2000; 275: 747073.
  • 116
    Wang X, McCullough K, Franke T, Holbrook N. Epidermal growth factor receptor-dependent Akt activation by oxidative stress enhances cell survival. J Biol Chem 2000; 275: 14 624–31.
  • 117
    Takeda K, Matsuzawa A, Nishitoh H, Ichijo H. Roles of MAPKKK ASK1 in stress-induced cell death. Cell Struct Funct 2003; 28: 239.
  • 118
    Tobiume K, Matsuzawa A, Takahashi T et al. ASK1 is required for sustained activations of JNK/p38 MAP kinases and apoptosis. EMBO Rep 2001; 2: 2228.
  • 119
    Noguchi T, Takeda K, Matsuzawa A et al. Recruitment of tumor necrosis factor receptor-associated factor family proteins to apoptosis signal-regulating kinase 1 signalosome is essential for oxidative stress-induced cell death. J Biol Chem 2005; 280: 37 033–40.
  • 120
    Matsuzawa A, Saegusa K, Noguchi T et al. ROS-dependent activation of the TRAF6-ASK1-p38 pathway is selectively required for TLR4-mediated innate immunity. Nat Immunol 2005; 6: 58792.
  • 121
    Bienert G, Schjoerring J, Jahn T. Membrane transport of hydrogen peroxide. Biochim Biophys Acta 2006; 1758: 9941003.
  • 122
    Mitsushita J, Lambeth J, Kamata T. The superoxide-generating oxidase Nox1 is functionally required for Ras oncogene transformation. Cancer Res 2004; 64: 358085.
  • 123
    Mahadev K, Motoshima H, Wu X et al. The NAD(P)H oxidase homolog Nox4 modulates insulin-stimulated generation of H2O2 and plays an integral role in insulin signal transduction. Mol Cell Biol 2004; 24: 184454.
  • 124
    Azzi A, Ricciarelli R, Zingg J. Non-antioxidant molecular functions of alpha-tocopherol (vitamin E). FEBS Lett 2002; 519: 810.
  • 125
    Munteanu A, Zingg J, Ogru E et al. Modulation of cell proliferation and gene expression by alpha-tocopheryl phosphates: Relevance to atherosclerosis and inflammation. Biochem Biophys Res Commun 2004; 318: 31116.
  • 126
    Lee W, Akatsuka S, Shirase T et al. alpha-Tocopherol induces calnexin in renal tubular cells: Another protective mechanism against free radical-induced cellular damage. Arch Biochem Biophys 2006; 453: 16878.
  • 127
    Palmer R, Ferrige A, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987; 327: 5246.
  • 128
    Stamler J, Lamas S, Fang F. Nitrosylation: The prototypic redox-based signaling mechanism. Cell 2001; 106: 67583.
  • 129
    Yasinska I, Sumbayev V. S-nitrosation of Cys-800 of HIF-1alpha protein activates its interaction with p300 andstimulates its transcriptional activity. FEBS Lett 2003; 549: 1059.
  • 130
    Hara M, Agrawal N, Kim S et al. S-nitrosylated GAPDH initiates apoptotic cell death by nuclear translocation following Siah1 binding. Nat Cell Biol 2005; 7: 66574.
  • 131
    Matsushita K, Morrell C, Cambien B et al. Nitric oxide regulates exocytosis by S-nitrosylation of N-ethylmaleimide-sensitive factor. Cell 2003; 115: 13950.
  • 132
    Minetti M, Mallozzi C, Di Stasi A. Peroxynitrite activates kinases of the src family and upregulates tyrosine phosphorylation signaling. Free Radic Biol Med 2002; 33: 74454.
  • 133
    Otterbein L, Bach F, Alam J et al. Carbon monoxide has anti-inflammatory effects involving the mitogen-activated protein kinase pathway. Nat Med 2000; 6: 4228.
  • 134
    Suematsu M, Goda N, Sano T et al. Carbon monoxide: An endogenous modulator of sinusoidal tone in the perfused rat liver. J Clin Invest 1995; 96: 24317.
  • 135
    Hosoki R, Matsuki N, Kimura H. The possible role of hydrogen sulfide as an endogenous smooth muscle relaxant in synergy with nitric oxide. Biochem Biophys Res Commun 1997; 237: 52731.
  • 136
    Akatsuka S, Aung TT, Dutta KK et al. Contrasting genome-wide distribution of 8-hydroxyguanine and acrolein-modified adenine during oxidative stress-induced renal carcinogenesis. Am J Pathol 2006; 169: 132842.
  • 137
    Ohshima H, Tatemichi M, Sawa T. Chemical basis of inflammation-induced carcinogenesis. Arch Biochem Biophys 2003; 417: 311.
