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THE PARP-1 INHIBITOR INO-1001 FACILITATES HEMODYNAMIC STABILIZATION WITHOUT AFFECTING DNA REPAIR IN PORCINE THORACIC AORTIC CROSS-CLAMPING-INDUCED ISCHEMIA/REPERFUSION

Hauser, Balázs; Gröger, Michael*; Ehrmann, Ulrich*; Albicini, Maura; Brückner, Uwe Bernd; Schelzig, Hubert; Venkatesh, Balasubramanian#; Li, Hongshan**; Szabó, Csaba**††; Speit, Günter§; Radermacher, Peter*; Kick, Jochen

doi: 10.1097/01.shk.0000209561.61951.2e
Basic Science Aspects
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Inhibition of poly (ADP-ribose) polymerase 1 (PARP-1) improved hemodynamics and organ function in various shock models induced by sepsis or ischemia/reperfusion. PARP-1, however, is also referred to play a pivotal role for the maintenance of genomic integrity. Therefore, we investigated the effect of the PARP-1 blocker INO-1001 on hemodynamics, kidney function, and DNA damage and repair during porcine thoracic aortic cross-clamping. The animals underwent 45 min of aortic cross-clamping after receiving vehicle (n = 9) or i.v. INO-1001 (n = 9; total dose, 4 mg·kg−1, administered both before clamping and during reperfusion), data were recorded before clamping, before declamping, and 2 and 4 h after declamping. During reperfusion, continuous i.v. norepinephrine was incrementally adjusted to maintain blood pressure greater than or equal to 80% of the preclamping level. The plasma INO-1001 levels analyzed with high-pressure liquid chromatography were 1 to 1.4 μmol/L and 0.4 to 0.6 μmol/L before and after clamping, respectively. Although INO-1001-treated animals required less norepinephrine support, kidney function was comparable in the 2 groups. There was no intergroup difference either in the time course of DNA damage and repair (comet assay) as assessed both in vivo in whole blood before surgery, before clamping, before declamping, 2 h after declamping, and ex vivo in isolated lymphocytes (Ficoll gradient) sampled immediately before clamping and analyzed before, immediately, and 1 and 2 h after exposure to 4 bar 100% O2 for 2 h. There was no difference either in the expression of the cyclin-dependent kinase inhibitor gene, p27, in the kidney (immunohistochemistry). The reduced norepinephrine requirements during reperfusion suggest a positive inotropic effect of INO-1001, as demonstrated by other authors. In our model, INO-1001 proved to be safe with respect to DNA repair.

*Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung, Abteilung Thorax-und Gefäβchirurgie, Sektion Chirurgische Forschung, and §Abteilung Humangenetik, Universitätsklinikum, Ulm, Germany; ¦Aneszteziológiai és Intenzív Terápiás Klinika, Semmelweis Egyetem, Budapest, Hungary; Istituto di Anestesiologia e Rianimazione dell'Università degli Studi di Milano, Azienda Ospedaliera, Polo Universitario San Paolo, Milan, Italy; #Princess Alexandra and Wesley Hospitals, University of Queensland, Brisbane, Australia; **Inotek Pharmaceuticals Corporation, Beverly, Mass; and ††Klinikai Kísérleti Kutató-é Humán Élettani Intézet, Semmelweis Egyetem, Budapest, Hungarys

Received 22 Nov 2005; first review complete 7 Dec 2005; accepted in final form 6 Jan 2006

Address reprint requests to Peter Radermacher, MD, Sektion Anästhesiologische Pathophysiologie und Verfahrensentwicklung, Universitätsklinikum, D-89073 Ulm, Germany. E-mail: peter.radermacher@medizin.uni-ulm.de.

B. Hauser and M. Gröger equally contributed to this work.

This study was supported by the Deutsche Forschungsgemeinschaft (DFG Sche 899/2-1) and the Hungarian National Research Fund (OTKA; C.S.). Dr B. Hauser was the recipient of a Roman Herzog Research Fellowship of the Alexander von Humboldt Stiftung and the Gemeinnützige Hertie Stiftung. INO-1001 was kindly provided by Dr Garry Southan (Inotek Pharmaceuticals Corporation, Beverly, MA). Prof B. Venkatesh was supported by a study leave from the Princess Alexandra and Wesley hospitals in Brisbane, Australia, to the University of Ulm in Germany. Special thanks are dedicated to Wolfgang Siegler, Tanja Schulz, Ingrid Eble, Christian Maier, Martin Mergenthaler, and Matthias Wuttke for their skillful technical assistance.

