Introduction

Although open coronary artery by-pass grafting (CABG) surgery has become a routine procedure worldwide, patient morbidity and mortality due to adverse postoperative complications are still unacceptably high [1]. ROS, which are thought to be responsible from these adverse complications, including superoxide radical, hydroxyl radical and hydrogen peroxide have been shown to increase during CABG operation. Furthermore, several studies have proposed the essential role of ROS in the pathogenesis of myocardial ischemia–reperfusion injury [2, 3]. Mitochondria are the main source of ROS. Superoxide is the initial oxidant generated from NAD(P)H or other oxidases such as cytochrome P-450 and is also generated by the mitochondrial electron transport chain. ROS production is accompanied by the induction of natural defense and repair mechanisms and effect upon tissue’s vitality. Superoxide dismutase (SOD) is a first line of defense against oxidative stress under physiological and pathological conditions [4]. SOD catalyses the dismutation reaction of the toxic superoxide radical to molecular oxygen and hydrogen peroxide and thus forms a crucial part of the cellular antioxidant defence mechanism. In most mammalian species, SOD appears to be the most active antioxidant enzyme in the myocardium. Manganese superoxide dismutase (MnSOD), a key mitochondrial antioxidant enzyme, is one important repair enzyme for reactive oxidative stress (ROS)-induced damage. MnSOD polymorphisms in the 9 position of the signal sequence of the protein may lead to critical enzyme deficiency. In MnSOD polymorphism two genetic variants of MnSOD were identified. A structural mutation, a T to C substitution in the mitochondrial targeting sequence, was found that changes the amino acid codon at −9 position in the signal peptide from valine (GTT) to alanine (GCT) [5].

7,8-Dihydro-8-oxoguanine (8-OHdG) is one of the main DNA modifications produced by ROS. Because 8-OHdG can pair with cytosine and adenine bases during DNA synthesis, when 8-hydroxyguanine (8-OHG) is present in the DNA template, it causes G:C to T:A transversions [5]. On the other hand, 8-hydroxydeoxyguanine (8-OHdG) is one of the main DNA modifications produced by hydrogen peroxide. It is highly mutagenic in vitro and in vivo, yielding G:C to T:A transversion, since it directs the incorporation of cytosine and adenine nucleotides opposite the lesion during DNA replication and it induces A:T to C:G transversions when 8-hydroxy-dGTP in the nucleotide pool is incorporated into DNA [6, 7]. The hOGG1 gene encodes the DNA glycosylase that removes the mutagenic lesion 7,8-dihyro-8-oxoguanine (8-OHdG) from DNA. A frequently found polymorphism resulting in a serine to cysteine substitution at position 326 of the OGG1 protein has been associated in several molecular epidemiologic studies with cancer development [8, 9]. hMTH1 also removes 8-OHdG from the nucleotide pool [10]. Less is known about the consequences of CABG on the endogenous antioxidant capacity that is derived from the activity of antioxidant enzymes such as glutathione peroxidase (GPx) and SOD, responsible for the clearance of peroxides and superoxide, respectively.

In the present study, it was aimed (i) to measure the DNA damage (8-OHdG) and endogenous antioxidant activity (SOD) at the following stages of the intervention: before anesthesia (T1), after anesthesia (T2), after ischemia (T3), after reperfusion (T4) and after the surgery (T5) and reveal the differences among the time intervals (no individual susceptibility), (ii) to reveal individual susceptibility by investigating the impact of OGG1, MTH1 and MnSOD gene polymorphisms on serum 8-OHdG and SOD activity in patients undergoing coronary artery bypass grafting.

