reperfusion injury

Free Radical Biology & Medicine, Vol. 28, No. 1, pp. 1–12, 2000 Copyright © 2000 Elsevier Science Inc. Printed in the USA. All rights reserved 0891-5849/00/$–see front matter

PII S0891-5849(99)00211-7

Original Contribution DNA DAMAGE IN HUMAN LEUKOCYTES AFTER ISCHEMIA/REPERFUSION INJURY CHRISTIAN WILLY,* SASCHA DAHOUK,* CHRISTOPH STARCK,‡ WALTER KAFFENBERGER,‡ HEINZ GERNGROß,* ULLA G. PLAPPERT†

and

*Department of Surgery, Military Hospital Ulm, Ulm; †Department of Occupational, Social and Environmental Medicine, University of Ulm, Ulm; and ‡Institute of Radiobiology, Federal Armed Forces Medical Academy, Munich, Germany (Received 18 December 1998; Revised 9 August 1999; Accepted 18 August 1999)

Abstract—Leukocytes have been shown to play an important role in the development of tissue injury after ischemia and reperfusion (I/R). In the present study, the effects of tourniquet-ischemia on induction of DNA damage in peripheral leukocytes and on respiratory burst of neutrophils in humans were examined. The DNA damage was measured as increased migration of DNA using the single-cell gel-electrophoresis technique (comet assay). Intracellular production of reactive oxygen species by neutrophils was measured flow-cytometrically using dihydrorhodamine 123 as indicator. Postischemic, significantly increased migration of DNA was found in leukocytes of 20 patients (tourniquet-ischemia of the lower limb for 65–130 min, anterior-cruciate-ligament-reconstruction) and in 10 experiments (1 volunteer, repeated tourniquet-ischemia of the upper limb for 60 min, no operation). DNA effects were most pronounced 5–30 min after tourniquet release, and then declined over a 2 h period, but did not return to preischemic baseline values. A similar time course showed the oxidative status of unstimulated granulocytes during reperfusion. Simultaneously, opposing changes were measured in formyl peptide (f-MLP)- or phorbol ester (PMA)-stimulated granulocytes, which showed a significantly declined respiratory burst reaction after tourniquet-release indicating preactivation of neutrophils by I/R. Our data suggest that I/R induces genotoxic effects in human leukocytes presumably in response to oxidative stress during reperfusion. © 2000 Elsevier Science Inc. Keywords—Oxygen-derived free radicals, Ischemia/reperfusion-injury, Human leukocytes, DNA strand breaks, Comet assay, Respiratory burst, Free radical

INTRODUCTION

to play a pivotal role in the pathogenesis of I/R injury [4 – 8]. Mainly under discussion as potential biological sources of these free radicals are the enzymes (i) reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, found in the plasma and cell membrane of polymorphonuclear neutrophilic granulocytes (PMN) [9,10]; and (ii) the xanthine dehydrogenase/xanthine oxidase, located in vascular endothelial cells [11]. The central role of leukocyte activation, adhesion and the release of oxygen free radicals in the pathophysiological sequelae of reperfusion-associated microcirculatory disturbances has been inferred from several studies in which reperfusion injury was significantly reduced by depletion of neutrophils, by monoclonal antibodies preventing leukocyte adhesion, and by pharmacological inhibition of toxic oxygen radicals [12–15]. The cytotoxic effects of free radicals are the initiation of peroxidation of polyunsaturated fatty acids in membrane or plasma lipoproteins, the direct inhi-

Postischemic reperfusion injury represents a source of substantial morbidity and mortality in various fields of medicine, i.e., myocardial infarction, stroke, trauma, septic or hemorrhagic shock, multiple organ failure, coronary thrombolysis, bypass surgery, and organ transplantation. At the microcirculatory level, ischemia/ reperfusion (I/R) injury is thought to be initiated by chemotactic accumulation of circulating leukocytes, their activation and interaction with the endothelium of postcapillary venules [1–3]. In addition, oxygen free radicals, such as superoxide anion, hydrogen peroxide, peroxynitrite, and the highly reactive hydroxyl radical are thought Address correspondence to: Dr. med. Christian Willy, M.D., Department of Surgery, Military Hospital Ulm, Oberer Eselsberg 40, D-89081 Ulm, Germany; Tel: ⫹49-(0) 731/171-2021; Fax: ⫹49-(0) 731/ 553100; E-Mail: [email protected]. 1

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C. WILLY et al.

