Prolongation of oxidative stress by long-lived reactive protein species induced by X-ray radiation and their genotoxic action

Abstract The formation of long-lived reactive protein species of bovine serum albumin (BSA), ovalbumin, casein and casein hydrolyzate with a half-life of 3-5 hours was shown using chemiluminescence induced by X-ray radiation. It was found that long-lived reactive protein species are capable of generating reactive oxygen species (ROS) (H(2)O(2), OH(?), HO(2)(?, 1)O(2)) in the aquatic environment over a long period of time in vitro. The interaction of X-ray-irradiated BSA with DNA in vitro led to the formation of 8-oxoguanine (8-oxo-7,8-dihydroguanine), a biomarker of oxidative damage to DNA. Some natural antioxidants are effective scavengers of ROS (inosine, tryptophan, methionine and ascorbate). They protect DNA from the action of long-lived reactive protein species leading to ROS generation and the formation of 8-oxoguanine. The intravenous injection of X-ray radiation-induced, long-lived reactive protein species to rats, as well as the peroral and intraperitoneal administration of these products to mice, gave rise to cytogenetic injuries in the cells of their red bone marrow through the formation of micronuclei in polychromatophilic erythrocytes. The administration of the same natural antioxidants used for in vitro experiments soon after irradiation made it possible to effectively eliminate the genotoxic action of oxidative stress caused by radiation-induced, long-lived reactive protein species. Our data represent clear evidence that the oxidative damage to proteins induced by X-rays is directly involved in the induction of a response to DNA damage in rodents.     

Heat-induced formation of reactive oxygen species and 8-oxoguanine, a biomarker of damage to DNA

Heat-induced formation of 8-oxoguanine was demonstrated in DNA solutions in 10^–3 M phosphate buffer, pH 6.8, by enzyme-linked immunosorbent assays using monoclonal antibodies against 8-oxoguanine. A radiation-chemical yield of 3.7 × 10^–2 µmol J^–1 for 8-oxoguanine production in DNA upon γ-irradiation was used as an adequate standard for quantitation of 8-oxoguanine in whole DNA. The initial yield of heat-induced 8-oxoguanine exhibits first order kinetics. The rate constants for 8-oxoguanine formation were determined at elevated temperatures; the activation energy was found to be 27 ± 2 kcal/mol. Extrapolation to 37°C gave a value of k₃₇ = 4.7 × 10^–10 s^–1. Heat-induced 8-oxoguanine formation and depurination of guanine and adenine show similarities of the processes, which implies that heat-mediated generation of reactive oxygen species (ROS) should occur. Heat-induced production of H₂O₂ in phosphate buffer was shown. The sequence of reactions of thermally mediated ROS formation have been established: activation of dissolved oxygen to the singlet state, generation of superoxide radicals and their dismutation to H₂O₂. Gas saturation (O₂, N₂ and Ar), D₂O, scavengers of ¹O₂, O₂^–• and OH^• radicals and metal chelators influenced heat-induced 8-oxoguanine formation as they affected thermal ROS generation. These findings imply that heat acts via ROS attack leading to oxidative damage to DNA.     

Guanosine and inosine display antioxidant activity, protect DNA in vitro from oxidative damage induced by reactive oxygen species, and serve as radioprotectors in mice

The effect of ribonucleosides on 8-oxoguanine formation in salmon sperm DNA dissolved in 1 mM phosphate buffer, pH 6.8, upon exposure to gamma rays was examined by ELISA using monoclonal antibodies against 8-oxoguanine. Nucleosides (1 mM) decreased the radiation-induced yield of 8-oxoguanine in the order Guo > Ino > Ado > Thd > Urd > Cyd. Guanosine and inosine considerably reduced deamination of cytosine in the DNA solutions upon heating for 24 h at 80 degrees C. The action of nucleosides on the heat-induced generation of reactive oxygen species in the phosphate buffer was studied. The concentration of hydrogen peroxide was measured by enhanced chemiluminescence in a peroxidase-luminol-p-iodophenol system; the hydroxyl radical formation was measured fluorometrically by the use of coumarin-3-carboxylic acid. Guanosine and inosine considerably decreased the heat-induced production of both hydrogen peroxide and OH radicals. Guanosine and inosine increased survival of mice after a lethal dose of radiation. They especially enhanced the survival of animals when were administered shortly after irradiation. The results indicate that guanosine and inosine, natural antioxidants, prevent oxidative damage to DNA, decrease the generation of ROS, and protect mice against gamma-radiation-induced death.     