Materials and methods

Patient population

Sixty patients (18 women and 42 men) aged 30–75 years, with American Society of Anesthesiologists (ASA) physical status II and IV, who underwent elective CABG surgery were prospectively studied after obtaining approval from the Ataturk Training and Research Hospital ethics committee. The study was performed in accordance with the declaration of Helsinki. Their demographic data are reported in Table 2. Each patient was informed about the anaesthetic procedure and study protocol in detail at the preoperative visit and gave their written consent before inclusion in the study. Patients answered standardised health questionnaires about their medical history, occupational exposure, and lifestyle such as smoking and drug consumption to rule out confounding effects. Before surgery, patients underwent thorough cardiac diagnostics, including determination of ejection fraction. We excluded pregnant women and patients with hormone treatment, left ventricular ejection fraction of less than 40%, a history of previous cardiac operation, neuromuscular disorders, neurological, haematological, hepatic, renal, or severe pulmonary disease, a history of drug and alcohol abuse, patients undergoing combined operations (simultaneous valve repair, carotid endarterectomy, or left ventricular aneurysm repair) from the study.

Before surgery, patients underwent thorough cardiac diagnostics, including determination of ejection fraction. Age, body mass index (BMI), medical history, current medications, preoperative left ventricular ejection fraction (LVEF/%), and infarction time, if present, were recorded for each patient. All preoperative cardiac medication was continued until the morning of the surgery, except for angiotensin-converting enzyme (ACE) inhibitors.

Anaesthetic and surgical procedure

All patients were pre-medicated with oral diazepam (5 mg) the night before and intramuscular morphine sulphate (0.1 mg kg−1) 45 min before the surgery. Noninvasive monitoring was established and radial artery was cannulated under local anaesthesia. This procedure enables internal monitoring of arterial tension and patient’s metabolic and blood-gas status. It was also used to obtain blood samples required for polymorphism studies. Anaesthesia was similar for all patients, and consisted of 10 μg kg−1 fentanyl in combination with 0.3 mg kg−1 etomidate and 0.15 mg kg−1 cisatracurium besylate, given intravenously at induction. Intubation was performed after 2 min and anaesthesia was maintained by additional doses of fentanyl (5 μg kg−1) and cisatracurium besylate (0.03 mg kg−1), and by inhalation of 0.5–2% sevoflurane. The depth of anaesthesia during surgery was between 40 and 50, according to the bispectal index (BIS XP™; Aspect Medical Systems, Newton, MA). Ventilation during the anaesthesia was performed in a volume-controlled mode. In surgery, a median sternotomy was made, heparin was given (350 IU kg−1), aortic and right atrial venous cannula were inserted, and a standard cardiopulmonary bypass (CPB) with moderate systemic hypothermia (28 to 30°C of nasopharynx temperature) was instituted. Activated coagulation time (ACT) was kept above 450 s throughout the CPB period. Standard pump priming solution was used in each patient. Routine surgical technique and cardioprotective strategies were used in all patients. For myocardial protection, the aorta was cross-clamped and cold anterograde and retrograde intermittent cardioplegia (Plegisol® Abbott, North Chicago, IL, ABD) were used. Vasopressors and vasodilators were administered as necessary. Hematocrit concentrations were maintained between 20 and 25%. After the surgical procedure, by reperfusion and rewarming of the heart to temperature of 36°C, the heart was placed in atrioventricular mode at a rate of 90 bpm, and the patients were separated from CPB. After removal of the aortic cannula, heparin activity was neutralised with protamine at a ratio of 1 mg of protamine per 100 U heparin. Protamine administration was further guided by ACT measurements aiming at 140 s. When mean arterial pressure was below 60 mmHg, vasopressor therapy was started. During surgery, we recorded the duration of aortic clamping and CPB, volume of cardioplegic solution used, volume of haemodilution and infusions, diuresis, and minimum body temperature. At the end of the surgical procedure, patients were transferred to the intensive care unit. When haemodynamically stable and rewarmed, the patients were weaned from the ventilator and extubated.

