Fullerene derivative attenuates ischemia-reperfusion-induced lung injury

Life Sciences 72 (2003) 1271 – 1278 www.elsevier.com/locate/lifescie

Fullerene derivative attenuates ischemia-reperfusion-induced lung injury Y.-L. Lai a,*, P. Murugan b, K.C. Hwang b a

Department of Physiology, National Taiwan University College of Medicine, No. 1 Sec. 1, Jen-ai Road Taipei 100, Taiwan b Department of Chemistry, National Tsing Hua University, Hsinchu, Taiwan Received 27 March 2002; accepted 3 October 2002

Abstract Reactive oxygen species are the major contributing factors to lung ischemia-reperfusion (IR) injury. In this study, we tested whether a water soluble antioxidant fullerene derivative [C60(ONO2)7 F 2] attenuates IR lung injury. Young Wistar rats were divided into two groups: control and C60(ONO2)7 F 2. Under ventilation with 95% air-5% CO2 gas mixture and a 2.5 cm H2O end-expiratory pressure, the isolated lungs were perfused with a physiological solution. The experimental protocol included three periods: baseline (10 min), ischemia (45 min) and reperfusion (60 min, ventilated with 95% O2-5% CO2 gas mixture). Before and after ischemia, we measured pulmonary arterial pressure (Ppa), pulmonary venous pressure and lung weight (W). Then, pulmonary capillary pressure and filtration coefficient (Kfc) were calculated. Ischemia caused increases in Ppa, W and Kfc in the control group. For most cases, the above ischemia-induced increases were attenuated by the C60(ONO2)7 F 2 pretreatment. Our results suggest that the antioxidant C60(ONO2)7 F 2 attenuates IR-induced lung injury. D 2002 Elsevier Science Inc. All rights reserved. Keywords: Antioxidants; Pulmonary artery pressure; Pulmonary edema

Introduction Ischemia-reperfusion (IR)-induced lung vascular injury ultimately depends upon some balance between lung metabolic stress, the extent of the IR-induced inflammatory response, endogenous antioxidant levels, and the timing, magnitude, and duration of reactive oxygen species (ROS) generation during both periods of ischemia and reperfusion. Fisher et al. [1] showed that ROS generation occurs in

* Corresponding author. Tel.: +886-2-2393-8235; fax: +886-2-2396-4350. E-mail address: [email protected] (Y.-L. Lai). 0024-3205/02/$ - see front matter D 2002 Elsevier Science Inc. All rights reserved. PII: S 0 0 2 4 - 3 2 0 5 ( 0 2 ) 0 2 3 7 4 - 3

1272

Y.-L. Lai et al. / Life Sciences 72 (2003) 1271–1278

the IR rat lung by assessing biochemical markers of oxidant production. Importantly, oxidant production is significantly augmented during reperfusion [1]. Three major ROS sources are proposed during IR period [2]. First, reperfusion with oxygen will initiate the oxidation of xanthine and hypoxanthine by xanthine oxidase, which leads to production of large amount of superoxide and hydrogen peroxide. Hydrogen peroxide will be converted to hydroxyl radicals by reducing metal ions, such as Cu+ and Fe2 +. These ROS are believed to be heavily involved in the IR injuries [3]. Second, mitochondria damaged by ischemia may ‘leak’ more electrons than usual from their electron transport chain, forming more superoxide [2]. Third, post-ischemia tissues can generate increased amount of leukotriene B4, platelet activating factor and other chemoattractants for neutrophils, and up-regulate expression of adhesion molecule [4]. Neutrophils then adhere to endothelium and may be activated to release ROS. Subsequently, released ROS elicit vasoconstriction [5], which is the usual main feature of IR lung injury. One way that ROS could mediate IR lung injury is by generating the highly reactive hydroxyl radical, which has ability to abstract methylene hydrogen atoms from polyunsaturated fatty acids and to initiate lipid peroxidation [6]. Membrane peroxidation can increase fluidity and permeability, and enhance protein degradation. Several types of antioxidants attenuate IR lung injury [7–9]. In this study, we tested whether a water soluble antioxidant fullerene derivative C60(ONO2)7 F 2 attenuates IR lung injury.