  • 138
    Nakabeppu Y. Regulation of intracellular localization of human MTH1, OGG1, and MYH proteins for repair of oxidative DNA damage. Prog Nucleic Acid Res Mol Biol 2001; 68: 7594.
  • 139
    Vogelstein B, Kinzler KW. The Genetic Basis of Human Cancer. New York: McGraw-Hill, 1998.
  • 140
    Nishiyama Y, Suwa H, Okamoto K, Fukumoto M, Hiai H, Toyokuni S. Low incidence of point mutations in H-, K- and N-ras oncogenes and p53 tumor suppressor gene in renal cell carcinoma and peritoneal mesothelioma of Wistar rats induced by ferric nitrilotriacetate. Jpn J Cancer Res 1995; 86: 115058.
  • 141
    Toyokuni S, Mori T, Dizdaroglu M. DNA base modifications in renal chromatin of Wistar rats treated with a renal carcinogen, ferric nitrilotriacetate. Int J Cancer 1994; 57: 1238.
  • 142
    Toyokuni S, Uchida K, Okamoto K, Hattori-Nakakuki Y, Hiai H, Stadtman ER. Formation of 4-hydroxy-2-nonenal-modified proteins in the renal proximal tubules of rats treated with a renal carcinogen, ferric nitrilotriacetate. Proc Natl Acad Sci USA 1994; 91: 261620.
  • 143
    Toyokuni S, Luo XP, Tanaka T, Uchida K, Hiai H, Lehotay DC. Induction of a wide range of C2-12 aldehydes and C7-12 acyloins in the kidney of Wistar rats after treatment with a renal carcinogen, ferric nitrilotriacetate. Free Radic Biol Med 1997; 22: 101927.
  • 144
    Ozeki M, Miyagawa-Hayashino A, Akatsuka S et al. Susceptibility of actin to modification by 4-hydroxy-2-nonenal. J Chromatogr B Analyt Technol Biomed Life Sci 2005; 827: 11926.
  • 145
    Lynch M, Walsh B. Genetics and Analysis of Quantitative Traits. Sunderland, MA, USA: Sinauer Associates, 1998.
  • 146
    Tanaka T, Iwasa Y, Kondo S, Hiai H, Toyokuni S. High incidence of allelic loss on chromosome 5 and inactivation of p15INK4B and p16INK4A tumor suppressor genes in oxystress-induced renal cell carcinoma of rats. Oncogene 1999; 18: 37937.
  • 147
    Kamijo T, Weber JD, Zambetti G, Zindy F, Roussel MF, Sherr CJ. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc Natl Acad Sci USA 1998; 95: 82927.
  • 148
    Chan FK, Zhang J, Cheng L, Shapiro DN, Winoto A. Identification of human and mouse p19, a novel CDK4 and CDK6 inhibitor with homology to p16ink4. Mol Cell Biol 1995; 15: 26828.
  • 149
    Hiroyasu M, Ozeki M, Kohda H et al. Specific allelic loss of p16INK4A tumor suppressor gene after weeks of iron-mediated oxidative damage during rat renal carcinogenesis. Am J Pathol 2002; 160: 41924.
  • 150
    Oikawa S, Kawanishi S. Site-specific DNA damage at GGG sequence by oxidative stress may accelerate telomere shortening. FEBS Lett 1999; 453: 3658.
  • 151
    Von Zglinicki T, Pilger R, Sitte N. Accumulation of single-strand breaks is the major cause of telomere shortening in human fibroblasts. Free Radic Biol Med 2000; 28: 6474.
  • 152
    Toyokuni S, Tanaka T, Hattori Y et al. Quantitative immunohistochemical determination of 8-hydroxy-2′-deoxyguanosine by a monoclonal antibody N45.1: Its application to ferric nitrilotriacetate-induced renal carcinogenesis model. Lab Invest 1997; 76: 36574.
  • 153
    Kawai Y, Furuhata A, Toyokuni S, Aratani Y, Uchida K. Formation of acrolein-derived 2′-deoxyadenosine adduct in an iron-induced carcinogenesis model. J Biol Chem 2003; 278: 50 346–54.
  • 154
    Cremer T, Cremer C. Chromosome territories, nuclear architecture and gene regulation in mammalian cells. Nat Rev Genet 2001; 2: 292301.
  • 155
    Tanabe H, Muller S, Neusser M et al. Evolutionary conservation of chromosome territory arrangements in cell nuclei from higher primates. Proc Natl Acad Sci USA 2002; 99: 44249.
  • 156
    Parada L, McQueen P, Misteli T. Tissue-specific spatial organization of genomes. Genome Biol 2004; 5: R44.
  • 157
    Meaburn KJ, Misteli T. Chromosome territories. Nature 2007; 445: 37981.
  • 158
    Dietrich L, Ungermann C. On the mechanism of protein palmitoylation. EMBO Rep 2004; 5: 10537.
