8-Hydroxydeoxyguanosine: Not mere biomarker for oxidative stress, but remedy for oxidative stress-implicated gastrointestinal diseases

Abstract

Reactive oxygen species (ROS) attack guanine bases in DNA easily and form 8-hydroxydeoxyguanosine (8-OHdG), which can bind to thymidine rather than cytosine, based on which, the level of 8-OHdG is generally regarded as a biomarker of mutagenesis consequent to oxidative stress. For example, higher levels of 8-OHdG are noted in Helicobacter pylori-associated chronic atrophic gastritis as well as gastric cancer. However, we have found that exogenous 8-OHdG can paradoxically reduce ROS production, attenuate the nuclear factor-κB signaling pathway, and ameliorate the expression of proinflammatory mediators such as interleukin (IL)-1, IL-6, cyclo-oxygenase-2, and inducible nitric oxide synthase in addition to expression of nicotinamide adenine dinucleotide phosphate oxidase (NOX)-1, NOX organizer-1 and NOX activator-1 in various conditions of inflammation-based gastrointestinal (GI) diseases including gastritis, inflammatory bowel disease, pancreatitis, and even colitis-associated carcinogenesis. Our recent finding that exogenous 8-OHdG was very effective in either inflammation-based or oxidative-stress-associated diseases of stress-related mucosal damage has inspired the hope that synthetic 8-OHdG can be a potential candidate for the treatment of inflammation-based GI diseases, as well as the prevention of inflammation-associated GI cancer. In this editorial review, the novel fact that exogenous 8-OHdG can be a functional molecule regulating oxidative-stress-induced gastritis through either antagonizing Rac-guanosine triphosphate binding or blocking the signals responsible for gastric inflammatory cascade is introduced.

Key Words: 8-hydroxydeoxyguanosine, Oxidative stress, Inflammation, Carcinogenesis, Prevention

INTRODUCTION

When we consume a soft drink or eat a meal, the gastrointestinal (GI) mucosa is continuously stressed with various antigens that we have ingested because the GI lumen is actually outside the body[1]. As far as the interaction between materials from outside and the physiological barriers of the GI tract is concerned, everything existing outside the GI lumen can cause stressful reactions at the cellular level through mechanisms including antigen challenge, concurring oxidative stress, and some inflammatory assaults. The GI lumen can come under attack by many factors, including solid food, Helicobacter pylori (H. pylori) and other commensal bacteria, non-steroidal anti-inflammatory drugs, and gastric acid[2-4]. In particular, one important means of attack is mediated by oxygen, which is also the most important metabolic substance in the body. In spite of being essential for life, oxygen can cause various cellular stresses called oxidative stress by generating reactive oxygen species (ROS)[5]. The definition of oxidative stress is a disturbance of oxidant-antioxidant homeostasis, leading to potential cellular damage. Even though the existence of ROS and their pathological implications were discovered < 50 years ago, it is surprising that many diseases can be explained by oxidative stress and its subsequent dysregulation. Although ROS are a crucial regulator of cellular signal transduction and energy transmission, disturbance of the balance between generating and scavenging capability of ROS might lead to cell damage. ROS can react with cellular proteins or lipids, transforming them into oxidized forms, or bind with nucleic acids, turning them into mutated forms. It is particularly interesting that oxidative stress is closely associated with carcinogenesis.

Fortunately, to cope with these harmful effects of oxidative stress, cells may endeavor to enhance defensive factors of various types. The common examples of defense factors are reduced glutathione/oxidized glutathione, superoxide dismutase, catalase, heme oxygenase 1, G protein gamma-like, and nicotinamide adenine dinucleotide phosphate (NADPH) dehydrogenase, quinine 1[6-8]. However, disease can occur if there are insufficient defense factors or overwhelming offensive factors. Therefore, if we set up a sensitive marker that predicts the degree of oxidative stress, we can prevent the initiation or progression of disease by measuring this marker[9]. Moreover, if the level of this marker reflects the severity of disease, appropriate levels of scavenging agents can prevent complications of the disease, because the extreme is organ dysfunction or cancer. Several biomarkers to estimate oxidative stress have been suggested, but most of them have failed to reach clinical significance. One successful discovery of the late 1980s was the level of 8-hydroxydeoxyguanosine (8-oxo-7, 8-dihydroguanosine, 8-OHdG), because it has been proven to be increased in serum or urine of patients who have oxidative-stress-associated disease[10].