Materials and Methods Synthesis of C60(ONO2)7

F 2

The preparation of C60(ONO2)7 F 2 was achieved by converting C60 to polyhydroxylated C60 followed by nitration of the polyhydroxylated C60. The procedure for polyhydroxylation of C60 is basically similar to the previously reported processes [10,11]. In brief, C60 in benzene was reacted with nitrogen dioxide radical (SNO2) to produce polynitro C60, which was then hydrolyzed by aqueous NaOH to generate polyhydroxyl C60 in appreciable yield. Desorption chemical ionization mass showed that the polyhydroxyl C60 contains up to 9 hydroxyl moieties. The polyhydroxyl C60 was converted into polynitrated C60 by dissolving the polyhydroxyl C60 in fuming nitric acid (98%) and stirring at 40–50 jC for 30 h. Then the excess nitric acid was removed under reduced high pressure, to give yellow solids, and was dried in vacuum at 45 jC to furnished yellow solids of poly nitrated C60 in appreciable yield. Element analysis shows that the poly nitrated C60 contains 7 F 2 nitrogens in each C60 cage. Ischemia-reperfusion lung injury Young Wistar rats were divided into two groups: control and fullerene derivative C60(ONO2)7 F 2. Animals in the contol group were intraperitoneally injected with saline for 3 days. The antioxidant C60(ONO2)7 F 2 was dissolved in saline and injected intraperitoneally (10 mg/kg) once per day for 3 days prior to the study. This administration of C60(ONO2)7 F 2 was modified from our previous method [12]. On the day of the study, each animal was anesthetized with sodium pentobarbital (40 mg/kg, i.p.). Then, the isolated-perfused lungs were prepared according to our previous method [13], with some modifications. Briefly, after insertion of a tracheal cannula, the chest was opened and the lungs were ventilated with a humidified 95% air-5% CO2 gas mixture under an end-expiratory pressure of 2.5 cm H2O. After the right ventricle was injected with heparin (150 IU), the pulmonary artery was cannulated

Y.-L. Lai et al. / Life Sciences 72 (2003) 1271–1278

1273

and perfused with a perfusate. The perfusate was a mixture of bovine serum albumin (4 mg/100 ml) with Krebs-Henseleit buffer solution containing (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl 2H2O, 1.2 MgSO4 7 H2O, 1.2 KH2PO4, 25 NaHCO3, and 10 glucose. A wide-bore cannula was placed in the left atrium through the left ventricle to collect the effluent perfusate for recirculation. About 50 ml of initial perfusate was discarded to clear the blood before initiating recirculation. Perfusion rate of 3 ml/min/100 g body weight was maintained by a roller pump through an air bubble trap. The heart and the lungs were removed en bloc and were placed on a weighing pan, which was mounted on a Grass force transducer for detecting the change in lung weight, and was suspended in a constant-temperature (37 jC), humidified chamber. The weighing system was calibrated by placing a 2-g weight on the pan and adjusting the output to 5 cm of chart deflection. The pulmonary arterial (Ppa) and venous pressures (Pv) were continuously monitored with Statham pressure transducers, which were placed at the same height as the heart. The distance between the pressure transducers and the pulmonary artery and vein were 29 and 50 cm, respectively. Resistances of the connecting catheters were measured and then the above measured Ppa and Pv were corrected for these resistances of connecting tubings. Changes in lung weight, Ppa, and Pv were continuously recorded with a Grass recorder. In addition, the isolated-perfused lungs were continuously ventilated with the 95% air-5% CO2 gas mixture. For determination of capillary pressure (Pc) with a constant-flow perfusion, venous outflow was momentarily stopped for 3–4 sec at end expiration. There was a rapid rise in Pv followed by a slower but steady rise. Pc was obtained by extrapolating the slow rising component back to zero time [14]. In addition, filtration coefficient (Kfc) was determined by the gravimetric method of Drake et al. [15]. Upon achieving an isogravimetric state, we raised the Pv rapidly by 10 cm H2O for 10 min. This hydrostatic pressure caused the lung to gain weight promptly. This was followed by a slow but steady rise in lung weight. The rapid component represents the expansion of pulmonary blood vessels, whereas the slow component is due to fluid filtration into the interstitial space. The initial rate of fluid filtration was estimated by extrapolating the slow component to zero time, in a semilog plot. The value of y-intercept was divided by the hydrostatic pressure challenge (DPc) and normalized to 100 g of lung weight. At first, the isolated-perfused rat lung was ventilated with 95% air-5% CO2 gas mixture during the equilibration (the baseline) period of 20 min. Continuously with the same ventilation as before, the perfusion was then stopped (the ischemia period) for 45 min. Subsequently, reperfusion (the reperfusion period) began right after the ischemia period. The lungs were ventilated with a gas mixture of 95% O25% CO2 during the reperfusion period. During the whole experimental period, the lungs were ventilated with a positive end-expiratory pressure of 2.5 cm H2O. Ppa, Pv, Pc and weight gain were determined before as well as 1, 6, 16, 36 and 66 min after the reperfusion. Kfc was determined before (the baseline period) and 66 min after the reperfusion. Furthermore, arterial resistance (Ra) and venous resistance (Rv) were separately calculated. Values are means F SE. A nonpaired t-test was used to compare differences in parameters between the control and the fullerene derivative groups. Differences between values before and after ischemia were analyzed by paired t-test. Differences were considered significant if p < 0.05.