Blood sampling

The serum samples were collected at the following stages of the intervention: the reference level (T1) was obtained immediately after cannulation of the arterial system and before anaesthesia; the second sample (T2) was taken 20 min after anaesthesia was introduced; the third sample (T3) was taken after 20 min of the aortic cross-clamp; the fourth sample (T4) was taken 20 min after the removal of the aortic cross-clamp; and the final sample (T5) was obtained by venipuncture 24 h after the surgery. At the same time points, hemoglobin, sodium, potassium, glucose, lactate and bicarbonate were determined in blood samples taken from the radial artery. Venous blood samples, taken in EDTA for genotyping analysis, were obtained during routine blood sampling for biochemical and hematological analyses from the patients. OGG1 Ser326Cys, MTH1 Val83Met and MnSOD Ala9Val polymorphisms were genotyped by restriction fragment length polymorphism–polymerase chain reaction (RFLP–PCR).

DNA isolation

Genomic DNA was isolated from blood using a commercial DNA extraction kit according to manufacturer’s recommendations (Blood DNA Extraction Kit, Cat No: GF-BD-050, Vivantis).

OGG1, MTH1 and MnSOD Genotyping

A 207 bp fragment was amplified by PCR in a 30 μl reaction volume that contained 100 ng genomic DNA, 0.2 mM of dNTP, 1.5 mM of MgCl2, 0.3 pmol of each primer and 1 U of Taq DNA polymerase for OGG1 genotyping. A 247 bp fragment was amplified by PCR in a 25 μl reaction volume that contained 100 ng genomic DNA, 0.2 mM of dNTP, 1.5 mM of MgCl2, 0.2 μM of each primer and 1.5 U of Taq DNA polymerase for MTH1 genotyping. For MnSOD genotyping, a 172 bp fragment was amplified by PCR in a 25 μl reaction volume that contained 100 ng genomic DNA, 0.25 mM of dNTP, 1.5 mM of MgCl2, 0.3 μM of each primer and 1 U of Taq DNA polymerase. Primers, cycling conditions, restriction enzymes and incubation conditions for gene polymorphisms are presented in Table 1. All digested products were electrophoresed on a 3% agarose gels and visualized under UV light with ethidium bromide.

Table 1 RFLP–PCR genotyping analysis for polymorphisms on OGG1, MTH1 and MnSOD genes
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Measurement of SOD activity

SOD activity in serum was measured by a colorimetric method with a commercial kit (SOD Activity Assay Kit, Cat No: K335-100, Biovision) according to the manufacturer’s instructions.

Measurement of the levels of serum 8-hydroxy-2′-deoxyguanosine (8-OHdG)

The levels of serum 8-OHdG were measured by Enzyme Linked Immunosorbent Assay (ELISA) (StressXpress DNA Damage ELISA Kit, EKS-350, Stressgen) according to the manufacturer’s instructions.

Statistical analysis

Data analysis was performed by using SPSS for Windows, version 11.5 (SPSS Inc., Chicago, IL, United States). Whether the distributions of continuous variables were normally or not was determined by using Shapiro–Wilk test. Data were shown as median (IQR). The differences between groups were compared by using Mann–Whitney U test. A P value less than 0.05 was considered statistically significant. Moreover, whether the differences among time intervals were statistically significant or not was evaluated by Friedman test. When the P value from the Friedman test is statistically significant to know which time differs from which others, Bonferroni Adjusted Wilcoxon Sign Rank test was used.

Results

The frequencies of OGG1, MTH1 and MnSOD allele and genotype were estimated with CABG surgery in this study. Table 2 displays the baseline characteristics and operative data of 60 CABG patients. The participants were predominantly male (n = 42, 70%) and nonsmokers (n = 39, 65%). OGG1 Ser326Cys, MTH1 Val83Met and MnSOD Ala16Val genotype distributions and their allele frequencies in the entire study population are summarized in Table 3. We found that the frequencies of OGG1 Ser/Ser, Ser/Cys and Cys/Cys genotypes were 55, 42 and 3%, respectively; while the frequencies of MnSOD Ala/Ala, Ala/Val and Val/Val genotypes were 52.6, 42.1 and 5.3%, respectively. The frequencies of MTH1 Val83Val and Val83Met genotypes also are 93.3 and 6.7%.