Efficacy and site specificity of hydrogen abstraction from DNA 2-deoxyribose by carbonate radicals

The carbonate radical anion CO₃^?? is a potent reactive oxygen species (ROS) produced in vivo through enzymatic one-electron oxidation of bicarbonate or, mostly, via the reaction of CO₂ with peroxynitrite. Due to the vitally essential role of the carbon dioxide/bicarbonate buffer system in regulation of physiological pH, CO₃^?? is arguably one of the most important ROS in biological systems. So far, the studies of reactions of CO₃^?? with DNA have been focused on the pathways initiated by oxidation of guanines in DNA. In this study, low-molecular products of attack of CO₃^?? on the sugar–phosphate backbone in vitro were analyzed by reversed phase HPLC. The selectivity of damage in double-stranded DNA (dsDNA) was found to follow the same pattern C4′ > C1′ > C5′ for both CO₃^?? and the hydroxyl radical, though the relative contribution of the C1′ damage induced by CO₃^?? is substantially higher. In single-stranded DNA (ssDNA) oxidation at C1′ by CO₃^?? prevails over all other sugar damages. An approximately 2000-fold preference for 8-oxoguanine (8oxoG) formation over sugar damage found in our study identifies CO₃^?? primarily as a one-electron oxidant with fairly low reactivity toward the sugar–phosphate backbone.     

Phloroglucinol (1,3,5-trihydroxybenzene) protects against ionizing radiation-induced cell damage through inhibition of oxidative stress in vitro and in vivo

Exposure of cells to γ-rays induces the production of reactive oxygen species (ROS) that play a main role in ionizing radiation damage. We have investigated the radioprotective effect of phloroglucinol (1,3,5-trihydroxybenzene), phlorotannin compound isolated from Ecklonia cava, against γ-ray radiation-induced oxidative damage in vitro and in vivo. Phloroglucinol significantly decreased the level of radiation-induced intracellular ROS and damage to cellular components such as the lipid, DNA and protein. Phloroglucinol enhanced cell viability that decreased after exposure to γ-rays and reduced radiation-induced apoptosis via inhibition of mitochondria mediated caspases pathway. Phloroglucinol reduced radiation-induced loss of the mitochondrial membrane action potential, reduced the levels of the active forms of caspase 9 and 3 and elevated the expression of bcl-2. Furthermore, the anti-apoptotic effect of phloroglucinol was exerted via inhibition of mitogen-activated protein kinase kinase-4 (MKK4/SEK1), c-Jun NH₂-terminal kinase (JNK) and activator protein-1 (AP-1) cascades induced by radiation exposure. Phloroglucinol restored the level of reduced glutathione (GSH) and protein expression of a catalytically active subunit of glutamate-cysteine ligase (GCL), which is a rate-limiting enzyme in GSH biosynthesis. In in vivo study, phloroglucinol administration in mice provided substantial protection against death and oxidative damage following whole-body irradiation. We examined survival with exposure to various radiation doses using the intestinal crypt assay and determined a dose reduction factor (DRF) of 1.24. Based on our findings, phloroglucinol may be possibly useful as a radioprotective compound.     

Cellular protection of morin against the oxidative stress induced by hydrogen peroxide

Flavonoids are a class of secondary metabolites abundantly found in fruits and vegetables. In addition, flavonoids have been reported as potent antioxidants with beneficial effects against oxidative stress-related diseases such as cancer, aging, and diabetes. The present study was carried out to investigate the cytoprotective effects of morin (2′,3,4′,5,7-pentahydroxyflavone), a member of the flavonoid group, against hydrogen peroxide (H₂O₂)-induced DNA and lipid damage. Morin was found to prevent the cellular DNA damage induced by H₂O₂ treatment, which is shown by the inhibition of 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation (a modified form of DNA base), inhibition of comet tail (a form of DNA strand breakage), and decrease of nuclear phospho histone H2A.X expression (a marker for DNA strand breakage). In addition, morin inhibited membrane lipid peroxidation, which is detected by inhibition of thiobarbituric acid reactive substance (TBARS) formation. Morin was found to scavenge the intracellular reactive oxygen species (ROS) generated by H₂O₂ treatment in cells, which is detected by a spectrofluorometer, flow cytometry, and confocal microscopy after staining of 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Morin also induces an increase in the activity of catalase and protein expression. The results of this study suggest that morin protects cells from H₂O₂-induced damage by inhibiting ROS generation and by inducing catalase activation.     