bition of mitochondrial respiratory chain enzymes, the inactivation of membrane sodium channels, and other oxidative modifications of proteins [16 –19]. A variety of experimental models of ischemic injury have also shown that free radicals induce postischemic oxidative damage to DNA [20 –23]. Although several human studies have revealed that oxygen radicals generated by other mechanisms like irradiation, hyperbaric oxygen (HBO) therapy, and physical exercise lead to oxidative damage to DNA [24 – 28], only few data exist that demonstrate the influence of reperfusion-associated oxygen radicals on DNA in humans [29,30]. For example, DNA strand breaks were detected post mortem in reperfused myocardial samples of patients who died of acute myocardial infarction after initially successful thrombolysis [29]. However, up to now, the course of I/R-induced DNA damage in humans in vivo remains unknown. This study was, therefore, designed to investigate the genotoxic effects in peripheral white blood cells of humans during the reperfusion period after tourniquet-ischemia of the lower and upper limbs in vivo. The study was performed in patients undergoing an elective reconstructive operation of the knee joint, and in an additional set of experiments in a volunteer to investigate the effects provoked by a “pure” I/R injury of the upper limb without surgical trauma and anesthesia. Published studies have shown that the complex interactions of agents inducing DNA damage by oxygen radicals can be investigated readily in human leukocytes using the alkaline single-cell gel-electophoresis assay (comet assay) [31, 32]. Therefore, our experiments applied this genotoxicity test, which has been utilized to detect a broad spectrum of DNA damage and to ascertain repair of strand breaks in human DNA with high sensitivity [26,31,33,34]. For preparation, samples are embedded in agarose on microscopic slides, lysed, placed in alkali, an electric current is applied and the samples are stained with a fluorescent DNA-binding dye. In its alkaline version, DNA strand breaks and alkali-labile sites become apparent, and the amount of DNA migration indicates the amount of DNA damage in the cell [31]. Although undamaged DNA remains within the nucleus, DNA containing strand breaks streams out from the nucleus towards the anode generating a “comet tail” [31]. Simultaneously, to study the intracellular production of oxygen metabolites, the respiratory burst in activated PMN was determined to correlate these data with DNA damage.

(age range: 21– 48 years, median: 29 years), which had to undergo an anterior cruciate ligament reconstruction, DNA damage was determined before and after tourniquet-ischemia of the lower limb. Between January 1997 and June 1998, in one volunteer in an experiment repeated 10 times, DNA strand breaks were investigated before and after the tourniquet-ischemia of the upper limb. Tourniquet-ischemia of the lower limb. Venous blood samples of 20 men were collected before and after tourniquet-ischemia both at the operated and the contralateral limb. The samples (v. dorsalis pedis) were drawn before tourniquet-ischemia (cuff-pressure: 400 mmHg), 45 min later and 0 –2, 5, 15, 30, and 120 min after release of the tourniquet. The ischemic period during the operation was not interrupted and lasted about 90 min (range: 65–130 min). Other genotoxic effects (radiological or chemical effects, smoking habits, sports) could be ruled out by the history of the patients. To exclude a possible effect of anesthesia, cells of 15 additional patients (age range: 19 –53 years, median: 30 years) were investigated before operation. Five of them were anesthetized by endotracheal anesthesia (duration: 9 –16 min), the other 10 patients were anesthetized by spinal block (duration: 12–20 min). All patients gave informed, written consent. The study was approved by the local institutional review board. Tourniquet-ischemia of the upper limb. Additionally, to investigate effects provoked by a “pure” I/R injury without surgical trauma and anesthesia, a tourniquet-ischemia of the upper limb (cuff-pressure: 260 mmHg) lasting 1 h was used 10 times with the same volunteer (one of the authors, 35 years old, intervals between experiments: 3–7 weeks). Blood samples were taken at the same time points as above (v. cubitalis). In this set of experiments, more blood could be taken, and therefore, additionally, the respiratory burst of peripheral PMN could be investigated, too. Lactate level In order to monitor the metabolic response to I/R as well as the extent of ischemia, serum lactate levels were determined (sodium fluoride-anticoagulated blood). A lactate level of 0.5–2.2 mmol/l was considered to be normal.

MATERIALS AND METHODS

Subjects and study protocol

Alkaline comet assay

The study consists of two different sets of investigations. From March 1996 to December 1997, in 20 men

Heparinized whole blood was kept at 4°C during the period of operation and thereafter until freezing.