Table 2 Baseline characteristics and operative data of 60 CABG patients
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Table 3 OGG1 Ser326Cys, MTH1Val83Met and MnSOD Ala16Val Genotype Distributions and their Allele Frequencies
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Figure 1 summarizes the levels of 8-OHdG, as independently from genotypes, at 5 predetermined time intervals (as T1: before anesthesia induction, T2: after anesthesia induction, T3: at aortic cross-clamping, T4: during reperfusion and T5: at 24 h of operation). The levels of 8-OHdG at T3, T4 time periods were decreased significantly compared to T1, while the difference between T2 and T3, T4 time periods as well as between T3–T5 and T4–T5 were significant, pointing out that the lowest 8-OHdG level obtained at T3 might be the result of repair induced by ischemia for compensation of oxidative stress (P < 0.001). However, an upward incline at T4–T5 time periods is also remarkable. The enzyme activity of the antioxidant SOD in full blood at 20 min after reperfusion (T4) reached a maximum rise compared to the levels obtained at T1, T2, and T3 (P < 0.001), however, afterwards this increase showed a decline until the end of 24 h (P < 0.001) (Fig. 2).

Fig. 1
figure 1

Serum 8-OHdG (ng/ml) levels in five different time intervals (T1–T5) not taking account into genotypes (T1–T5). *P < 0.001; T1–T3, T1–T4; § P < 0.001; T2–T3, T2–T4; □ P < 0.001, T3–T5, T4–T5 Anova

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Fig. 2
figure 2

The activities of SOD in five different time intervals (T1–T5) not taking account into genotypes (T1–T5). *P < 0.001; T1–T4; # P < 0.001; T2–T4; °P < 0.001, T3-T4; □ P < 0.001, T4–T5 Anova

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When it was checked whether genotypes had impacts on the levels of 8-OHdG, for OGG1, 8OHdG activities which are measured in homozygotes at T1, T2, T3, T4 and T5 were not significantly different from those in heterozygous/mutant ones (P > 0.05) (Table 4). Based on the genotype, we obtained the lowest 8-OHdG level at T3 as a result of ischemia induced by aortic cross-clamping.

Table 4 The levels of serum 8-OHdG (ng/ml) and the activity of SOD in five different time intervals not taking account into genotypes (T1–T5)
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Table 5 summarizes the effect of genotype on the levels of 8-OHdG for MTH1 Val83Met. The levels at predetermined time intervals are not different between the homozygote and heterozygous/mutant individuals (P > 0.05). The levels of 8OHdG in patients carrying heterozygous/mutant variant at T3 were higher than in patients carrying homozygotes, but this increase was not statistically significant. It suggested that the lack of repair by OGG1 for 8-OHdG caused the increase of the levels of 8-OHdG. This finding showed the importance of genetic background of patients. For gene–gene (OGG1-MTH1), in contrary of our expectations, no inter-group (T1-T5) differences were found with respect to the levels of 8OHdG, for both OGG1 Ser326Cys and MTH1 Val83Met genotypes which play role in repair of the DNA damage (8-OHdG) (P > 0.05) (Table 6). Table 7 shows the activity of SOD in five different time intervals (T1–T5) in terms of MnSOD Ala16Val genetic polymorphism. The activities of SOD in patients carrying heterozygous/mutant variants were decreased in predetermined time intervals compared to patients carrying wild, except T2, however these decreases were not statistically significant. A highest decrease was observed in T4, reperfusion consumed SOD activity even if that was not statistically significant, this trend might give an idea about the consuming of SOD, especially at T4 and provide comparable data at other time points (Table 8).

Table 5 The levels of serum 8-OHdG (ng/ml) in five different time intervals (T1–T5) in terms of OGG1 Ser326Cys genotype
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Table 6 The levels of serum 8-OHdG (ng/ml) in five different time intervals (T1–T5) in terms of MTH1 Val83Met genotype
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Table 7 The levels of serum 8-OHdG (ng/ml) and the activity of SOD in five different time intervals (T1–T5) in terms of OGG1 Ser326Cys plus MTH1 Val83Met genotypes
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Table 8 The activity of SOD in five different time intervals (T1–T5) in terms of MnSOD Ala16Val genetic polymorphism
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Discussion

This study aimed at gaining further insight into the short-term effects of oxidative stress on the changes in endogenous antioxidant capacity and 8-OHG, which is a sensitive biomarker of DNA damage, in patients undergoing CABG by sevoflurane anesthesia and moreover, the impact of individual susceptibility on ischemia–reperfusion injury in terms of their genetic polymorphisms was revealed by comparisons among time intervals.

