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Increased Nitrosative Stress and Oxidative DNA Damage in Patients with Carbon Monoxide Poisoning

Fatma Midik Ertosun1, Suat Zengin2, Basrı Can2, Hasan Ulusal1, Arzu Yucel1, and Seyithan Taysi1 

1Department of Medical Biochemistry, Medical School, Gaziantep University, Gaziantep, Turkey; 2Department of Emergency medicine, Medical School, Gaziantep University, Gaziantep, Turkey 

Correspondence: (S.T.) 

Ertosun FM et al. Reactive Oxygen Species 9(27):136–143, 2020; ©2020 Cell Med Press

(Received: October 12, 2019; Revised: January 3, 2020; Accepted: January 7, 2020) 

ABSTRACT | Carbon monoxide (CO) remains the most common cause of lethal poisoning around the world. Influence of the brain, which is the most susceptible organ of hypoxia in patients with CO poisoning (COP), is a determining factor of the severity of the clinical condition and mortality. In this study, we aimed to investigate nitric oxide (NO), peroxynitrite (ONOOˉ), 8-hydroxydeoxyguanosine (8-OHdG) levels and nitric oxide synthase (NOS) activity in the serum of patients with COP during admission and treatment processes. The study was conducted prospectively on 36 patients who were admitted to Gaziantep University Medical Faculty Emergency Medicine Department between November 2015 and March 2016 due to COP. The serum samples were prepared for all the COP patients on admission. They were repeated at 180th min of treatment. The samples were taken once from the control group. We showed that admission levels of NO˙, ONOOˉ, and 8-OHdG, and NOS activity in patients with COP were higher than those observed at 180th min. These values were also higher in COP patients compared to control group. These findings suggested that nitrosative stress might play a role in the pathophysiology of COP and increase DNA damage. 

KEYWORDS | Antioxidants; Carbon monoxide poisoning; Free radicals; Nitrosative stress; Oxidative DNA damage; Oxidative stress 

ABBREVIATIONS | CO, carbon monoxide; COP, carbon monoxide poisoning; NO˙, nitric oxide; 8-OHdG, 8-hydroxydeoxyguanosine; NOS, nitric oxide synthase; ROS, reactive oxygen species; RNS, reactive nitrogen species 


  1. Introduction
  2. Materials and Methods

2.1. Study Design

2.2. Biochemical Analysis

2.2.1. Determination of NOS Activity

2.2.2. Determination of Nitric Oxide

2.2.3. Determination of Peroxynitrite

2.2.4. Determination of 8-OHdG

2.3. Statistical Analysis

  1. Results
  2. Discussion


Carbon monoxide (CO) is a smell-free, tasteless, colorless, and nonirritant toxic gas produced primarily as a result of the incomplete combustion of any carbonaceous fossil fuel [1, 2]. Carbon monoxide poisoning (COP) is one of the important reasons of morbidity and mortality in toxicological cases [3]. CO binds to hemoglobin with an affinity of more than 240 times that to oxygen, and for this reason, total oxygen-carrying capacity of the hemoglobin reduces. This competitive binding shift combined with the CO inhibition of cellular respiration produces tissue hypoxia, anaerobic metabolism, and lactic acidosis. The nonspecific symptoms of CO exposure include headache, nausea, vomiting, palpitations, dizziness, and confusion [4]. 

Oxidative/nitrosative damage in living organism is a well-established general mechanism of cell and tissue injury that is primarily caused by free radicals, reactive oxygen species (ROS), and reactive nitrogen species (RNS) [5–7]. ROS/RNS are implicated in the pathogenesis of many diseases [5, 8–10]. Nitric oxide (NO˙) is an important biological messenger that plays an important role in the physiology of the central nervous system (CNS). NO˙ is produced from L-arginine by a family of isoenzymes called nitric oxide synthases and acts as an important physiological signaling molecule mediating a large variety of cellular functions [11]. However, its overproduction induces cytotoxic and mutagenic effects. When present in excess, NO˙reacts rapidly with superoxide radical (O2˙ˉ) to form peroxynitrite (ONOOˉ), which is itself cytotoxic and readily decomposes into the highly reactive and toxic hydroxyl radical (OH) and nitrogen dioxide. ONOOˉis much more reactive than NO˙or O2˙ˉ, which causes diverse chemical reactions in biological system including nitration of tyrosine residues of proteins, triggering of lipid peroxidation, inactivation of aconitase, inhibition of the mitochondrial electron transport, oxidation of biological thiol compounds, and DNA damage [12]. 

The purpose of this study was to evaluate the variations of pre- and post-treatment NOS activity, and NO˙, ONOOˉ, and 8-hydroxideoxiguanosine (8-OHdG), a marker of oxidative DNA damage, levels in COP and to learn more about the pathophysiology of COP.


2.1. Study Design 

The study was carried out at the Departments of Emergency and Clinical Biochemistry of Gaziantep University. The study protocol conformed to the principles of the Helsinki Declaration, and the study was approved by the Medical Ethics Committee of Gaziantep University. 