8-OHdG: GENERATION AND SIGNIFICANCE AS A BIOMARKER FOR OXIDATIVE STRESS

Generation and metabolism of 8-OHdG

When DNA is attacked by oxidative stress such as ROS, ultraviolet light, or genotoxic agents, guanine is easily oxidized into 8-oxo-7,8-dihydroguanine (8-oxo-Gua)[11]. The existence of this oxidized guanine in genomic DNA can cause transversion mutation such as G-T or G-A binding, accumulation of which can lead to detrimental consequences[12-14]. Fortunately, mammalian cells have multiple repair systems such as base excision repair enzymes or nucleotide excision repair (NER) enzymes, which counteract the hazardous effects of 8-oxo-Gua. Consequently, 8-OHdG, a nucleoside form of 8-oxo-Gua, is generated from either damaged oligomer which contains 8-oxo-Gua by NER or from cytoplasmic oxidized nucleotides like 8-hydroxy-deoxyguanosine triphosphate (8-hydroxy-dGTP). Fortunately, exogenously administered 8-OHdG cannot reincorporate into genomic DNA because the activity of deoxynucleotide kinase which converts 8-OHdG into 8-hydroxy-dGTP is very low, although wild deoxyguanosine can be actively converted to deoxyguanosine triphosphate which can be used as a substrate of DNA polymerase[15-22].

Biological significance of 8-OHdG

8-OHdG can cross the cell membrane unlike any other species that contains oxidized guanine, thus, it is usually detected in the urine or serum of patients who have diseases associated with oxidative stress[23]. Examples of application of 8-OHdG as a disease-associated clinical marker are summarized in Table 1.

Table 1 Application of 8-hydroxydeoxyguanosine as a potential biomarker for various clinical diseases.

Diseases	References
Helicobacter pylori infection	Hahm et al[58], Baik et al[59]
Colorectal tumor	Sato et al[60], Gushima et al[61]
Breast cancer	Matsui et al[62], Djuric et al[63], Musarrat et al[64]
Bladder/prostate cancer	Chiou et al[65]
Lung cancer	Erhola et al[66]
Atherosclerosis	Martinet et al[67]
Diabetes	Kanauchi et al[68]
Smoking	Asami et al[69], Kiyosawa et al[70]

8-OHdG: NEW FOCUS ON PARADOXICALLY RELIEVING ACTION OF OXIDATIVE STRESS

Anti-oxidative and anti-inflammatory actions of 8-OHdG

Although many studies have shown increased levels of 8-OHdG in oxidative-stress-associated diseases, the exact biological role of 8-OHdG has not been investigated. Oxidized deoxyguanosine is notorious for inducing mutagenesis, therefore, most researchers have felt that 8-OHdG might have mutagenic or at least harmful effects in cells, and that is why mammalian physiology tries hard to excrete this oxidized guanosine. However, under the innovative hypothesis that the generation of this molecule can be one of the defense mechanisms of cells against oxidative-stress-induced inflammation, we have tried to obtain evidence that oxidized guanosine can interact with the GTPase family, which is broadly involved in cytoskeleton modification, triggering inflammation, regulating apoptosis, and carcinogenesis[24-27]. Interestingly, genetically modified oxidized GTP, 8-oxo-GTPγS, seems to interact with the small GTPase family such as Ras, Rho, Rac and cdc42[28]. Among these, we have focused on the role of Rac1 in inflammatory cascades[29,30] because Rac1 activation is crucial for aggregating NADPH oxidase (NOX) complex and subsequent ROS production[31]. As a result, we have concluded that inhibition of Rac1 by exogenous 8-OHdG, which is a transmittable form of oxidized guanosine, can significantly block ROS-mediated inflammation. Compared with other nucleoside products, 8-OHdG has a potent anti-inflammatory effect by inhibiting the activity of Rac1 on lipopolysaccharide (LPS)-stimulated microglial cells, chemokine-activated neutrophils, and inflammatory mediator-stimulated macrophages[21,32-34]. Moreover, since endogenously produced 8-OHdG is much lower than exogenously treated concentrations of 8-OHdG, we propose the biological role of the antioxidative and anti-inflammatory actions of 8-OHdG, implying that 8-OHdG formation can be a defense mechanism against oxidative stress, and enrichment with exogenous 8-OHdG can be a strategy to prevent the initiation or progression of inflammatory disease, backed up with additional fact that only pretreatment or earlier administration of 8-OHdH is effective. To clarify and compare the cellular effect of 8-OHdG with existing anti-inflammatory agents, we have also investigated several animal models of acute inflammation. Intraperitoneal LPS injection to mice causes severe inflammation in lung tissues by inducing tumor necrosis factor (TNF)-α, interleukins, and myeloperoxidase activity and recruiting neutrophils. Simultaneous treatment with 8-OHdG significantly decreases the level of the above markers, and the efficacy of 8-OHdG is even more potent than that of aspirin; a conventional anti-inflammatory agent[35]. We also have investigated the antiallergic effects of 8-OHdG in ovalbumin-sensitized mice[36,37]. One of the major phenomena of oxidative stress is stress-related mucosal disease (SRMD), the mechanism of which is mucosal damage induced by ROS involved in reperfusion injury after local ischemia. We established a water-immersion restraint stress model, which mimics SRMD, causing severe ulceration and hemorrhagic lesions in gastric mucosa. Treatment with 8-OHdG reduces the pathological lesions, as well as other angiogenesis mediators such as TNF-α and vascular endothelial growth factor[21]. Recently, we have established an animal model of H. pylori-infected gastric inflammation by infecting four times with bacteria, followed by ingestion of a high-salt diet for > 16 wk. The degree of gastric inflammation induced by H. pylori is significantly decreased by continuous ingestion of 8-OHdG-dissolved water ad libitum. Moreover, dysplastic and precancerous lesions are observed in this animal model with chronic H. pylori infection, but curiously, the carcinogenesis that results from chronic H. pylori infection is apparently ameliorated with exogenous administration of 8-OHdG (Figure 1).