The systemic increase in oxidative stress during CABG is well-documented but the various components of the oxidant–antioxidant balance and the contribution of the various mechanisms involved have not been fully evaluated yet [14]. Extracorporeal circulation, by increasing contact of blood with foreign substances, will induce systemic inflammatory responses associated with complement activation, cytokine release and cellular activation of neutrophils [15]. Protective and repair mechanisms are normally present in the cell to prevent DNA damage and the negative impact on endogenous antioxidant capacity from ROS during CABG process and these defense and repair mechanisms include the antioxidant enzymes (such as SOD, catalase and GPx) and DNA repair systems (such as BER, NER, etc.). Single Nucleotide Polymorphisms (SNPs) on genes, which play role in these mechanisms, can give rise to individual gene variants that can alter susceptibility to many pathological and physiological states. Given that most individuals show extensive sequence variation in their DNA repair and antioxidant genes, it is likely that susceptibility will vary between individuals depending on the particular combination of alleles inherited. We found that the distribution of the OGG1 Ser326Cys, MTH1 Val83Met and MnSOD Ala16Val genotypes in the study population were as 42, 6.7 and 42.1%, respectively. The frequencies of these genotypes are similar to the results obtained from studies of Caucasians [11–13].

Nagayoshi et al. [16] found that urinary levels of 8-OHG were higher in cardiac patients than in non-cardiac patients after assessment of serial changes in oxidative stress of patients with acute myocardial infarction. However, they only evaluated systemic changes in 8-OHdG, not the changes at different time intervals. In our study, we found a slight but not statistically significant decrease in the levels of serum 8-OHG and the activity of SOD after ischaemia that may be associated with oxygen radicals formed. The enzyme activity of SOD showed a maximum rise at 20 min after reperfusion. It suggested that large quantities of ROS released during reperfusion damage proteins responsible for intracellular homeostasis producing tissue injury. Verma et al. [17] demonstrated that this injury can be further exacerbated during open heart surgery when the myocardium is exposed to global ischemic cardioplegic arrest. Furthermore, several studies have pointed to strategies attenuating reperfusion injury and systemic inflammatory response during open heart surgery to improve clinical outcome [18, 19]. Acute preoperative hemodilution may be associated with reduced effects of aortic cross-clamping and improved myocardial recovery in these patients. However, study in higher-risk patients with poor ventricular function and those requiring complex cardiac surgery, which are not the interest of our study, could contribute to the results.

OGG1 is the key enzyme for recognition and initial excision of the most common form of oxidative DNA base damage, 8-OHG. OGG1 gene is highly polymorphic with the most studied SNP being at codon 326 (Ser326Cys). It is not clear whether the contribution of this polymorphism affects the catalytic properties of the enzyme, and thus limited knowledge is available on the association between a variety of susceptibilities to different disease states and SNPs in this critical DNA repair gene. Homozygous carriers of the variant form of the OGG1 Ser326Cys gene appear to have reduced repair capacity for oxidized DNA lesions [20].