Thirty-six patients (14 males) with COP comprised the patient group, age range 19–80 years (mean ± SD, 37.3 ± 13.4 years), and 20 healthy subjects were used as a control group, age range 22–65 years (mean ± SD, 35.7 ± 10.6 years). The patient and control groups were free of acute or chronic medical disorders and were of normal body habitus. All the participants submitted a detailed medical history and underwent a physical examination by the investigating physicians. Subjects with coronary artery disease, hypercholesterolemia, hypertension, neurological disorders, diabetes mellitus, liver and kidney diseases, peripheral vascular disease, lung disease, multiple sclerosis, iron deficiency, anemia, and obesity found after medical history, physical examinations, and laboratory examinations were excluded from the study. 

The carboxyhemoglobin (COHb) level was measured using a signal extraction pulse CO-oximetry device (Masimo SET Rainbow, Irvine, CA, USA). All the patients were given 100% oxygen using a non-breather face mask. No patients received hyperbaric oxygen therapy. 

COP and healthy control subjects were recruited into the study after obtaining their informed consent. Blood samples for analysis of biochemical parameters were taken once from the control group and twice from the patients: on admission (group 1) and at 180th min (group 2) of treatment. Venous blood was collected in vacutainers without additive, allowed to clot for 30 min at room temperature, and centrifuged at 3000 g for 5 min to separate the serum. The serum aliquots were stored at –80oC until the analysis date. Hemolyzed samples were excluded. 

2.2. Biochemical Analysis 

2.2.1. Determination of NOS Activity 

NOS activity was determined according to the procedure described before [13]. Results were expressed as U/ml. 

2.2.2. Determination of Nitric Oxide 

NO˙ levels were measured using the Griess reagent as previously described [14]. Results were expressed as µmol/L. 

2.2.3. Determination of Peroxynitrite 

Peroxynitrite was determined according to the method described by Vanuffelen et al. [15] and modified by Al-Nimer et al. [16]. Results were expressed as µmol/L. 

2.2.4. Determination of 8-OHdG 

Serum 8-OHdG levels were measured using the competitive enzyme-linked immunosorbent assay (Rel Assay DC, Gaziantep, Turkey) and the results were expressed as ng/ml. 

2.3. Statistical Analysis 

Kolmogorov–Smirnov test was used for conformity to normal distribution. One-way variance analysis and least significant difference (LSD) multiple comparison tests were used to compare the variables with normal distribution. Paired-samples t-test was used before and after treatment. Pearson test was used for correlation analysis. Data are expressed as mean ± standard deviation (SD) and statistical difference was considered at p < 0.05. Statistical analysis was performed with Statistical Package for the Social Sciences for Windows (SPSS, version 11.5, Chicago, IL, USA). Orange canvas version 3.23 program was used for Histogram graphs.


The parameters measured in all groups are shown in Table 1, and the histograms are presented in Figures 1–4. The activity of NOS and the levels of NO˙, ONOOˉ, and 8-OHdG in group 1 and 2 were higher than those in the control group, and the increase was statistically significant (p < 0.0001 for NOS activity, NO˙, and ONOOˉ; p < 0.01 for 8-OHdG). Also, the activity of NOS and the levels of NO˙, ONOOˉ, and 8-OHdG in COP patients upon their arrival (group 1) to the emergency department were higher than those in group 2, and this increase was also statistically significant. On the other hand, there was no correlation between COHb levels and the activity/levels of NOS, NO˙, ONOOˉ, and 8-OHdG.  

TABLE 1. The levels of COHb, NO, and ONOOˉ, and the activity of NOS in various groups
Parameter Control Admission (group 1) 180th min (group 2)
NO˙ (μmol/L) 3.7 ± 1.79 8.8 ± 3.5b 7.46 ± 2.3b,c
NOS (U/ml) 4.8 ± 1.39 19.7 ± 9.9b 12.2 ± 5.9b,e
ONOOˉ (μmol/L) 93.2 ± 35.0 149.0 ± 58.5b 100.1 ± 41.48b,e
8-OHdG (ng/ml) 5.3 ± 1.6 6.7 ± 1.69a 5.9 ± 1.76a,d
COHb (%) Undetectable 27.2 ± 7.5 9.1±5.2
Note: Data represent mean ± SD. a, p < 0.01 and b, p < 0.0001 vs. control group; c, p < 0.05, d, p < 0.01, and e, p < 0.0001 vs. group 1.


FIGURE 1. Histogram display of NO values in all groups. p < 0.0001, group 1 (on admission) and group 2 (180th min) vs. control group; p < 0.05, group 1 (on admission) vs. group 2 (180th min).


FIGURE 2. Histogram display of NOS values in all groups. p < 0.0001, group 1 (on admission) and group 2 (180th min) vs. control group; p < 0.0001, group 1 (on admission) vs. group 2 (180th min).