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INTRODUCTION

Thoracic aortic cross-clamping is a typical example of ischemia/reperfusion (I/R) injury that affects those organs, which receive their blood supply from below the level of cross-clamping. I/R-induced O2 radical formation causes oxidative DNA strand breakage, which, in turn, activates the nuclear enzyme poly (ADP-ribose) polymerase (PARP; (1, 2)). Activation of PARP results in an inefficient cellular metabolic cycle with transfer of the ADP-ribosyl moiety of NAD+ to protein acceptors, which, in turn, results in depletion of cellular energy-rich phosphates and necrotic-type cell death. The major pathophysiological consequences of this cellular energy failure are vascular hyporeactivity, myocardial failure, and gut epithelial dysfunction (1, 2). Consequently, highly potent PARP inhibitors were developed (1, 2), which attenuated organ dysfunction in various large animal shock models (3-10). In particular, these compounds improved heart function in sepsis (3), endotoxemia (4), or after I/R injury associated with cardiopulmonary bypass (8, 9). Given these encouraging results, we investigated the effect of the novel potent PARP-1 inhibitor INO-1001 (11) on hemodynamics and kidney function in a recently established porcine model of thoracic aortic cross-clamping-induced I/R injury (12). Swine were investigated because of their striking similarity with humans with respect to both their susceptibility to oxidative stress and tissue antioxidant profiles (13, 14).

The pathophysiological consequences of PARP-1 activation, however, are opposed to its vital role in the maintenance of genomic integrity through its function in base-excision repair (1), a phenomenon referred to as the "Yin Yang of PARP activation" (1). Because inconclusive data are available in the literature on the effects of PARP-1 inhibition on DNA damage and repair (15-20), we also evaluated the effect of INO-1001 on both the induction and the repair of DNA strand breaks using the comet assay (single-cell gel electrophoresis; (21)). Finally, we additionally assessed the effects of INO-1001 on the expression of the cyclin-dependent kinase inhibitor (CDKI) gene, p27 (Kip1) (p27), which is referred to as a marker of cell senescence in the kidney (22) after I/R injury (23, 24).

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MATERIALS AND METHODS

The experiments were performed in adherence to the National Institutes of Health Guidelines on the Use of Laboratory Animals. The experimental protocol for this study was approved by the university Animal Care Committee and the federal authorities for animal research (Regierungspräsidium Tübingen, Baden-Württemberg, Germany). Eighteen 3-month-old domestic pigs (German Landrace) with a median body weight of 47 kg (range, 43-60 kg) of either sex were used.

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Animal preparation

The preparation of the animals before surgery, induction, maintenance of anesthesia, and placement of catheters have been described in detail previously (12). During the surgical instrumentation, hydroxyethylstarch was infused as needed to maintain constant cardiac filling pressures. Body temperature was monitored rectally and kept normal (range, 37.5-38.5°C) with heating pads or external cooling. From a left anterior thoracotomy, the descending thoracic aorta was exposed, and a polyester surgical retraction tape was placed around the aorta immediately below the origin of the left subclavian artery. The retraction tape was directed through a plastic tube out of the thoracic cavity and a drain was put in place to prevent tension pneumothorax.