  • 159
    Fratelli M, Demol H, Puype M et al. Identification by redox proteomics of glutathionylated proteins in oxidatively stressed human T lymphocytes. Proc Natl Acad Sci USA 2002; 99: 350510.
  • 160
    Ishii T, Uchida K. Induction of reversible cysteine-targeted protein oxidation by an endogenous electrophile 15-deoxy-delta12,14-prostaglandin J2. Chem Res Toxicol 2004; 17: 131322.
  • 161
    Levine R, Williams J, Stadtman E, Shacter E. Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 1994; 233: 34657.
  • 162
    Oh-Ishi M, Ueno T, Maeda T. Proteomic method detects oxidatively induced protein carbonyls in muscles of a diabetes model Otsuka Long-Evans Tokushima Fatty (OLETF) rat. Free Radic Biol Med 2003; 34: 1122.
  • 163
    Toyokuni S, Miyake N, Hiai H et al. The monoclonal antibody specific for the 4-hydroxy-2-nonenal histidine adduct. FEBS Lett 1995; 359: 18991.
  • 164
    Gayarre J, Sanchez D, Sanchez-Gomez F, Terron M, Llorca O, Perez-Sala D. Addition of electrophilic lipids to actin alters filament structure. Biochem Biophys Res Commun 2006; 349: 138793.
  • 165
    Okada K, Wangpoengtrakul C, Osawa T, Toyokuni S, Tanaka K, Uchida K. 4-Hydroxy-2-nonenal-mediated impairment of intracellular proteolysis during oxidative stress. Identification of proteasomes as target molecules. J Biol Chem 1999; 274: 23 787–93.
  • 166
    Montine T, Amarnath V, Martin M, Strittmatter W, Graham D. E-4-hydroxy-2-nonenal is cytotoxic and cross-links cytoskeletal proteins in P19 neuroglial cultures. Am J Pathol 1996; 148: 8993.
  • 167
    Montine K, Olson S, Amarnath V, Whetsell W, Graham D, Montine T. Immunohistochemical detection of 4-hydroxy-2-nonenal adducts in Alzheimer's disease is associated with inheritance of APOE4. Am J Pathol 1997; 150: 43743.
  • 168
    Humphries K, Szweda L. Selective inactivation of alpha-ketoglutarate dehydrogenase and pyruvate dehydrogenase: Reaction of lipoic acid with 4-hydroxy-2-nonenal. Biochemistry 1998; 37: 15 835–41.
  • 169
    Lauderback C, Hackett J, Huang F et al. The glial glutamate transporter, GLT-1, is oxidatively modified by 4-hydroxy-2-nonenal in the Alzheimer's disease brain: The role of Abeta1-42. J Neurochem 2001; 78: 41316.
  • 170
    Ji C, Kozak K, Marnett L. IkappaB kinase, a molecular target for inhibition by 4-hydroxy-2-nonenal. J Biol Chem 2001; 276: 18 223–8.
  • 171
    Toyokuni S, Yamada S, Kashima M et al. Serum 4-hydroxy-2-nonenal-modified albumin is elevated in patients with type 2 diabetes mellitus. Antioxid Redox Signal 2000; 2:6815.
  • 172
    Perluigi M, Fai Poon H, Hensley K et al. Proteomic analysis of 4-hydroxy-2-nonenal-modified proteins in G93A-SOD1 transgenic mice: A model of familial amyotrophic lateral sclerosis. Free Radic Biol Med 2005; 38: 96068.
  • 173
    Tanito M, Haniu H, Elliott M, Singh A, Matsumoto H, Anderson R. Identification of 4-hydroxynonenal-modified retinal proteins induced by photooxidative stress prior to retinal degeneration. Free Radic Biol Med 2006; 41: 184759.
  • 174
    Carbone D, Doorn J, Kiebler Z, Ickes B, Petersen D. Modification of heat shock protein 90 by 4-hydroxynonenal in a rat model of chronic alcoholic liver disease. J Pharmacol Exp Ther 2005; 315: 815.
  • 175
    Eddy S. Non-coding RNA genes and the modern RNA world. Nat Rev Genet 2001; 2: 91929.
  • 176
    Sevignani C, Calin G, Siracusa L, Croce C. Mammalian microRNAs: A small world for fine-tuning gene expression. Mamm Genome 2006; 17: 189202.
  • 177
    Mason R. Using anti-5,5-dimethyl-1-pyrroline N-oxide (anti-DMPO) to detect protein radicals in time and space with immuno-spin trapping. Free Radic Biol Med 2004; 36: 121423.
  • 178
    Ramirez D, Mejiba S, Mason R. Immuno-spin trapping of DNA radicals. Nat Methods 2006; 3: 1237.