DNA damage in human leukocytes after ischemia/reperfusion injury

Blood samples were mixed with a freezing medium [20% dimethyl sulfoxide (DMSO) and 80% of cell culture medium (RPMI 1640); 1:1] and aliquoted for final storage at ⫺140°C in liquid nitrogen. Before starting the actual assay, samples were defrosted in a water bath (37°C), washed (10% fetal calf serum and 90% RPMI 1640 medium) and spun twice for 10 min (300 ⫻ g) at room temperature. Then, the cells were resuspended in 600 ␮l of RPMI 1640 medium. The alkaline single-cell gel-electophoresis assay (comet assay) for the measurement of DNA single- and double-strand breaks and alkali-labile sites was used as developed by Singh and coworkers [31] with slight modifications according to Plappert et al. [26]. Of each aliquot, 10,000 –15,000 cells (resuspended leukocytes) were mixed with 85 ␮l of 0.5% low-melting point agarose (LMP-agarose, Gibco BRL; Eggenstein, Germany) at 37°C and added on a fully frosted microscope slide (Labcraft, Curtin Matheson Scientific Inc., TX, USA), which had been coated with 300 ␮l of 0.6% agarose (Gibco BRL), the so-called “bottomlayer”. The slides were kept at 4°C for 5 min in order to allow the agarose to solidify. Next, the slides were immersed in lysing solution (100 mM Na2-ethylenediaminetetraacetate acid [EDTA], 10 mM Tris (hydroxymethyl)-aminomethan (TRIS), 2.5 M NaCl, 1% Na-sarcosinate, 1% Triton X-100 and 10% DMSO; pH 10) for 90 min to lyse the embedded cells and to permit DNA unfolding. After the lysis, the slides were placed in a horizontal gel electrophoresis chamber filled with alkaline buffer (1 mM Na2-EDTA, 300 mM NaOH; pH 13). The cells were exposed to alkali for 60 min in order to allow DNA unwinding. To electrophorese the DNA, an electric field of 0.8 V/cm for 30 min was applied (25 V; 300 mA). After electrophoresis the slides were washed with neutralization buffer (3 ⫻ 15 min; 0.4 M TRIS; pH 7.5). Finally, the slides were stained with 80 ␮l ethidium bromide in PBS (20 ␮g/ml), covered with a coverslip and kept in a humidified box until evaluation. The extent of DNA migration was visualized at 200-fold magnification using a Zeiss fluorescence microscope equipped with an excitation filter of 515–560 nm and a barrier filter of 590 nm. The microscope was connected to a Pulnix TM 765 camera and a personal computer-based analysis system (Comet Analysis Software, Version 2.4, Kinetik Imaging Ltd.; Liverpool, UK). Data are based on 60 randomly selected cells per sample (30 cells each from two replicate slides). For analysis the tail moment (TM ⫽ percentage of DNA in the tail ⫻ tail length) was chosen [35]. Heterogeneity of TM were calculated using the dispersion coefficient (H) where H ⫽ variance/mean.

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Cell respiratory burst assay The respiratory burst (RB) activity of isolated neutrophils was determined using dihydrorhodamine (DHR) 123 (Molecular Probes; Eugene, OR, USA) as an indicator for intracellular hydrogen peroxide production [36]. Neutrophils were isolated from peripheral blood (lithium-heparin anticoagulated) by Ficoll-Hypaque density gradient centrifugation (900 ⫻ g; 10 min, room temperature) followed by lysis of erythrocytes. The isolated cells were washed and resuspended in either phosphate-buffered saline [(PBS), pH 7.3, without Ca⫹⫹ and Mg⫹⫹ (Boehringer Mannheim; Mannheim, Germany)] for phorbol myristate acetate (PMA) stimulation or Hanks’ balanced salt solution (HBSS) for cytochalasin B/formyl-methionyl-leucyl-phenylalanine (CytB/f-MLP) stimulation, respectively. For dye uptake, cells were incubated in PBS (supplemented with 5 mM glucose) or HBSS with 40 ␮M DHR 123 [stock solution: 40 mM in dimethyl formamide (DMF); stored in aliquots at ⫺80°C] at 37°C for 15 min. After reading the baseline DHR 123 fluorescence intensity of PMN flow cytometrically, cells were stimulated at 37°C for 15 min either with PMA (100 ng/ml in PBS; stock solution: 1 mg/ml in DMF) or f-MLP (1 ␮M in HBSS; stock solution: 10-4 M in DMSO) or remained unstimulated. CytB (10 ␮M; stock solution: 10 mM in DMSO) was added 1 min before stimulation with f-MLP. Cell suspensions were analyzed on a FACScan flow cytometer using the CONSORT 30 data analysis system and the FACScan Research Software (all from Becton Dickinson; Heidelberg, Germany). Debris and dead cells were eliminated by “live gating” on events with forward and side scatter characteristics of viable neutrophils. Cell viability after gating was consistently ⬎95% as determined by propidium iodide dye exclusion measured flow cytometrically. Fluorescence data (“green”: 530 ⫾ 15 nm) derived from RB activity (DHR 123 fluorescence) were collected using 4-decades logarithmic signal amplifications; light scatter signals were displayed on linear scales. Results from the data of 3000 –5000 intact cells were expressed as changes in mean channel fluorescence intensity (MCFI) of unstimulated neutrophils (spontaneous “respiratory burst”) during the reperfusion period. Additionally, comparing MCFI in stimulated neutrophils to unstimulated cells, increase of MCFI was calculated by applying the formula MCFI(stimulated cells) ⫺ MCFI(unstimulated cells) MCFI(unstimulated cells) thereby obtaining MCFI as a factor of increase over the MCFI of the unstimulated sample at the same time point.

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C. WILLY et al. Table 1. Values of Lactate Plasma Levels (mmol/l) of Humans Before and 0 to 120 Min After Tourniquet-Ischemia of the Lower and Upper Limb Upper limb (volunteer)a (tourniquet: 60 min)

Lower limb (patients) [tourniquet: 90 (65–130) min]

Systemic circulation

Reperfused limb

Timepoints

No. of patients

Baseline 0–2 min Peak(5–30 min) 120 min baseline 0–2 min Peak(5–30 min) 120 min

20 20 20 20 20 20 20 20

Median lactate level (25%/75% Quartile) 1.3 1.6 2.9 1.7 1.3 4.9 5.8 1.9

No. of Experiments

(1.1/1.5) (1.2/1.9) (2.1/3.6)‡ (1.5/2.2) (1.1/1.5) (3.9/5.6)‡,‡‡ (4.6/6.3)‡,‡‡ (1.6/3.1)*