DNA Damage Responses and Oxidative Stress in Dyskeratosis Congenita

Dyskeratosis congenita (DC) is an inherited multisystem disorder of premature aging, cancer predisposition, and bone marrow failure caused by selective exhaustion of highly proliferative cell pools. DC patients also have a poor tolerance to chemo/radiotherapy and bone marrow transplantation. Although critically shortened telomeres and defective telomere maintenance contribute to DC pathology, other mechanisms likely exist. We investigate the link between telomere dysfunction and oxidative and DNA damage response pathways and assess the effects of antioxidants. In vitro studies employed T lymphocytes from DC subjects with a hTERC mutation and age-matched controls. Cells were treated with cytotoxic agents, including Paclitaxel, Etoposide, or ionizing radiation. Apoptosis and reactive oxygen species (ROS) were assessed by flow cytometry, and Western blotting was used to measure expression of DNA damage response (DDR) proteins, including total p53, p53S15, and p21^WAF. N-acetyl-cysteine (NAC), an antioxidant, was used to modulate cell growth and ROS. In stimulated culture, DC lymphocytes displayed a stressed phenotype, characterized by elevated levels of ROS, DDR and apoptotic markers as well as a proliferative defect that was more pronounced after exposure to cytotoxic agents. NAC partially ameliorated the growth disadvantage of DC cells and decreased radiation-induced apoptosis and oxidative stress. These findings suggest that oxidative stress may play a role in the pathogenesis of DC and that pharmacologic intervention to correct this pro-oxidant imbalance may prove useful in the clinical setting, potentially alleviating untoward toxicities associated with current cytotoxic treatments.     

Selective oxidative stress induces dual damage to telomeres and mitochondria in human T cells

Oxidative stress caused by excess reactive oxygen species (ROS) accelerates telomere erosion and mitochondrial injury, leading to impaired cellular functions and cell death. Whether oxidative stress‐mediated telomere erosion induces mitochondrial injury, or vice versa, in human T cells—the major effectors of host adaptive immunity against infection and malignancy—is poorly understood due to the pleiotropic effects of ROS. Here we employed a novel chemoptogenetic tool that selectively produces a single oxygen (¹O₂) only at telomeres or mitochondria in Jurkat T cells. We found that targeted ¹O₂ production at telomeres triggered not only telomeric DNA damage but also mitochondrial dysfunction, resulting in T cell apoptotic death. Conversely, targeted ¹O₂ formation at mitochondria induced not only mitochondrial injury but also telomeric DNA damage, leading to cellular crisis and apoptosis. Targeted oxidative stress at either telomeres or mitochondria increased ROS production, whereas blocking ROS formation during oxidative stress reversed the telomeric injury, mitochondrial dysfunction, and cellular apoptosis. Notably, the X‐ray repair cross‐complementing protein 1 (XRCC1) in the base excision repair (BER) pathway and multiple mitochondrial proteins in other cellular pathways were dysregulated by the targeted oxidative stress. By confining singlet ¹O₂ formation to a single organelle, this study suggests that oxidative stress induces dual injury in T cells via crosstalk between telomeres and mitochondria. Further identification of these oxidation pathways may offer a novel approach to preserve mitochondrial functions, protect telomere integrity, and maintain T cell survival, which can be exploited to combat various immune aging‐associated diseases.     