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Measurement and calculations

Blood pressure (peripheral mean arterial pressure [pMAP], diastolic mean arterial pressure), mean pulmonary arterial pressure, and central venous pressure were continuously recorded; pulmonary arterial occlusion pressure and cardiac output were also intermittently recorded (12). Intrathoracic blood volume was assessed via thermal dye double-indicator dilution using indocyanine green. Renal blood flow was continuously recorded with a precalibrated ultrasound flow probe (12). Arterial, pulmonary arterial, and renal venous blood samples were analyzed for blood gases and acid-base status and for total hemoglobin content and hemoglobin O2 saturation (12). Systemic and regional oxygen transport data were calculated from the hemodynamic and blood gas results using standard formula. Arterial and renal venous lactate and pyruvate concentrations were measured with commercially available kits using a chemiluminescence whole-blood analyzer (12). The creatinine clearance was calculated from the serum and urine creatinine levels, and the urine volume was collected from the beginning of the 45-min period of clamping until the end of the subsequent 4 h of reperfusion (i.e., over a total 285 min (12). Serum creatinine levels were normalized per g total plasma protein content to correct for any dilutional effects of intravenous fluids (25). INO-1001 plasma and intracellular concentrations in the isolated lymphocytes, as shown in the succeeding section, were measured by high-pressure liquid chromatography, as described previously (26).

Surgery-and-I/R injury-related DNA damage in vivo was assessed in whole-blood samples-collected before starting the surgical instrumentation, before clamping, before declamping, and 2 h after declamping-measuring the "tail moment" in the alkaline version of the comet assay (single-cell gel electrophoresis; (27-29)). Induction and repair of DNA damage was evaluated ex vivo in isolated lymphocytes before and after exposure to hyperbaric oxygen (HBO; 2 h at 4 bar), as described previously (29). For this purpose, the cells were separated on Ficoll gradients from whole-blood samples taken immediately before cross-clamping (to ensure that intracellular INO-1001 concentrations had reached levels consistent with PARP-1= blockade). Thereafter, they were denaturated with alkali, and electrophoresis was performed (27-29). Measurements were made by image analysis (Comet Assay II; Perceptive Instruments, Haverhill, Suffolk, UK), determining the mean tail moment of 50 cells per slide (27-29).

Immunohistochemical examination of kidney tissue samples for p27 gene expression was performed using specific antibodies, as described previously (23, 24). Briefly, the specimens were fixed in formalin. After that, the samples were embedded in paraffin, cut, and fixed on microscope slides. Tissue samples were then deparaffinated. Endogenous peroxidase was blocked by incubation in hydrogen peroxide dissolved in methanol. The specimen was incubated with anti-p27 (p27 (C-19) sc-528 rabbit polyclonal IgG; Santa Cruz Biotechnology, Inc, Santa Cruz, Calif) antibodies. Tissue sections were revealed by EnVision monoclonal system (EnVision+ System-labeled Polymer-HRP Anti-Rabbit; DAKO Cytomation, Carpinteria, Calif), developed with liquid diaminobenzidine DAB+ substrate-chromogen system (Liquid DAB+ Substrate Chromogen System; DAKO Cytomation, Carpinteria, Calif) and counterstained with hematoxylin. Slides were evaluated by an investigator blinded for the group assignment, and only nuclear staining was considered positive and counted. Scores were performed for glomerular, tubular, interstitial, and vascular p27 expression. To ensure maximum compatibility and validity of results, rectangles of the kidney cortex measuring 0.5 × 0.5 cm were marked in each slide, and all counts were performed within that area.