10 10 10 10 10 10 10 10

Median lactate level (25%/75% Quartile) 0.8 1.2 1.2 1.0 0.8 3.6 3.1 1.0

(0.6/1.2) (1.0/1.7)* (1.0/1.9)‡ (0.8/1.1) (0.6/1.2) (3.3/4.2)‡,‡‡ (2.7/4.1)‡,‡‡ (0.7/1.7)

a

This set of experiments was performed in 1 volunteer using 10 repeated experiments. *,†,‡Significantly elevated versus preischemic baseline (*p ⬍ .05, † p ⬍ .01, ‡ p ⬍ .001; Kruskal-Wallis test with Bonferroni correction). **,††,‡‡Significantly elevated compared with corresponding values of systemic circulation (**p ⬍ .05, †† p ⬍ .01, ‡‡ p ⬍ .001; Kruskal-Wallis test with Bonferroni correction).

White blood cell count and cell viability Venous blood samples (EDTA-anticoagulated) were taken during reperfusion in both sets of experiments and 500 cells were counted on air-dried, Unna-Pappenheimstained smears by either of two experienced technicians. Additionally, cell viability was determined by using the fluorescein diacetate/ethidium bromide assay to control the effects of freezing and defrosting [37]. Freshly prepared staining solution consisted of 30 ␮g/ml fluorescein diacetate and 8 ␮g/ml ethidium bromide in PBS. Resuspended cells were mixed with staining solution (1:1), spread on a microscope slide and covered with a coverslip. Viable cells appear green-fluorescent, whereas orange-stained nuclei indicate dead cells. Five hundred cells per data point were counted.

Statistics

duced by I/R without any additional tissue trauma and anesthesia. Lactate plasma levels In both experimental settings, lactate levels significantly increased in the early reperfusion period after tourniquet-ischemia (Table 1). The peak value in the reperfused limb showed more than three times higher lactate levels compared to baseline values. Lactate levels gradually declined over a 2 h period after tourniquetischemia without returning to preischemic values in the group of patients. Compared with the contralateral limb representing the systemic circulation, postischemic lactate levels were significantly more pronounced in the reperfused limb at the peak time point (5–30 min) after tourniquet release (p ⬍ .001). DNA migration (tail moment)

Results are expressed as median values, lower 25% quartile (Q25%) and upper 75% quartile (Q75%). Data were analyzed using the nonparametric Kruskal-Wallis test with the Bonferroni correction for multiple comparisons where necessary. Statistical significance was set at *p ⬍ .05; †p ⬍ .01; ‡p ⬍ .001 (ns ⫽ not significant, vs. ⫽ versus). RESULTS

In this study, genotoxic effects were analyzed in two sets of investigations, first, in patients after I/R of the lower limb, and second, in 10 experiments in one volunteer after I/R of the upper limb. This second set of experiments was performed to analyze the effects in-

I/R of the lower limb. I/R injury combined with an operation increased the median tail moment in peripheral blood cells (Table 2, Fig.1A). While under ischemic conditions no significantly elevated tail moment was observed, immediately after tourniquet release DNA migration in white blood cells increased. Postischemic tail moment was significantly elevated 5–30 min after tourniquet release, and then declined over the 2 h period, but did not return to preischemic baseline values. Compared with the contralateral limb (systemic circulation), postischemic tail moment was significantly more pronounced in the reperfused limb at the peak time point (5–30 min) after tourniquet release (p ⬍ .05). The extent of heterogeneity within a sample for tail moment is presented in Table 3. Postischemic heterogeneity was significantly

DNA damage in human leukocytes after ischemia/reperfusion injury

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Fig. 1. DNA migration (tail moment) of human peripheral white blood cells before ischemia and at defined intervals during reperfusion at the lower (A; n ⫽ 20) and upper (B; n ⫽ 10) limbs (from 0 to 120 min; sys ⫽ systemic circulation, loc ⫽ reperfused limb, bl ⫽ baseline, p(5-30) ⫽ peak value of time-points 5, 15, and 30 min after tourniquet release). DNA migration is given as increase of tail moment compared to baseline, individually for each patient (A) resp. each experiment (B). The upper side of the gray area represents the 75% quartile, the lower side the 25% quartile. The dark gray bar represents the median. *p ⬍ .05, †p ⬍ .01, ‡p ⬍ .001 vs. baseline; **p ⬍ .05 vs. corresponding values in systemic circulation (Kruskal-Wallis test with Bonferroni correction).

elevated 0 –30 min after tourniquet release in both the reperfused and the contralateral limb. Heterogeneity returned over the 2 h period to preischemic baseline values. Compared with the contralateral limb (systemic circula-

tion) there were no significant differences. Figure 2 presents examplary in four patients the individual response within a sample for tail moment and demonstrates the interindividual differences of the increase of tail mo-

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C. WILLY et al. Table 2. Values of DNA Migration (Median Tail Moment) in the Comet Assay With Human Leukocytes Before and 0 to 120 Min After Tourniquet-Ischemia of the Lower and Upper Limb Lower limb (patients) [tourniquet 90 (65–130) min]