Reactive oxygen species in vascular biology: implications in hypertension

Reactive oxygen species (ROS), including superoxide (·O₂^–), hydrogen peroxide (H₂O₂), and hydroxyl anion (OH-), and reactive nitrogen species, such as nitric oxide (NO) and peroxynitrite (ONOO^–), are biologically important O₂ derivatives that are increasingly recognized to be important in vascular biology through their oxidation/reduction (redox) potential. All vascular cell types (endothelial cells, vascular smooth muscle cells, and adventitial fibroblasts) produce ROS, primarily via cell membrane-associated NAD(P)H oxidase. Reactive oxygen species regulate vascular function by modulating cell growth, apoptosis/anoikis, migration, inflammation, secretion, and extracellular matrix protein production. An imbalance in redox state where pro-oxidants overwhelm anti-oxidant capacity results in oxidative stress. Oxidative stress and associated oxidative damage are mediators of vascular injury and inflammation in many cardiovascular diseases, including hypertension, hyperlipidemia, and diabetes. Increased generation of ROS has been demonstrated in experimental and human hypertension. Anti-oxidants and agents that interrupt NAD(P)H oxidase-driven ·O₂^– production regress vascular remodeling, improve endothelial function, reduce inflammation, and decrease blood pressure in hypertensive models. This experimental evidence has evoked considerable interest because of the possibilities that therapies targeted against reactive oxygen intermediates, by decreasing generation of ROS and/or by increasing availability of antioxidants, may be useful in minimizing vascular injury and hypertensive end organ damage. The present chapter focuses on the importance of ROS in vascular biology and discusses the role of oxidative stress in vascular damage in hypertension.     

Photo-protective properties of Lomentaria hakodatensis yendo against ultraviolet B radiation-induced keratinocyte damage

This study aims to investigate the photoprotective properties of a Lomentaria hakodatensis ethanol extract (LHE) against ultraviolet B (UVB) radiation-induced cellular damage in human HaCaT keratinocytes. LHE exhibited scavenging activity against intracellular reactive oxygen species (ROS), which were generated by either hydrogen peroxide (H₂O₂) or UVB radiation. Moreover, LHE scavenged superoxide anion generated by the xanthine/xanthine oxidase system and hydroxyl radical generated by the Fenton reaction (FeSO₄ + H₂O₂). Furthermore, LHE exhibited UVB absorptive properties and attenuated injury to cellular components (e.g., lipids, proteins and DNA), resulting from UVB-induced oxidative stress. In addition, LHE reduced apoptosis in response to UVB, as shown by decreased DNA fragmentation and the formation of apoptotic bodies. These results suggest that LHE protects human keratinocytes against UVB-induced oxidative stress by scavenging ROS and absorbing UVB rays; thereby reducing damage to biological components.     

Pyrimido[1,2-a]-purin-10(3H)-one, M1G, is less prone to artifact than base oxidation

Pyrimido[1,2-a]-purin-10(3H)-one (M₁G) is a secondary DNA damage product arising from primary reactive oxygen species (ROS) damage to membrane lipids or deoxyribose. The present study investigated conditions that might lead to artifactual formation or loss of M₁G during DNA isolation. The addition of antioxidants, DNA isolation at low temperature or non-phenol extraction methods had no statistically significant effect on the number of M₁G adducts measured in either control or positive control tissue samples. The number of M₁G adducts in nuclear DNA isolated from brain, liver, kidney, pancreas, lung and heart of control male rats were 0.8, 1.1, 1.1, 1.1, 1.8 and 4.2 M₁G/10⁸ nt, respectively. In rat liver tissue, the mitochondrial DNA contained a 2-fold greater number of M₁G adducts compared with nuclear DNA. Overall, the results from this study demonstrated that measuring M₁G is a reliable way to assess oxidative DNA damage because the number of M₁G adducts is significantly affected by the amount of ROS production, but not by DNA isolation procedures. In addition, this study confirmed that the background number of M₁G adducts reported in genomic DNA could have been overestimated by one to three orders of magnitude in previous reports.     

Assessing protein oxidation by inorganic nanoparticles with enzyme‐linked immunosorbent assay (ELISA)

Growth in the nanotechnology industry is leading to increased production of engineered nanoparticles (NPs). This has given rise to concerns about the potential adverse and toxic effects to biological system and the environment. An important mechanism of NP toxicity is oxidative stress caused by the formation of reactive oxygen species (ROS) or via direct oxidation of biomolecules. In this study, a protein oxidation assay was developed as an indicator of biomolecule oxidation by NPs. The oxidation of the protein, bovine serum albumin (BSA) was evaluated with an enzyme‐linked immunosorbent assay (ELISA) to measure the protein carbonyl derivatives formed from protein oxidation. The results showed that some NPs such as Cu(0), CuO, Mn₂O₃, and Fe(0) caused oxidation of BSA; whereas, many of the other NPs tested were not reactive or very slowly reactive with BSA. The mechanisms involved in the oxidation of BSA protein by the reactive NPs could be attributed to the combined effects of ROS‐dependent and direct protein oxidation mechanisms. The ELISA assay is a promising method for the assessment of protein oxidation by NPs, which can provide insights on NP toxicity mechanisms. Biotechnol. Bioeng. 2013; 110: 694–701. © 2012 Wiley Periodicals, Inc.     