In the current literature, most epidemiological studies of the OGG1 Ser326Cys polymorphism are in relation to cancer. In the aspect of lung cancer, some authors could not demonstrate any association due to the polymorphisms of OGG1 Ser326Cys [21–23]. However, Karahalil et al. found a significant decrease in the risk of lung cancer for the Ser326Cys genotype, disappearing after adjustments for age, sex, and smoking as reported similarly by De Ruyck et al. [24]. The authors explained this as a result of proteins involved and up-regulated by oxidative stress for the repair and prevention of 8-OHG-derived mutations. Paz-Elizur et al. [25] also demonstrated that low OGG is associated with high risk of head and neck cancer. The studies on association with ischemia–reperfusion injury induced 8-OHG and the DNA-repair capacity of an individual are limited. In a study by You et al. [26], in the ischemic-reperfused rat hearts exposed to oxidative stress induced by high levels of oxygen free radicals, both the formation of 8-OHG in DNA and the level of its repair process, 8-OHG endonuclease activity was found to be increased. In another experimental model, the accumulation of 8-OHG in nuclear DNA and altered level of OGG1 expression, as a pathogenic response to necrosis of renal tubular cells during ischemia–reperfusion injury of the kidney demonstrated the association between OGG1 and 8-OHG [27].

We also studied MTH1 Val83Met polymorphism. Consequently, it may be difficult to interpret the findings of our study in the aspect of MTH polymorphism. One reason for this might be the limited number of studies because of the infrequent number of variants of MTH. However, in our study, we tried to investigate the combined effect of the two polymorphisms on the detection of oxidative DNA damage biomarker and cellular SOD activity. Nevertheless, we examined the two DNA repair enzymes responsible from the same activity. The hypothesis of our study was to determine whether this combined action could act synergistically in oxidative DNA damage etiology. We have to acknowledge that the study did not yield statistically significant results, but none of the reports up to date have attempted to show the relationship between OGG1, MTH1 and MNSOD gene polymorphisms on 8-OHG and SOD activity in myocardial ischemia reperfusion research on clinical basis. Thus, to our knowledge, this is the first study to evaluate the prevalence of three polymorphisms in a Caucasian population or to examine the associations between the polymorphisms and ischemia reperfusion injury.

SOD, is the key enzyme involved in the detoxification of superoxide radicals. There are a few studies investigating the association of cancer and chronic illnesses with genetic polymorphisms in MnSOD gene [28]. Isbir et al. investigated the effect of MnSOD genetic polymorphism on cytokine release and MnSOD dismutase in patients undergoing CABG with CPB. Their results showed that baseline IL-6 did not differ between patients with different MnSOD genotypes but IL-6 levels were significantly higher in all patients but more significantly in V(VV + AV) carriers (P = 0.003). The MnSOD ValVal genotype was associated with significantly lower preoperative MnSOD levels compared to the AA carriers (P < 0.05). They concluded that MnSOD Ala16Val polymorphism influences IL-6 production and baseline MnSOD activity [29]. These epidemiological studies have yielded mixed results with either a weak or strong association in between. Since there is no study addressing the association between ischemia–reperfusion injury and polymorphisms of OGG1, MTH1 and MnSOD in open heart surgery patients, we conducted this study to investigate the role of individual susceptibility in these patients. We did not find significant differences in the levels of serum 8-OHG and the activity of SOD between sampling times. We assumed that active repair of endogenous oxidative DNA lesions took place in the myocardium, which survived the insults of ischemia and reperfusion. We further believe that repair of oxidative base lesions in the myocardium was associated with induced repair activity, which is attributed to the over expression and activation of some repair enzymes, especially OGG1. Results from these analyses may be affected by sources of bias and low participation rates that are common to clinical based studies. Of concern are the limited sample numbers in this study.

Conclusions

In conclusion, our experiments performed in a small number of selected patients, could not show a significant association between the OGG1 Ser326Cys, MTH1 Val83Met, MnSOD Ala16Val polymorphisms and oxidative response to ischemia–reperfusion injury. These polymorphisms cannot explain individual variation in susceptibility in ischemia–reperfusion related tissue injury in humans. However, it is well documented that a close relationship existing between the DNA damaging activity of endogenous reactive ROSs and their effects on the microcirculation results in rapid increases of both serum 8-OHG content and SOD activity during CABG operations. Based on the data obtained in this study and in the light of relevant observations in the literature, larger case–control and prospective epidemiologic studies are needed to prove that reduced OGG1, MTH1 and MnSOD are indeed risk factors for patients in situations in which ischemia and reperfusion occur, such as cardioplegia during cardiac surgery.