Systemic circulation

Reperfused limb

Timepoints

No. of patients

Baseline Intraischemic 0–2 min 5 min 15 min 30 min 120 min Peak(5–30 min) Baseline Intraischemic 0–2 min 5 min 15 min 30 min 120 min Peak(5–30 min)

20 20 20 20 20 20 20 20 20 20 20 20 20 20

Upper limb (volunteer)a (tourniquet: 60 min)

Median tail moment (25%/75% quartile) 2.83 3.07 4.24 3.77 3.73 3.86 3.35 4.09 2.83 n.v. 4.28 4.07 4.80 4.25 3.49 4.88

No. of experiments

(2.34/3.27) (2.68/3.44) (3.40/4.83)‡ (3.49/4.41)‡ (3.23/4.62)‡ (2.99/4.54)‡ (2.84/3.89)* (3.65/5.20)‡ (2.34/3.27) n.v. (3.60/4.80)‡ (3.58/4.80)‡ (3.91/6.02)‡ (3.65/4.98)‡ (3.07/4.30)† (4.35/6.43)‡,⫹

10 10 10 10 10 10 10 10 10 10 10 10 10 10 10

Median tail moment (25%/75% quartile) 3.49 3.29 3.86 4.81 4.54 3.63 3.87 4.81 3.49 n.v. 3.99 4.94 3.63 4.28 4.02 4.94

(2.65/3.70) (2.67/4.95) (3.15/6.31)* (3.47/5.84)‡ (3.84/5.36)‡ (2.98/4.36) (2.41/5.09) (3.87/5.92)‡ (2.65/3.70) n.v. (3.31/5.06)† (3.71/6.17)‡ (3.37/5.71)† (2.67/4.95)* (2.65/4.54)* (3.81/6.38)‡

a This set of experiments was performed in 1 volunteer using 10 repeated experiments. *,†,‡Significantly elevated versus preischemic baseline (*p ⬍ .05, † p ⬍ .01, ‡ p ⬍ .001; Kruskal-Wallis test with Bonferroni correction). ⫹ Significantly elevated compared with corresponding values of systemic circulation ( ⫹ p ⬍ .05; Kruskal-Wallis test with Bonferroni correction). n.v. ⫽ no value.

ment. Some patients showed a marked increase in heterogeneity (Fig. 2, e.g., patient acsc, sapo) whereas in other patients (Fig. 2, e.g., alfr, chku¨) only minimal changes could be observed. Figure 3 shows the relationship between migration of DNA (peak value of the reperfused limb) and duration of

ischemia. Not significant, but clear in its tendency, the correlation coefficient r was 0.314 for DNA migration with respect to the duration of ischemia (p ⫽ .1809; 95% confidence interval: ⫺0.150 to 0.664). The described genotoxic effects of I/R injury of the lower limb were not secondary to influences by anesthesia because neither

Table 3. Values of Heterogeneity within the Sample for Tail Moment in the Comet Assay With Human Leukocytes Before and 0 to 120 Min After Tourniquet-Ischemia of the Lower Limb Lower limb [Tourniquet: 90 (65–130) min]

Systemic circulation

Reperfused limb

Timepoints

No. of patients

Baseline Intraischemic 0–2 min 5 min 15 min 30 min 120 min Baseline Intraischemic 0–2 min 5 min 15 min 30 min 120 min

20 20 20 20 20 20 20 20 20 20 20 20

Heterogeneity (25%/ 75% quartile) 5.86 5.91 6.13 7.72 7.36 6.48 6.45 5.86 n.v. 6.66 6.71 6.93 7.69 5.99

(3.00/7.91) (5.91/8.45) (5.55/9.83)‡ (4.67/9.21)‡ (5.64/9.84)‡ (4.21/8.46)* (4.06/7.57) (3.00/7.91) n.v. (5.40/9.64)‡ (4.99/9.14)‡ (5.82/9.35)‡ (6.04/9.16)‡ (4.56/7.13)

n.v. ⫽ no value. *,†,‡: Significantly elevated versus preischemic baseline (*p ⬍ .05, † p ⬍ .01, ‡ p ⬍ .001; Kruskal-Wallis test with Bonferroni correction).

DNA damage in human leukocytes after ischemia/reperfusion injury

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Fig. 2. Distribution of DNA migration (tail moment) among human peripheral white blood cells from four examplary choosen patients (acsc, alfr, chku¨, sapo). Data based on 54 cells per sample (from 0 to 120 min after tourniquet release; s ⫽ systemic circulation, f ⫽ reperfused limb, bl ⫽ baseline).