Globular adiponectin-induced RAW 264 apoptosis is regulated by a reactive oxygen species-dependent pathway involving Bcl-2

Globular adiponectin (gAd), a truncated form of adipocyte-derived cytokine, stimulates RAW 264 cells to produce reactive oxygen species (ROS), which trigger an apoptotic cascade. In this study, we investigated the generation of intracellular and mitochondrial ROS in gAd-stimulated RAW 264 cells. Treatment with gAd efficiently induced the generation of intracellular and mitochondrial ROS, as detected by dichlorodihydrofluorescein diacetate and MitoSOX fluorescence, respectively. Furthermore, gAd treatment significantly increased 8-oxoguanine, a specific indicator of oxidative DNA damage. The transfection of RAW 264 cells with iNOS- and gp91^phox-specific small interfering RNA reduced markedly the generation of intracellular, but not mitochondrial, ROS. Quantitative PCR revealed that the expression ratio of Bcl-2 to Bax was reduced in a time-dependent manner in gAd-treated RAW 264 cells. The overexpression of Bcl-2 markedly inhibited gAd-induced apoptosis in RAW 264 cells and also reduced both the intracellular and the mitochondrial ROS generation induced by gAd treatment. Moreover, the overexpression of Bcl-2 significantly suppressed gAd-induced NO secretion and NOS activity. In addition, the inhibition of NOS activity partially reduced the oxidative DNA damage induced by gAd. Taken together, these results demonstrate that the gAd-induced apoptotic pathway acting via ROS/RNS generation involves Bcl-2.     

Protective effect of 1,2,4-benzenetriol on LPS-induced NO production by BV2 microglial cells

Summary Hydroxyhydroquinone or 1,2,4-benzenetriol (BT) detected in the beverages has a structure that coincides with the water-soluble form of a sesame lignan, sesamol. We previously showed that sesame antioxidants had neuroprotective abilities due to their antioxidant properties and/or inducible nitric oxide synthase (iNOS) inhibition. However, studies show that BT can induce DNA damage through the generation of reactive oxygen species (ROS). Therefore, we were interested to investigate the neuroprotective effect of BT in vitro and in vivo. The results showed that instead of enhancing free radical generation, BT dose-dependently (10–100 μM) attenuated nitrite production, iNOS mRNA and protein expression in lipopolysaccharide (LPS)-stimulated murine BV-2 microglia. BT significantly reduced LPS-induced NF-κB and p38 MAPK activation. It also significantly reduced the generation of ROS in H₂O₂-induced BV-2 cells and in H₂O₂-cellfree conditions. The neuroprotective effect of BT was further demonstrated in the focal cerebral ischemia model of Sprague–Dawley rat. Taken together, the inhibition of LPS-induced nitrite production might be due to the suppression of NF-κB, p38 MAPK signal pathway and the ROS scavenging effect. These effects might help to protect neurons from the ischemic injury.     

Increased Reactive Oxygen Species Formation and Oxidative Stress in Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is an autoimmune inflammatory disorder. Highly reactive oxygen free radicals are believed to be involved in the pathogenesis of the disease. In this study, RA patients were sub-grouped depending upon the presence or absence of rheumatoid factor, disease activity score and disease duration. RA Patients (120) and healthy controls (53) were evaluated for the oxidant—antioxidant status by monitoring ROS production, biomarkers of lipid peroxidation, protein oxidation and DNA damage. The level of various enzymatic and non-enzymatic antioxidants was also monitored. Correlation analysis was also performed for analysing the association between ROS and various other parameters.MethodsIntracellular ROS formation, lipid peroxidation (MDA level), protein oxidation (carbonyl level and thiol level) and DNA damage were detected in the blood of RA patients. Antioxidant status was evaluated by FRAP assay, DPPH reduction assay and enzymatic (SOD, catalase, GST, GR) and non-enzymatic (vitamin C and GSH) antioxidants.ResultsRA patients showed a higher ROS production, increased lipid peroxidation, protein oxidation and DNA damage. A significant decline in the ferric reducing ability, DPPH radical quenching ability and the levels of antioxidants has also been observed. Significant correlation has been found between ROS and various other parameters studied.ConclusionRA patients showed a marked increase in ROS formation, lipid peroxidation, protein oxidation, DNA damage and decrease in the activity of antioxidant defence system leading to oxidative stress which may contribute to tissue damage and hence to the chronicity of the disease.     