Results One min after the forty-five-min ischemia caused a marked increase in pulmonary arterial pressure (Ppa) in the control group (Fig. 1). However, the increase in Ppa returned gradually toward the

1274

Y.-L. Lai et al. / Life Sciences 72 (2003) 1271–1278

Fig. 1. Ischemia-reperfusion-induced alteration in pulmonary arterial pressure (Ppa) in two groups of rats. Statistical differences compared to the control group: *p < 0.05, **p < 0.01. ##Statistical differences (p < 0.01) compared to the baseline value.

baseline level at five min after the ischemia. Similarly, IR-induced alterations in Ra followed the trend of Ppa (Fig. 2). These IR-induced increases in Ppa and Ra were not significantly altered, although Ppa and Ra were reduced, by the fullerene derivative. Ischemia induced a gradual elevation in lung weight with time until the end of the experiment (66 min after the ischemia) (Fig. 3). This type of lung weight gain was significantly reduced by the fullerene derivative during 36–66 min following the

Fig. 2. Ischemia-reperfusion-induced alteration in pulmonary arterial resistance (Ra) in two groups of rats. **Statistical differences (p < 0.01) compared to the control group. ##Statistical differences (p < 0.01) compared to the baseline value.

Y.-L. Lai et al. / Life Sciences 72 (2003) 1271–1278

1275

Fig. 3. Ischemia-reperfusion-induced alteration in lung weight in two groups of rats. *Statistical differences (p < 0.05) compared to the control group. Statistical differences compared to the baseline value: #p < 0.05, ##p < 0.01.

ischemia. Also, IR caused a marked increase in Kfc (from 0.46 F 0.07 to 0.62 F 0.09 ml/mmHg/100 g lung weight) in the control group; and this increase was attenuated in the the fullerene derivative group (from 0.54 F 0.04 to 0.55 F 0.04 ml/mmHg/100 g lung weight) (Fig. 4). At the end of the

Fig. 4. Ischemia-reperfusion-induced alteration in pulmonary filtration coefficient (Kfc) in two groups of rats. #Statistical differences (p < 0.05) compared to the baseline value.

1276

Y.-L. Lai et al. / Life Sciences 72 (2003) 1271–1278

Fig. 5. Ischemia-reperfusion-induced alteration in lung wet weight to dry weight (W/D) ratio in two groups of rats. *Statistical differences (p < 0.05) compared to the control group.

experiment, the fullerene derivative significantly decreased the IR-induced increase in lung W/D ratio (Fig. 5).

Discussion One min after a 45-min ischemia there was a marked increase in Ppa. The IR caused also a gradual increase in lung weight with time, as well as an increase in filtration coefficient. The IR-induced increases in lung weight and Kfc were markedly attenuated by the fullerene derivative C60(ONO2)7 F 2. Several features of the relationship between IR-induced vascular alterations and the fullerene derivative will be discussed below. Ischemia-reperfusion lung injury is generally characterized by inflammation [16], edema [17,18] and physiological dysfunction, including significant disorders of vascular function [19,20]. There is evidence that significant reperfusion injury predicts a worse outcome for long-term graft survival [21,22]. As mentioned in the Introduction section, ROS play an important role in IR-induced lung injury. In order to antagonize ROS, antioxidants have been found to attenuate this type of lung injury. We [12,23] and others [24,25] found that fullerene derivatives are potent antioxidants. Therefore, we tested here if C60(ONO2)7 F 2 attenuates IR-induced pulmonary vascular disorders. A C60 molecule contains 30 C = C double bonds [26]. A bridged type substitution group will use up 1 C = C double bond. For C60 and its derivatives, the chemical reactivity is due to its double bonds. C60 is known to be able to react with up to 34 methyl radicals [25]. The antioxidant effect of C60 is related to: the number of reactive sites and the distant location to the reactive site. The higher the number of reactive sites and the closer the distance, will turn out to be the more effective antioxidant effect of fullerene derivatives. In addition, C60(ONO2)7 F 2 can release nitric oxide (NO) (Hwang KC. Unpublished observations) and this action is similar to trinitroglycerol [27]. It is possible that the released NO induces decreases in Ppa (Fig. 1) and Ra (Fig. 2). Our results confirmed that there is a close relationship between ROS and IR-induced vascular dysfunction in lungs [1]. This confirmation is mainly based on the fact that IR caused marked increases