spinal nor general anesthesia induced increased median tail moments in peripheral white blood cells (data not shown). I/R of the upper limb. Compared with the effects seen in investigations of the lower limb, the same postischemic tail moment course was seen during I/R of the upper limb (Table 2, Fig. 1B). Respiratory burst I/R injury of the upper limb induced the intracellular production of oxygen metabolites in peripheral neutrophils, which is a sign of the intracellular NADPH oxidase activation (Table 4). Postischemic spontaneous respiratory burst had its peak level at 5–30 min after tourniquet release (systemic circulation and reperfused limb, both p ⬍ .001), and then declined over the 2 h period without returning to preischemic baseline values (Table 4). Oxidative response of isolated PMN was evaluated as increase in mean fluorescence intensity after stimulation

with f-MLP or PMA. In comparison with unstimulated neutrophils at the same time point, the f-MLP- or PMAinduced respiratory burst reaction significantly declined at 5–30 min after tourniquet-release indicating preactivation of neutrophils by I/R (Table 4). The relationship between “spontaneous” respiratory burst and the factor of increased respiratory burst due to stimulation by fMLP (identical time point) showed a significant correlation coefficient r ⫽ ⫺0.656 (p ⫽ .0374; 95% confidence interval: ⫺0.910 to ⫺0.046). No correlation could be detected in our study by analyzing the relation between individual tail moment courses and the corresponding oxidative responses of neutrophils.

White blood cell count and cell viability I/R of the lower limb. White blood cell count of fresh EDTA-blood samples showed in the reperfused lower limb a marked increase for PMN up to 199.8% of baseline values (Q25%/Q75%: 147.8%/242.8%; p ⬍ .001 vs. baseline) and 149.4% in the systemic circulation (Q25%/

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C. WILLY et al. DISCUSSION

The present study was carried out to gain further insight into the effects of ischemia-reperfusion (I/R) on DNA of leukocytes in humans. The principle finding is that after tourniquet-ischemia genotoxic effects were found in human peripheral leukocytes both locally in the reperfused limb and systemically in the contralateral, not affected limb even in the early reperfusion period. I/R-induced DNA effects in human leukocytes during the early reperfusion period

Fig. 3. Relationship between DNA migration of human peripheral white blood cells (tail moment of the peak value of the reperfused limb) and duration of ischemia (n ⫽ 6 tourniquet ⬍ 80 min; n ⫽ 8 tourniquet from 80 to 100 min; n ⫽ 6 tourniquet ⬎ 100 min). Each black dot represents the peak value of one patient of the time points 5, 15, and 30 min after tourniquet release in the reperfused limb. The upper side of the gray rectangle represents the 75% quartile, the lower side the 25% quartile. The dark gray bar represents the median of each group (no significant differences, Kruskal-Wallis test).

Q75%: 124.8%/235.9%; p ⬍ 0.001 vs. baseline) 120 min after release of the tourniquet. At the same time point, in the reperfused lower limb a pronounced decrease of lymphocytes was observed to 69.3% (Q25%/Q75%: 63.2%/104.4%; p ⬍ .01 vs. baseline) and 76.5% (Q25%/ Q75%: 58.3%/107.1%; p ⬍ .01 vs. baseline). Defrosted leukocytes evaluated in the comet assay showed the following constant blood differential independent of the time point of venipuncture: 65.1% lymphocytes, 11.4% monocytes, and 23.5% granulocytes. This unphysiological differential is caused by freezing and thawing. So, freezing and thawing caused the sticking together of granulocytes resulting in a loss for viability measurement and in the comet assay. Cell viability measured by the fluorescein diacetate/ethidium bromide method was ⬎ 97% in every experiment. I/R of the upper limb. Also, 120 min after release of the tourniquet blood samples showed a significant increase of PMN up to 141.7% in the reperfused limb (Q25%/ Q75%: 108.9%/162.7%; p ⬍ .001 vs. baseline) and 145.0% in the systemic circulation (Q25%/Q75%: 106.7%/ 162.4%; p ⬍ .001 vs. baseline). Simultaneously, a pronounced decrease of lymphocytes to 87.0% in the reperfused limb (Q25%/Q75%: 67.1%/90.0%; p ⬍ .01 vs. baseline) and to 87.5% in the systemic circulation (Q25%/ Q75%: 74.1%/96.0%; p ⬍ .01 vs. baseline) could be observed.

In vitro studies have demonstrated that I/R induces DNA damage in cultured cells [38] or isolated tissue [20,39]. These findings have been confirmed in animal studies in which I/R was found to affect DNA and to result in double- and single-strand breaks [21–23,40 – 42] A postmortem human study investigating I/R-induced effects after acute myocardial infarction demonstrated DNA strand breaks in reperfused myocytes [29]. These observations are extended by our findings obtained in human white blood cells, which demonstrate significant effects on DNA during the first 2 h after tourniquetischemia of the upper or lower limbs (Fig. 1). Numerous studies have shown that this I/R-associated DNA damage could be due to the generation of oxygen radicals during the reperfusion period. In fact, reports of animal experiments demonstrated that DNA could be a target of free radicals released after ischemia and reperfusion. So, DNA lesions that are characteristics of DNA damage mediated by free radicals (e.g., 8-hydroxy-2⬘-deoxyguanosine) were detected at a significantly increased level during reperfusion [23, 42]. Additionally, in vitro experiments showed that chromosomal damage in human lymphocytes depends on xanthine oxidase concentration and on the duration of exposition to this potent source of oxygen free radicals (OFR) [43]. In cultured cells DNA damage after reperfusion could be prevented by genetic induction of Cu,Zn-superoxide-dismutase cDNA [44]. Our findings, that the oxidative status of granulocytes increased simultaneously with elevated DNA effects (Table 4), confirm the potential pivotal role of OFR inducing DNA damage during reperfusion. Considering this relation between DNA damage and the well-known oxidative response to I/R, it remains unclear why we observed no significant correlation between DNA strand breaks and the respiratory burst of neutrophilic cells analyzing each of the individuals (analysis not shown). It is intriguing to speculate that apart from the respiratory burst of neutrophils various mechanisms not assessed in the present study are operative in influencing I/R-mediated genotoxic effects. In fact, there