Figure 1 Antitumorigenic action of exogenous 8-hydroxydeoxyguanosine on Helicobacter pylori-induced gastric tumorigenesis. Helicobacter pylori (H. pylori) infection with additional administration of high-salt diet resulted in gastric tumorigenesis in interleukin-10 knockout mice. Continuous administration of exogenous 8-hydroxydeoxyguanosine (8-OHdG) led to significant prevention of H. pylori-induced gastric tumorigenesis as well as amelioration of gastritis. H. pylori infection followed with high-salt diet resulted in significant tumorigenesis as well as atrophic changes in gastric mucosa, whereas the gastric mucosa co-treated with 8-OHdG was free from tumorigenesis or atrophic changes.

Molecular mechanisms to impose anti-inflammatory action of 8-OHdG

Although the possible mechanism to explain how 8-OHdG inhibits the activity of Rac1 has not been clearly documented yet, we hypothesize that 8-OHdG can interfere with the GTP-binding pocket of Rac1, because GTP and 8-OHdG share a similar conformation with a guanine base. 8-OHdG does not affect the activity of phosphoinositide 3-kinase/AKT or that of Rac1-guanosine exchange factor, which is an upstream pathway of Rac1 activation by exchanging Rac1-bound-GDP into GTP, turning inactive Rac1 into the active form[38-40]. However, treatment with 8-OHdG dramatically decreases the portion of Rac1-GTP, implying the specific molecular target of 8-OHdG might be Rac1 inactivation[21]. Rac1 is a crucial mediator of activating NOX complex[41,42], therefore, inactivating Rac1 could decrease the generation of ROS, and block the redox-sensitive nuclear factor (NF)-κB pathway. Rac1 also directly binds to signal transducer and activator of transcription (STAT)3 and regulates its activity[43], therefore, the ratio of phosphorylated to total STAT3 would be decreased by treatment with 8-OHdG[32]. The regulation of Rac1-mediated ROS, NF-κB and STAT3, which are the important mediators of inflammatory cascades, is the latest possibility of how 8-OHdG exerts an anti-inflammatory action.