Y.-L. Lai et al. / Life Sciences 72 (2003) 1271–1278

1277

in Ppa, weight gain, and Kfc, and that most of these increases were profoundly attenuated by the employed ROS scavenger, the fullerene derivative C60(ONO2)7 F 2. The above reasoning is mainly based on the idea that antioxidants suppress oxygen radicals [28]. Compared to weight gain and Kfc, there was a less effect of the fullerene derivative on the temporal trend of Ppa (Figs. 1, 3, and 4). Also, the treatment of the fullerene derivative caused a trend of non-significant increase in capillary pressure (Pc) following IR (data not shown). This non-significant increase in Pc could not explain the decrease in Kfc in the fullerene derivative group (Fig. 4). These results might imply that IR-generated ROS cause an endothelial damage [29,30] which, in turn, elicited vascular leakage. Therefore, the fullerene derivativeinduced marked suppressions in both lung weight gain and Kfc did not seem to relate to its effect on Ppa or Pc. In summary, one min after a 45-min ischemia caused marked increase in Ppa. The IR caused also a gradual increase in lung weight with time, as well as an increase in filtration coefficient. Most of the above IR-induced increases were markedly attenuated by the fullerene derivative. Our results suggest that the fullerene derivative C60(ONO2)7 F 2 is an effective antioxidant which can attenuate IR-induced lung weight gain and filtration coefficient. Due to its antioxidant characteristic, the derivative might be used to prevent oxidant-induced pulmonary diseases such as respiratory distress syndrome, fibrosis, emphysema, and asthma. More investigations are needed to demonstrate its potential benefits, however.

Acknowledgements We thank J.-H. Lin and C.-F. Huang for their technical assistants. This investigation was supported by the National Science Council (NSC89-2320-B002-078).

References [1] Fisher A, Dodia C, Ayene I, Al-Mehdi A. Ischemia-reperfusion injury to the lung. Annals of the New York Academy of Sciences 1994;723:197 – 207. [2] Nakazawa H, Genka C, Fujishima M. Pathological aspects of active oxygen/free radicals. Japanese Journal of Physiology 1996;46:15 – 32. [3] Lynch MJ, Grum CM, Gallagher KP, Bolling SF, Deeb M, Morganroth ML. Xanthine oxidase inhibition attenuates ischemic-reperfusion lung injury. Journal of Surgical Research 1988;44:538 – 44. [4] Seekamp A, Ward PA. Ischemia-reperfusion injury. Agents and Actions 1993;41:137 – 52. [5] Yu CC, Lai Y-L. In vivo pressure-flow curve in unilateral rat lung ischemia-reperfusion injury. Journal of Applied Physiology 2001;90:1865 – 70. [6] Moore TM, Khimenko PL, Taylor AE. Endothelial damage caused by ischemia and reperfusion and different ventilatory strategies in the lung. Chinese Journal of Physiology 1996;39:65 – 81. [7] Akins WK, Taylor AE. Role of xanthine oxidase and neutrophils in ischemia-reperfusion injury in the rabbit lung. Journal of Applied Physiology 1990;69:2012 – 8. [8] Grosso MA, Brown JM, Viders DE, Mulvin DW, Banerjee A, Velasco SE, Repine JE, Harken AH. Xanthine oxidasederived oxygen radicals induce pulmonary edema via direct endothelial cell injury. Journal of Surgical Research 1989; 46:355 – 60. [9] Koyama I, Toung TJK, Rogers MC, Curtner GH, Traystman RJ. O2 radicals mediate reperfusion lung injury in ischemic O2-ventilated canine pulmonary lobe. Journal of Applied Physiology 1987;63:111 – 5. [10] Chiang LY, Bhonsle JB, Wang LY, Shu SF, Chang TM, Hwu JR. Efficient one-flask synthesis of water-soluble [60]fullerenols. Tetrahedron 1996;52:4963 – 72.