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Experimental protocol

After surgical preparation, 120 min of recovery time was allowed. Thereafter, the animals were randomly assigned to receive either the vehicle (glucose 5%) or the PARP inhibitor INO-1001 (dose, 2 mg·mL−1 diluted in glucose 5%), starting at 90 min before clamping. After administering an initial INO-1001 dose of 2 mg·kg−1·h−1 after 30 min, 1 mg kg−1·h−1 INO-1001 was infused for 60 min until the time immediately before clamping. Glycemia was controlled to stay between 60 and 120 mg·dL−1. Even the highest INO-1001 or vehicle infusion rate of 100 mL/h during the first 30 min of drug administration (i.e., a total of 2.5 g glucose infused for 30 min) did not affect blood glucose so that insulin was never needed to achieve the target glycemia values. The INO-1001 infusion was stopped during the clamping period and restarted again after declamping during the remaining 4 h until the end of the experiment (dosage, 0.5 mg·kg−1·h−1). As a result, the animals received a total of 4 mg·kg−1 during the whole experiment. This approach was chosen so that plasma levels were higher at the time immediately before clamping to provide a sufficient loading up of the tissues with the PARP-1 inhibitor. Furthermore, total doses of 1 mg kg−1 and 4 mg kg−1 of INO-1001 in dogs (7-9) and pigs (30), respectively, had virtually completely abolished poly (ADP-ribose) staining in the heart, lung, and intestine after postcardiopulmonary bypass reperfusion. After baseline data collection, the retraction tape around the aorta was tightened for 45 min, and complete aortic occlusion was verified by the disappearance of the blood pressure distal to the clamping. This clamping period was chosen because, in a previous investigation, 45 min of juxtarenal aortic cross-clamping had resulted in a 50% drop in the creatinine clearance affiliated with a comparable increase of blood creatinine concentrations (12). During the clamping period, a combination of nitroglycerine (dosage, 1.7 mg·min−1), esmolol (dosage, 16.5 mg·min−1), and adenosine-5'-triphosphate (dosage range, 2-10 mg·min−1) was infused and adjusted to maintain pMAP between 80% and 120% of the baseline value. The choice of these drugs is based on experience in our previous study (12). To ensure constant fluid administration, all animals received 10 mL·kg−1·h−1 of Ringer solution from the beginning of the surgical instrumentation until the end of the experiment. In addition, 1500 mL of hydroxyethylstarch was infused during the clamping period to optimize preload before the declamping. Five min before declamping, a second set of data was obtained and the retraction tape was gradually released after 1 min. To prevent declamping-associated hypotension, another 1500 mL of hydroxyethylstarch was administered during the first 30 min of reperfusion. In addition, continuous i.v. norepinephrine was incrementally adjusted as long as needed to maintain a pMAP level greater than 80% of baseline value. Further data sets were obtained at 120 and 240 min after declamping. At the end of the experiment, the animals were killed under deep anesthesia with an additional dose of sodium pentobarbital and intravenous potassium chloride, and biopsies were taken from the right kidney for immunohistochemistry.

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Statistical analysis

After exclusion of normal distribution using the Kolmogorov-Smirnov test, the Friedman repeated measures analysis of variance on ranks with post hoc multiple comparison procedure (Dunn method) and subsequent Bonferroni correction were used for comparison of data between measurement points. Mann-Whitney test was performed to compare data between treatment groups at identical measurement points. A P value less than 0.05 was regarded as statistically significant.

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RESULTS

One animal died in the INO-1001 group because of acute cardiac failure immediately after the third data collection, 2 h after declamping. Because the subsequent autopsy did not reveal any plausible cause of death other than accidental acute pulmonary air embolism, data from this animal are included in the analysis. Median INO-1001 plasma levels were 1.35 μmol/L before clamping and 0.48 μmol/L during reperfusion (Fig. 1), whereas the median plasma levels in the intracellular concentrations in the isolated lymphocytes were 72 nmol/L (range, 40-118 nmol/L) and 38 nmol/L (range, 24-44 nmol/L) before and 2 h after exposure to HBO, respectively.

Fig. 1

Fig. 1

Systemic hemodynamics, oxygen exchange, and metabolism data are summarized in Table 1. Aortic cross-clamping and the vasodilator infusion to maintain blood pressure resulted in a significantly increased cardiac output. Neither preload parameters nor metabolic or oxygen-exchange variables showed significant differences between the 2 groups at any time point. However, a significantly shorter norepinephrine infusion time and reduced total norepinephrine doses (Fig. 2) were needed to achieve the target blood pressure in the INO-1001-treated animals during reperfusion.

Table 1

Table 1

Fig. 2

Fig. 2

Data on renal hemodynamics, oxygen exchange, and metabolism are presented in Table 2. Urine production was similar in the 2 groups, whereas renal function, as defined by serum creatinine and creatinine clearance, remained impaired until the end of the observation period, again without intergroup difference.