DNA damage in human leukocytes after ischemia/reperfusion injury

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Table 4. Values of Spontaneous “Respiratory Burst” (MCFI) in % Baseline and of the Increase of Respiratory Burst (MCFI) in % Baseline After Stimulation by f-MLP and PMA in Human Peripheral Neutrophils Before and 0 to 120 Min After Tourniquet-Ischemia of the Upper Limba Increase of respiratory burst in % baseline Spontaneous respiratory burst

Timepoints Systemic Circulation

Reperfused Limb

Baseline 0–2 min Peak(5–30 min) 120 min Baseline 0–2 min Peak(5–30 min) 120 min

Median (25%/75% quartile) 100.0 115.4 139.0 121.5 100.0 115.7 131.8 109.4

— (92.9/134.6) (118.4/161.5)‡ (117.4/144.2)† — (88.8/130.8) (111.1/155.0)‡; ns (102.5/137.6)*; ns

Stimulation by f-MLP

Timepoints Baseline 0–2 min Nadir(5–30 min) 120 min Baseline 0–2 min Nadir(5–30 min) 120 min

Median (25%/75% quartile) 100.0 93.4 65.5 77.6 100.0 103.4 72.0 88.4

— (76.8/118.1) (63.3/78.3)† (63.7/109.1) — (79.0/106.5) (61.8/93.0)†; ns (63.6/117.0)

Stimulation by PMA Median (25%/75% quartile) 100.0 111.7 88.6 68.4 100.0 103.7 69.5 102.1

— (80.9/116.8) (71.1/98.5) (62.5/109.9) — (78.6/111.1) (64.7/103.7)*; ns (63.6/155.4)

This set of experiments was performed in 1 volunteer using 10 repeated experiments (n ⫽ 10). MCFI ⫽ mean channel fluorescence intensity; ns ⫽ Not significantly elevated compared with corresponding values of systemic circulation (Kruskal-Wallis test with Bonferroni correction). *,†,‡: Significantly elevated vs. preischemic baseline (*p ⬍ .05, † p ⬍ .01, ‡ p ⬍ .001, Kruskal-Wallis test with Bonferroni correction). a

is a growing body of evidence, that (i) other oxidative mediators like peroxynitrite, a potent trigger of DNA strand breakage, produced from nitric oxide (NO) [19] and the endothelial xanthine-oxidase-system [11,43]; (ii) several subsequent cellular responses like the DNA damage-inducible expression of the GADD45 gene [45] as well as the activation of nuclear enzymes (e.g., endonuclease, poly(ADP ribose)-synthetase (PARS)) [46,47]; and (iii) protective antioxidative mechanisms [27,48] are implicated in reperfusion-associated DNA damage. Therefore, our results demonstrating an increased respiratory burst in granulocytes and reperfusion-associated DNA effects could not prove a causal relationship between these two phenomena. Nevertheless, against the background of the literature presented above it seems plausible that the DNA effects observed in our study are due to postischemic oxidative stress. Further studies investigating additionally oxidized DNA bases, like 8-hydroxy-2⬘-deoxyguanosine, could contribute to further insight into the real intranuclear interactions between OFR and DNA during the reperfusion period. Local ischemia resulted in early systemically detectable genotoxic effects and marked oxidative response in peripheral white blood cells Previous animal studies described DNA single-strand breaks in neurons after as little as 1 min of reperfusion after 1 h of middle cerebral artery occlusion [22]. Other authors found significantly increased lesions (8-hydroxy2⬘-deoxyguanosine) in cortical DNA during 10 –20 min of reperfusion in mice after 30 min of forebrain ischemia [40] and a significant increase of DNA single-strand breaks in gerbils after 15 min of ischemia and 60 min of

recirculation [21]. Our findings demonstrate that in humans, too, after 60 –130 min of ischemia genotoxic effects appear immediately after tourniquet release (Table 2). These effects are relatively heterogeneous among cells as a function of sample time (Table 3). The majority of cells are unaffected and therefore the increased tail moment reflects differences both in the extent of migration and the proportion of damaged cells. In addition, our results (Fig. 3) accord with observations in animal studies, that the intensity of DNA damage increased proportionally with the duration of ischemia [49,50]. This may be due to an increase of radical-producing substrates during ischemic periods of longer duration [51]. Compared with the baseline, DNA damage, oxidative response (“respiratory burst”) and the concentration of neutrophils showed a significant increase in the systemic circulation. This demonstrates that even short-time ischemia of a limb lasting only 60 min as is commonly used, e.g., for “minor” hand surgery results in numerical, morphologic, and biochemical alterations of all circulating white blood cells in the human organism. It is well known, that particularly PMNs release substantial amounts of oxygen radicals and other reactive agents [9,10,12]. This suggests that the locally restricted ischemic event is extended to the whole body and possibly results in secondary tissue damage through activated inflammatory cells and in induction of prolonged DNA damage in interacting cells (e.g., endothelial cells of the systemic circulation) [52]. There have been no investigations in humans up to now, which could confirm or disprove our findings. Apart from this, peak values of DNA damage showed a significant difference between leukocytes from the reperfused lower limb and those from the contralateral

