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Bia'himJca et Biophysica Acta. 107311991)374-379 © 1991 ElsevierSciencePublishers B.V.0304-4165/91/$03.50 ADONIS 030441659100105C

Inhibition of carrageenin-induced paw edema by a superoxide dismutase derivative that circulates b o u n d to albumin Yukio Ando, Masayasu lnoue, Shukuro Araki and Yoshimasa Morino Departments of Bi~hemistry and Medicine, Kumamoto University Medical School, Kumamolo (Japan)

Although the possible involvement of suporoxlde radical and its metabollts(s) in the pathogenesls of various types of edema have been suggested, direct evidence supporting this concept is lacking. Since intravenously adminigered Cu2+ZnZ+-Iype supemxide dismntase (SOD) rapidly disappeared from the circulation with a half.life of 4 mln, the enzyme could not be used to test wbetber suporoxide radicals played a critical role in the modulation of vascular permeability. We previously synthesized a S O D derivative (SM-SOD) by linking poly(styrene co-maleie acid butyl ester) (SM) to the enzyme (Ogino, T., lunne, M., Ando, V., Awal, M., Maeda, H. and Morino Y. (19~8) Int. d. Pept. Protein Res. 32,1583-1588); SM-SOD circulates bound to albumin with a boll-life of 6 h. To test whether suportrxlde radicals play an important role in the regulation of vascular permeability, the effect of SM-SOD on experimental paw edema was studied in the rat. Subcutaneous injections of carrageenin to the paw rapidly induced local edema by increasing vascular permeability. Intravenous administration of S M - S O D markedly inhibited d~e carrageenln-tnduced increase in vascular permeability and suppressed the development of paw edema. In contrast, the same dose of SOD showed no such inhibitory effect. These results suggested that superoxlde radical a n d / o r its metabolite(s) might play a critical role in the pathogenesls of carrageenin-ioduced vasogenic edema.

Reactive oxygen species have been postulated to play an important role in the pathogenesis of vasogenic edema [1-5]. However, the mechanism and reactive oxygen species by which vascular permeability is increased remains to be elucidated. Since peroxidative degradation of polyunsaturated fatty acids in cell membranes might be triggered by reactive oxygens [6,7], scavenging supernxide radical and its metabolite(s) might be important in protecting tissues from oxidative injury. Hence, saperoxide dismutase (SOD) has been used to decrease oxygen toxicity [8,9]. However, intravenously injected SOD disappears from the circulation with a half-life of 4 rain predominantly due to glomerular filtration. Thus, superoxide radicals in an injured tissue can not be dismutated effectively by a single dose

Abbreviations: SOD, superoxide dismutase: SM, poly(styrene ¢omalelc acid butyl ester); PMN, polymorphonuelear leukocytes. Correspondence: M. lnoue, The Department of Biochemistry, Kumamoto Universit3 Medical School, 2-2-1 Honjo, Kumamoto, Japan.

of SOD [10]. To increase the half-life of SOD in the circulation, the enzyme derivatives cross linked with macromolecules, such as albumin and Ficoll, have been synthesized [11-13]. However, the enzyme activity significantly decreased by such chemical modification. Since the apparent molecular size of these derivatized eagymes are significantly larger than that of unmodified SOD, their delivery to extravaseular compartment(s) of injured tissues would be limited. Liposomally entrapped SOD has also been used for decreasing oxygen toxicity in various inflammatory diseases [14,15]. However, mainly due to its xenobiotic nature, a significant fraction of the injected lyposomal SOD is trapped by reticuloendothelial systems, particularly those in lung and liver [14-16]. To overcome such frustrating situations, we synthesized a SOD derivative (SM-SOD) by linking poly(styrene eo-maleic acid butyl ester) ( M r 1600) to the enzyme [17]. SM-SOD (M~ 36000) reversibly binds to the warfarin site on albumin, circulates with half-life of 6 h and accunmlates in an injured site of a tissue whose local pH is decreased [18]. The present work demonstrates that carrageenin-induced paw edema of the rat was effectively prevented by intravenous administration of SM-SOD.