Table 2

Table 2

The time course of the surgery-and-I/R injury-related DNA damage in vivo in whole-blood samples and the oxidative DNA damage in isolated lymphocytes exposed to HBO ex vivo are presented in Fig. 3, A and B, respectively. The surgical instrumentation and the clamping/declamping procedure doubled the DNA damage when compared with the values immediately after induction of anesthesia. Importantly, there was, however, no intergroup difference. HBO exposure of lymphocytes, isolated immediately before clamping (i.e., after infusing 2 mg·kg−1 INO-1001 or vehicle, respectively), caused a 2-fold to 3-fold increase of the DNA damage, which had returned to baseline 2 h after the HBO exposure, indicating rapid and efficient repair. Again, there was no intergroup difference. In addition, INO-1001 did not modify the immunohistochemical expression of the cyclin-dependent kinase inhibitor gene, p27, in the kidney (Fig. 4).

Fig. 3

Fig. 3

Fig. 4

Fig. 4

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DISCUSSION

These are the cardinal findings of the present study: (1) INO-1001 significantly reduced the need for vasopressor support required to maintain hemodynamic target values during the early reperfusion period, but (2) did not influence postclamping kidney function, and, likewise, (3) did not affect induction and repair of DNA damage nor the expression of the cyclin-dependent kinase inhibitor gene, p27, indicating that selective PARP-1 inhibition is safe with respect to cellular genome integrity.

Aortic cross-clamping is a typical example of I/R injury where overproduction of reactive oxygen species may lead to both DNA damage (31-33) and/or organ injury (34, 35). In swine, postischemic oxidative DNA damage was found after transplantation of the liver and the small intestine (36), whereas organ injury presented as acute renal failure (37), paraplegia (38, 39), and impairment of intestinal barrier function (40). Our animals did not develop acute renal failure but presented with a 50% to 75% increase in plasma creatinine concentrations at the end of the experiment, thus with comparable degree of renal dysfunction as induced by juxtarenal aortic cross-clamping of equal duration (12). Two phenomena may explain this result: On the one hand, we only performed 45 min of aortic cross-clamping, whereas 60 to 120 min of clamping were investigated in all other studies. On the other hand and in sharp contrast with the studies mentioned previously (36-40), to mimic the clinical scenario of surgical interventions that comprise cross-clamping of the thoracic and/or abdominal aorta, we kept MAP within tight limits of the preclamping level both during the clamping period (using continuous i.v. esmolol, nitroglycerine, and adenosine triphosphate [ATP]) and during the early reperfusion phase (by aggressive fluid resuscitation and vasopressor support). The ATP administration may have assumed particular importance to attenuate kidney dysfunction in our study: ATP infusion markedly improved organ function in various models of I/R injury (41, 42). In contrast with the moderate renal dysfunction, we found, using the comet assay, that the surgical manipulation, aortic cross-clamping, and reperfusion was associated with a 3-fold to 4-fold increase in DNA damage in whole-blood samples, which agrees well with the amount of DNA damage observed in human leukocytes after tourniquet ischemia (43) and coronary angioplasty (44) when using the same methodology.

INO-1001 reduced both the norepinephrine amount and the norepinephrine application time required to maintain mean blood pressure within the target limits during the early reperfusion period. Heart rate, stroke volume, and filling pressures were comparable in the 2 groups during the norepinephrine infusion. Thus, although we did not directly assess the parameters of myocardial function, it is conceivable that INO-1001 allowed for a quicker and easier recovery of the myocardial contractility and/or the vascular vasoconstrictor responsiveness. This finding is consistent with previous studies in dogs, after cardiopulmonary bypass (8, 9), and swine, after regional myocaradial ischemia induced by left anterior descending coronary artery occlusion (45): PARP inhibition with PJ34 (45) or INO-1001 (8, 9, 30) improved the time derivative of both left and right ventricular pressures, elastance, and coronary perfusion, ultimately resulting in reduced infarct size (30, 45).