1278

Y.-L. Lai et al. / Life Sciences 72 (2003) 1271–1278

[11] Anantharaj V, Bhonsle J, Canteenwala T, Chaing LY. Synthesis and characterization of nitrated [60]fullerenols derivatives. Journal of the Chemical Society-Perkin Transactions I 1999;(22):31 – 6. [12] Lai Y-L, Chiang LY. Water-soluble fullerene derivatives attenuate exsanguination-induced bronchoconstriction of guinea pigs. Journal of Autonomic Pharmacology 1997;17:229 – 35. [13] Lai Y-L, Chu S-J, Ma M-C, Chen C-F. Temporal increase in the reactivity of pulmonary vasculature to substance P in chronically hypoxic rats. American Journal of Physiology 2002;282:R858 – 64. [14] Dawson CA, Linehan JH, Rickaby DA. Pulmonary microcirculatory hemodynamics. Annals of the New York Academy of Sciences 1982;384:90 – 106. [15] Drake R, Gaar KA, Taylor AE. Estimation of the filtration coefficient of pulmonary exchange vessels. American Journal of Physiology 1978;234:H266 – 74. [16] Moore TM, Khimenko PL, Adlins WK, Miyasaka M, Taylor AE. Adhesion molecules contribute to ischemia and reperfusion-induced injury in the isolated rat lung. Journal of Applied Physiology 1995;78:2245 – 52. [17] Barie PS, Hakim TS, Malik AB. Effect of pulmonary artery occlusion and reperfusion on extravascular fluid accumulation. Journal of Applied Physiology 1981;50:102 – 6. [18] Horgan MJ, Lum H, Malik A. Pulmonary edema after pulmonary artery occlusion and reperfusion. American Review of Respiratory Disease 1989;140:1421 – 8. [19] Korthuis RJ, Smith JK, Carden DL. Hypoxic reperfusion attenuates postischemic microvascular injury. American Journal of Physiology 1989;256:H315 – 9. [20] Krug AW, Rochemont M, Krob G. Blood supply of the myocardium after temporary coronary occlusion. Circulation Research 1966;19:57 – 62. [21] Pham S, Yoshida Y, Aeba R, Hattler BG, Iwaki Y, Zeevi A, Hardesty RL, Griffith BP. Interleukin-6, a marker of preservation injury in clinical lung transplantation. Journal of Heart and Lung Transplantation 1992;11:1017 – 24. [22] Okuda M, Furuhashi K, Nakai Y, Muneyuki M. Decrease of ischemia-reperfusion related lung oedema by continuous ventilation and allopurinol in rat perfusion lung model. Scandinavian Journal of Clinical and Laboratory Investigation 1993;53:625 – 31. [23] Wang IC, Tai LA, Lee DD, Kanakamma PP, Shen CK-F, Luh T-Y, Cheng CH, Hwang KC. C60 and water-soluble fullerene derivatives as antioxidants against radical-initiated lipid peroxidation. Journal of Medicinal Chemistry 1999;42:4614 – 20. [24] Krusic PJ, Wasserman E, Keizer PN, Morton JR, Preston KF. Radical reactions of C60. Science 1991;254:1183 – 5. [25] McEwen CN, McKay RG, Larsen CB. C60 as a adical scavenger. Journal of American Chemical Society 1992;114: 4412 – 4. [26] Kroto HW, Allaf AW, Balm SP. C60: Buckminsterfullerene. Chemical Reviews 1991;91:1213 – 35. [27] Artz JD, Toader V, Zavorin SI, Bennett BM, Thatcher GRJ. In vitro activation of soluble guanylyl cyclase and nitric oxide release: a comparison of NO donors and NO mimetics. Biochemistry 2001;40:9256 – 64. [28] Christie NA, Smith DE, Decampos KN, Slutsky AS, Patterson GA, Tanswell AK. Lung oxidant injury in a model of lung storage and extended reperfusion. American Journal of Respiratory and Critical Care Medicine 1994;150:1032 – 7. [29] Halliwell B. Reactive oxygen species and the central nervous system. Journal of Neurochemistry 1992;59:1609 – 23. [30] Steinberg H, Greenwald RA, Sciubba J, Das DK. The effect of oxygen-derived free radicals on pulmonary endothelial cell function in the isolated perfused rat lung. Experimental Lung Research 1982;3:163 – 73.