Aging in vertebrates,and the effect of caloric restriction: a mitochondrial free radical production–DNA damage mechanism?

Oxygen is toxic to aerobic animals because it is univalently reduced inside cells to oxygen free radicals. Studies dealing with the relationship between oxidative stress and aging in different vertebrate species and in caloric-restricted rodents are discussed in this review. Healthy tissues mainly produce reactive oxygen species (ROS) at mitochondria. These ROS can damage cellular lipids, proteins and, most importantly, DNA. Although antioxidants help to control this oxidative stress in cells in general, they do not decrease the rate of aging, because their concentrations are lower in long- than in short-lived animals and because increasing antioxidant levels does not increase vertebrate maximum longevity. However, long-lived homeothermic vertebrates consistently have lower rates of mitochondrial ROS production and lower levels of steady-state oxidative damage in their mitochondrial DNA than short-lived ones. Caloric-restricted rodents also show lower levels of these two key parameters than controls fed ad libitum. The decrease in mitochondrial ROS generation of the restricted animals has been recently localized at complex I and the mechanism involved is related to the degree of electronic reduction of the complex I ROS generator. Strikingly, the same site and mechanism have been found when comparing a long- with a short-lived animal species. It is suggested that a low rate of mitochondrial ROS generation extends lifespan both in long-lived and in caloric-restricted animals by determining the rate of oxidative attack and accumulation of somatic mutations in mitochondrial DNA.     

Diabetes and mitochondrial oxidative stress: A study using heart mitochondria from the diabetic Goto-Kakizaki rat

Increasing evidence shows that the overproduction of reactive oxygen species, induced by diabetic hyperglycemia, contributes to the development of several cardiopathologies. The susceptibility of diabetic hearts to oxidative stress, induced in vitro by ADP-Fe²⁺ in mitochondria, was studied in 12-month-old Goto-Kakizaki rats, a model of non-insulin dependent diabetes mellitus, and normal (non-diabetic) Wistar rats. In terms of lipid peroxidation the oxidative damage was evaluated on heart mitochondria by measuring both the O₂ consumption and the concentrations of thiobarbituric acid reactive substances. Diabetic rats display a more intense formation of thiobarbituric acid reactive substances and a higher O₂ consumption than non-diabetic rats. The oxidative damage, assessed by electron microscopy, was followed by an extensive effect on the volume of diabetic heart mitochondria, as compared with control heart mitochondria. An increase in the susceptibility of diabetic heart mitochondria to oxidative stress can be explained by reduced levels of endogenous antioxidants, so we proceeded in determinating -tocopherol, GSH and coenzyme Q content. Although no difference of -tocopherol levels was found in diabetic rats as compared with control rat mitochondria, a significant reduction in GSH (21.5% reduction in diabetic rats) and coenzyme Q levels of diabetic rats was observed. The data suggest that a significant decrease of coenzyme Q₉, a potent antioxidant involved in the elimination of mitochondria-generated reactive oxygen species, may be responsible for an increased susceptibility of diabetic heart mitochondria to oxidative damage.     

Phytol has antibacterial property by inducing oxidative stress response in Pseudomonas aeruginosa

Phytol, isolated from Aster yomena, is widely distributed as a constituent of chlorophyll. In the present study, we confirmed the antibacterial activity of phytol and its mechanism inducing oxidative cell death in Pseudomonas aeruginosa. In phytol-treated cells, elevated level of intracellular reactive oxygen species (ROS) and transient NADH depletion were observed. These results demonstrated that phytol induced ROS accumulation and that the electron transport chain was involved in increase of ROS. Due to this ROS generation, the imbalance developed between intracellular ROS and the antioxidant defense system, leading to decrease of reduced glutathione (GSH). Moreover, severe DNA damage was shown after treatment with phytol. DNA electrophoresis and a terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay were conducted with pretreatment with the antioxidant N-acetylcysteine (NAC) to evaluate the cause of DNA damage. In NAC-pretreated cells, alleviated damage was confirmed and it supports that phytol induces oxidative stress-mediated DNA damage. In conclusion, phytol exerts the antibacterial property via inducing oxidative stress response in P. aeruginosa.