FIGURE 3. Histogram display of ONOOˉvalues in all groups. p < 0.0001, group 1 (on admission) and group 2 (180th min) vs. control group; p < 0.01 group 1 (on admission) vs. group 2 (180th min).


FIGURE 4. Histogram display of 8-OHdG values in all groups. p < 0.01, group 1 (on admission) and group 2 (180th min) vs. control group; p < 0.01 group 1 (on admission) vs. group 2 (180th min).


The pathophysiology of COP was previously thought to be due exclusively to the cellular hypoxia caused by replacing oxyhemoglobin with COHb, while producing a relative anemia. COP is far more complex than expected and its pathophysiology is beyond the formation of COHb. The pathophysiological effects of CO lead to problems in four areas: hemoglobin binding, direct cellular toxicity, heme-containing proteins binding, and increases in oxidants, such as NO˙. These effects help to explain why COHb levels do not correlate to the severity of the clinical situation. If early and appropriate treatment is not provided, the combination of hypoxia/ischemia and direct cellular toxicity and the effects of oxidative/nitrosative damage may initiate a chain of events resulting in severe disability or death [2, 4, 17]. 

In the study, we found that NOS activity and the levels of NO˙, ONOOˉ and 8-OHdG in blood samples taken first and at the 180th min in the patient group were higher than those in the control group. Furthermore, the activity/levels measured in COP patients upon their arrival to the emergency department were higher than those at the 180th min. To the best of our knowledge, this is the first study that investigated simultaneously nitrosative stress parameters and 8-OHdG levels, a marker of oxidative DNA damage, in COP. 

Wang et al.[18] reported that CO-mediated delayed neuronal damage might be related to an increase of lipid peroxidation and a decrease of antioxidative status. Kavakli et al. [3] demonstrated that COP increased the total oxidant status but did not change the total antioxidant status. Zengin et al. [4]. showed that admission levels of total oxidative stress (TOS) and oxidative stress index (OSI) in patients with COP were higher than those observed at the 90th min. The levels of the above parameters were also higher in COP patients compared to those in control group. A study done by Ischiropoulos et al. [19] reported that a 10-fold increase in nitrotyrosine, a major product formed from the reaction of ONOOˉwith proteins, was found in the brains of CO-poisoned rats. 

NO˙ production by inducible NOS in the cells of the immune system under pathological conditions occurs for prolonged periods. Enhanced levels of NO˙ and NO˙ derivatives create nitrosative stress within the organism. NO˙ reacts with oxygen radicals or is being oxidized in oxygenated environment, generating highly reactive species––RNS. Oxidative damage is the combination of damages originating from O2˙ˉ and NO˙-derived reactive species. Nitrosative stress is thought to be involved in a number of pathological conditions such as inflammation, neurotoxicity, and ischemia in the organism [6]. Notably, peroxynitrite is a biologically important molecule generated as a result of NO˙ and O2˙ˉ reaction, and the reaction rate is four-fold faster than that of superoxide dismutase (SOD)-catalyzed conversion of O2˙ˉ to H2O2. Under regular conditions, ONOOˉ formation is very low. However, ONOOˉ formation would significantly increase, when NO˙ and O2˙ˉconcentrations are increased and/or in pathological conditions, where SOD activity is decreased. Besides initiating radical reactions, ONOOˉ induces nitration of biomolecules. Additionally, ONOOˉ activates a nuclear enzyme known as poly(ADP-ribose) synthetase (PARS). The PARS enzyme uses an excessive amount of NAD as a substrate, which may result in depletion of adenosine 5′-triphosphate (ATP) and cell apoptosis. ONOOˉ is also a potent initiator of DNA single strand breakage, which is an obligatory stimulus for the activation of the PARS. PARS activation triggered by DNA single strand breakage occurs following exposure to a variety of environmental stimuli and oxidants, especially hydroxyl radical and ONOOˉ [20, 21]. 

In COP, the increased NO˙ level is due to the competitive binding of CO to intracellular heme protein binding sites. In this regard, CO has a stronger affinity than NO˙ for heme protein binding sites. The increased vascular NO˙ will subsequently promote oxidative/nitrosative stress and the leakage of reactive mediators, triggering phagocyte adherence and activation. Some studies have reported cerebral vasodilatation after exposure to CO, which is associated with a temporary loss of consciousness and increased NO˙ levels [17, 22]. NO˙ also seems to play a pivotal role in a series of events culminating in oxidative damage to the brain, being responsible for delayed neurologic sequelae [22]. 

In summary, this study demonstrated a significant increase in nitrosative stress parameters and 8-OHdG levels in patients with COP, suggesting that these patients had a suppressed antioxidative status. Imbalance in the redox status induced by CO may lead to the overproduction of ROS and RNS, thereby impairing cell functions in COP. 


This study was supported by Gaziantep University Scientific Research Projects Unit (TF.YLT.16.32). The authors declare no conflicts of interest. 


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