10

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limb representing the systemic circulation. Likewise, in comparison to the “systemic” limb a significantly increased peak value of serum lactate levels could be observed in both experimental setups demonstrating pronounced metabolic alterations in the affected postischemic tissue (Table 1). The difference in DNA damage between the systemic and local limb was not seen during reperfusion after tourniquet-ischemia of the upper limb (Fig. 1). One reason could be the additional effects due to anterior cruciate ligament reconstruction or the more severe I/R-injury of the more voluminous lower limb leading to a greater volume of affected tissue. These assumptions were confirmed by lower increases of serum lactate and neutrophil levels after tourniquet-ischemia of the upper limb. DNA effects in human white blood cells declined gradually after 2 h of reperfusion but did not return to preischemic baseline values In our study, the peak value of DNA effects in human leukocytes appeared 15 to 30 min after tourniquet release and the tail moment had not quite returned to baseline values after 2 h of reperfusion. Previous animal studies revealed similar time courses of DNA damage after I/R. They showed that postischemic effects on DNA peaked at 1 to 6 h after tourniquet release and declined gradually afterwards to the baseline level after 4 to 24 h [21,22, 50,53]. Against this background, it is noteworthy that after an acute ␥-irradiation the DNA damage of human leukocytes measured with the comet assay was almost repaired within 2 h [26,33,55]. This is in accordance with investigations showing that the half-life of DNA singlestrand breaks is 5 min and that of DNA double-strand breaks is 12 min [54]. The persistence of DNA damage after I/R can be explained by the fact that in contrast to the cytotoxic effects of irradiation, the production of OFR lasted over all the observed reperfusion period and did not stop after the initial reperfusion event. Apart from a repair process, the measured decrease in tail moment could also be a “mix-effect” due to new populations of leukocytes possibly released out of the storage pools (bone marrow, spleen, etc.). To gain additional insight into the biological significance of the DNA effects observed using the comet assay it is necessary to compare these effects with other endpoints of genotoxicity. As in animal studies reported earlier, I/R leads to apoptosis which was detected by an increased frequency of nucleosomal DNA fragments on agarose gels (“DNA ladder”) [56,57], nuclear chromatin condensation [57], by dUTP incorporation into damaged DNA (TUNEL assay) [21,22,29,39,40,44,49,57] or detected using in situ DNA polymerase I-mediated biotindATP nick-translation (PANT) [22]. In addition, it could

be shown that I/R increased the mutation frequency in a reporter lacI gene in cortical DNA of transgenic mice after 30 min of forebrain ischemia and 8 h of reperfusion from 1.5 to 7.7 (per 100,000) [42]. Human in vitro I/R studies also detected DNA damage using the in situ DNA end-labeling method in myocytes, hepatocytes, and sinusoidal endothelial cells [29,30]. It may, therefore, be assumed that I/R induces both apoptosis and increase of mutagenity of affected cells. Up to now, the comet assay has not been used to assess DNA damage after I/R. However, the principle genotoxicity of OFR has been investigated in several studies using the comet assay. [25,32]. Against this background, it is intriguing to speculate that most of the postischemic DNA lesions detected in this study may have undergone error-free repair and that probably only a minority of them induced deleterious effects. However, it is possible, that the detected DNA damage corresponds to apoptosis. But, despite the strong effects on DNA as seen in the comet assay in our study, the role of the genetic stress response during ischemia/reperfusion and the fate of the cells undergoing it remains unknown. In conclusion, our experiments have shown genotoxic effects and the simultaneous occurrence of intracellular OFR production after I/R. These effects were seen both locally in the reperfused limb and—to a less pronounced extent—in the systemic circulation throughout the body implicating possible secondary tissue damage. A close relationship exists between the DNA damaging activity of endogenous reactive oxygen species and their effects on the microcirculation. Because dietary antioxidants reduce the extent of I/R injury at the microcirculatory level, antioxidants should protect against DNA damage, too. Based on the results obtained in this study, and in the light of relevant observations in the literature an approach may be suggested to verify DNA effects of dietary antioxidants after I/R in additional studies using the comet assay which is a useful tool for examining issues related to oxidative stress in human leukocytes. Acknowledgements — This work was financially supported by a grant from the Ministry of Defense of the Federal Republic of Germany (25G3–S–129699). We are grateful to P. Schu¨tz and G. Baur, D. Leipert, and M. Zarrabi for their skillful technical assistance.

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