Antitumorigenic action of 8-OHdG based on an efficient anti-inflammatory action

8-oxoguanine DNA glycosylase 1 (OGG1) is an endogenous DNA repair enzyme that repairs 8-OH-Gua in genomic DNA, cutting it into 8-OHdG (Figure 2). Although 8-OHdG does not reincorporate into genomic DNA as explained above, exogenously administered 8-OHdG can increase the ratio of 8-OH-Gua in genomic DNA by increasing error-prone DNA polymerase in the specific types of cells that have mutated forms of OGG1[19,44]. Although wild-type OGG1 can correct and repair 8-OH-Gua, OGG1 mutation cannot remove this harmful oxidized guanine, causing mutation and apoptosis. Treatment with 8-OHdG induces G1 arrest and apoptosis in KG-1 leukemia cells, which have an OGG1 mutation, but not in U937 leukemia cells, which have wild-type OGG1[45-48]. The wide profile of OGG1 mutations in human cancer is now actively under investigation[49-54], therefore, treatment of cancer that has OGG1 mutations with 8-OHdG might be developed as a new model of targeted chemotherapy. For example, we have investigated models of colitis induced by dextran sodium sulfate (DSS) and colitis-associated cancer induced by DSS combined with azoxymethane (AOM + DSS)[55-57], and determined whether exogenous 8-OHdG has a preventive effect on colitis and colitis-associated cancer[57]. Daily injection of 8-OHdG significantly inhibits recruitment of inflammatory cells in DSS-induced colitis, and chronic ingestion of diet containing 8-OHdG exerts a chemopreventive role in AOM + DSS-induced colitis-associated cancer (Figure 3)[47].

Figure 2 Generation and metabolism of 8-hydroxydeoxyguanosine. 8-oxoguanine DNA glycosylase 1 (OGG1) is a DNA glycosylase enzyme involved in base excision repair (BER), and is the primary enzyme responsible for excision of 7,7-dihydro-8-oxoguanine that occurs as a result of exposure to reactive oxygen species (ROS). OGG1 is a bifunctional glycosylase, because it is able to both cleave the glycosidic bond of the mutagenic lesion, and cause strand breakage in the DNA backbone. Nucleotide excision repair (NER) is a DNA repair mechanism, which is a particularly important mechanism by which the cell can prevent unwanted mutations by removing the vast majority of ROS-induced DNA damage.

Figure 3 Ameliorating effect of exogenous 8-hydroxydeoxyguanosine against dextran sodium sulfate-induced colitis. Dextran sodium sulfate (DSS) administration resulted in moderate to severe colitis manifested as significantly shortened colon length. However, colon length was significantly preserved by exogenous administration of 8-hydroxydeoxyguanosine (8-OHdG) (see arrows to compare the colon length), as shown by the pathological observation that the degree of DSS-induced colitis was apparently improved, suggesting that exogenous 8-OHdG had a significant preventive effect against DSS-induced colitis[47]. Eighteen mice were divided into three groups of six: a non-treated control group (Normal group); 5% DSS in tap water ingestion for 1 wk (DSS group); and DSS with daily injection of 8-OHdG (DSS + 8-OHdG group). 8-OHdG powder was dissolved in PBS and the Normal and DSS groups were treated as a negative control. Clinical phenotypes such as hematochezia and rectal prolapse were investigated and charted daily. There was no mortality observed in any of the groups. After 7 d DSS ingestion, all mice were killed and colons were removed, opened longitudinally, and rinsed with phosphate buffer solution. The lengths of colon were measured, and isolated tissues were subjected to histological examination.

CONCLUSION

There is extensive experimental evidence that oxidative damage permanently occurs to lipids of cellular membranes, proteins and DNA. In nuclear and mitochondrial DNA, 8-OHdG or 8-oxodG is one of the predominant agents of free-radical-induced oxidative lesions. This is why 8-OHdG has been used widely in many studies as a biomarker for the measurement of endogenous oxidative DNA damage, and as a risk factor for many diseases including cancer, because urinary 8-OHdG is a good biomarker for risk assessment of various cancers and other degenerative diseases. Several lines of evidence[7,29-32,47] show that exogenous 8-OHdG can: inhibit allergy-induced inflammation; remodel airway and lung tissues through Rac inactivation; regulate oxidative-stress-induced gastritis through antagonizing Rac-GTP binding or blocking the signals responsible for gastric inflammatory cascade; play a role in anti-inflammatory actions via suppression of intercellular adhesion molecule-1 gene expression by blockade of the Toll-like receptor 4/STAT3 signal cascade in inflammation-enhanced brain microglia; and prevent colitis-associated carcinogenesis based on efficient TNF-α inhibition or H. pylori-associated gastric carcinogenesis based on inhibition of cytokine generation. All this clearly suggests that 8-OHdG could be used as a potential tool to modulate GI tract inflammation as well as allergy-related bronchial disease, and is especially applicable to diverse inflammation-based diseases including gastritis, colitis, and esophagitis, as well as GI cancer, including esophageal, gastric and colon cancer, for which more extensive and well-designed clinical trials are required.