A major limitation of our study is the fact that we performed neither tissue poly (ADP-ribose) staining nor direct measurements of PARP activity, so that we do not have a direct proof of efficient PARP blockade. It must be underscored, however, that the same micromolar range of INO-1001 plasma concentrations was found in blood and heart samples, which showed a marked degree of inhibition of cellular PARP activity after 4 weeks of 1.25 mg·kg−1·h−1 oral INO-1001 in mice (26). Furthermore, when injected intravenously, the same (4 mg·kg−1) or even a markedly lower (1 mg·kg−1) total amount of INO-1001 in pigs (30) and dogs (7-9), respectively, as in our experiment, had virtually completely abolished poly (ADP-ribose) staining in the heart, lung, and intestine after postcardiopulmonary bypass reperfusion. Finally, in Chinese hamster ovary cells (20) and human neuronal SK-N-MC cells (46), incubation with 0.05 μmol/L INO-1001 (i.e., the same drug concentration as we measured in the isolated lymphocytes) resulted in up to 90% blockade of PARP activity. Hence, it is likely that PARP activation was efficiently inhibited by the INO-1001 infusion in our animals. Nevertheless, neither the surgery-and-I/R injury-related DNA damage in vivo nor the induction and the repair of DNA damage in isolated lymphocytes exposed to HBO ex vivo were affected. DNA single-strand breaks were quantified using comet assay, which is well known as a sensitive genotoxicity test. In its alkaline version, which was used in the present work, DNA strand breaks, incomplete excision repair sites, and alkali labile sites may contribute to increased DNA migration (21). In addition, ex vivo exposure of isolated lymphocytes to HBO not only represents a well-established model of oxidative DNA damage (28, 29, 47), but also allows for the study of the time course and the efficiency of DNA repair (48-51). Consequently, the virtually identical time courses of DNA damage, both in vivo and ex vivo, in the 2 groups indicate that PARP-1 inhibition with INO-1001 did not affect DNA repair under the experimental conditions investigated. This finding is underlined by the comparable expression of the CDKI gene, p27, in the kidney biopsies: in rodent models of kidney I/R injury (52), the amount of DNA damage was directly related to the enhanced expression of another CDKI gene, p21. The increased expression of these cell cycle regulatory molecules is referred to facilitate DNA repair (53), and, consequently, an uninfluenced time course of I/R-related DNA damage and repair as detected by the comet assay is likely to be concomitant with similar CDKI gene expression. Our finding that PARP-1 blockade with INO-1001 did not affect DNA repair and, hence, agrees with a previous in vitro work stating that PARP-1, in fact, is not essentially necessary for efficient DNA repair (16), possibly because it can be substituted by the PARP-2 isoform (54).

Recent reports indicate that there is a sex difference in the efficacy of PARP inhibitors in shock and reperfusion: Although PARP inhibition or PARP deficiency is efficacious in male animals, the efficacy is reduced in female animals (whereas, at the same time, female animals enjoy a "baseline" degree of protection against reperfusion injury and shock-induced mortality; (55-57)). Although the current study was not designed and powered to investigate sex differences, it did use animals of both sexes. A post hoc analysis revealed that the norepinephrine infusion time was statistically and significantly reduced by PARP inhibition when all animals were analyzed, and the difference remained significant in the male-only subgroup (71 min [range, 61-83 min] in the vehicle versus 50 min [range, 23-60 min] in the INO-1001 group, P = 0.01). However, no difference was seen in the female subgroup with and without PARP inhibition (69 min [range, 41-81 min) in the vehicle versus 53 min [25-93 min] in the INO-1001 group). The above subgroup analysis may support the notion that in cross-clamping, similar to stroke and endotoxin shock (55-57), male animals enjoy preferential cardiovascular protection when treated with PARP inhibitors.

In summary, in our porcine model of thoracic aortic cross-clamping, the PARP-1 inhibitor INO-1001 markedly reduced the norepinephrine requirements needed to maintain stable hemodynamics during the early reperfusion period, thus confirming the beneficial effect on heart function observed by other authors. INO-1001, however, did not attenuate kidney dysfunction, presumably as a result of the rather short clamping time. The unaffected time course of the DNA damage, both in vivo and ex vivo, in isolated and stimulated cells and the comparable renal CDKI gene expression suggest that PARP-1 blockade under these experimental conditions does not interfere with DNA repair.

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Keywords:

Norepinephrine; renal function; DNA strand breaks; comet assay; cyclin-dependent kinase inhibitor gene; p27

©2006The Shock Society