Materials and Methods 28 Materials Evan's blue, bovine serum albumin, bovine erythroeyte-type SOD, xanthine, xanthine oxidase and diamine oxidase were purchased from Sigma Chemicals (St. Louis). Carragecaln was from Zushi Chemicals (Tokyo). Poly(styrene co-maleic acid butyl ester) (SM) was obtained from Kurare (Kurashiki); 1 real SM contained 1 real of reactive anhydride group. Human erythrocytetype SOD was purified by the method of Garmer et al. [19]. 12~I-Labeled Bolton-Hunter reagent (2.2 Ci/mol) was from New England Nuclear (Boston). Synthesis of SM.SOD and radioactive enzyme samples SM-SOD was synthesized as described previously [17,18] by linking 2 real SM per real SOD. Specific activity of SM-SOD was 2600 units per mg protein. SOD activity was measured by the method of Beauchamp and Fridovich [20]. Radioactive albumin and SOD samples were synthesized by using 1251inbeled-Bolton-Hunter reagent [21]. Specific radioactivity of SOD and SM-SOD was 33000 epm/0og protein. Protein concentration was determined by the method of Lowry et al. [22] using bovine 5OD as the standard. R.adioactivity was determined in a Packard autogammascintillation spcctrophotometer model-5130. Inactivation of SM-SOD Inactive SM-SOD was prepared by incubating SMSOD (20 mg/ml) with 30 mM H202 containing Tris-HCI buffer (pH 9.0) at 3 7 ° C for 18 h. After extensive ,:lialysis against saline solution, the enzyme activity was determined by the method of Beauchamp and Fridovieh [20]. The remaining acti,Aty of SM-SOD sample thus obtained was less than 0.01~ of intact SM-SOD. In viva experiments Animals, Male Spraguc-Dawiey ruts, weighing 200250 g, were used for experimer,ts after fasting for 16 h. In viva experiments were performed under light diethyl ether anesthesia. Edema was induced in the paw by subcutaneous injcctiun of 100 ul of carrageenin dissolved in saline solution (10 mg/kg) [23]. The in viva fate of SOD and SM-SOD in intact and carragecnin-treated animals was determined by radioactive enzyme samples as described previously [17]. The volume of the paw was measured by using a Plethysmometer (Unicorn Model TK-101). To determine the change in vascular permeability of the paw, 0.5 ml of Evan's blue solution ( 1 5 in saline) was intravenously injected. At varying times after injection, animals were killed by bleeding and the paw was excised. After extracting the tissue-associated Evan's blue by formamide for 24 h at room temperature, the amounts

Time ( h ) Fig. I. Fate of SOD and SM-SOD in lhc ciretdation. Under pent~ barbital an~th~ia (5fl mg/kg), heparini,ed animals (I00O units/kg body weight)were intravenouslyadministeredwith 12Sl-labeledSOD or SM-SOD in the tail vein 1 rain before giving carrageenin.The injected dose of the eazyn~s was 0.3 mg/kg (50O000cpm/kg). At the indicated times, 0.1 ml of blood sampl~ were coil=ted from the left femoral vein and determined for radioactivity.O, SOD; • SM-SOD.

of the extracted dye were determined speetrophotometricaUy at 600 am as described previously [17]. Histological examination 3 h, after carrageenin treatment, animals were killed by bleeding. The excised paws were fixed in 10% formalin solution at room temperature for overnight. Specimens thus fixed were subjected to histochemieal examination by staining with hematoxilin and eosin. Results Fate of enzyme samples in animals that were induced with paw edema Previous studies showed that intravenously injected SOD rapidly underwent renal glomerular fthration, while SM-SOD formed a complex with albumin and circulated with a half-life of 6 h in the intact rat [17,18]. We examined the fate of SOD and SM-SOD in the circulation of earrageanin-treated rats. Intravenously injected SM-SOD also disappeared from the circulation with a half-life of 6 h, while SOD disappeared with a half-life of 4 rain in carrageenin-treated animals (Fig1). This observation confirmed the previous observation in intact animals [17,18] and suggested that SM-SOD might also form a dis,sociable complex with albumin and effectively dismutare superoxide radicals in the circulation of carrageenin-treated animals. Effect of SM-SOD on vascular permeability of carrageenin-treated paw To assess the change in vascular permeability of the earrageenin-injected tissue, pa',~ volume was measured at varying times after treatment. The volume of carrageenin-injected paw increased with time and reached a

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( ) Fig. 2. Eff~t of SM-SOD on the increase in tissue volume. Under light di~lhyl ether anesthesia, rats (200 g) were intravenously injected with either 0.2 ml of saline (o), 20 mg/kg of H202-inactivated SM-SOD (*), or 2O mg/kg of SM-SOD 01). After 5 rain, 2 mg of carrageenin solution (100 pl) was injected in the paw. At the indicated time~ after carrasecnin injection, the paw volume was measured as described in the text. Values show the increased paw volume (~ of initial volume). Data were expressed as mean±S.D, derived from eight paws.

m a x i m u m at 3 h after t r e a t m e n t . T o k n o w w h e t h e r reactive o x y g e n species play a n i m p o r t a n t role in t h e p a t h o g e n e s i s of p a w e d e m a , v a r y i n g dose of g M - S O D w a s injected i n t r a v e n o u s l y 5 rain before t r e a t m e n t w i t h carrageeain. S M * S O D s h o w e d n o significant effect o n t h e c h a n g e in p a w v o l u m e d u r i n g t h e first 60 rain. H o w e v e r , t h e increase in p a w v o l u m e w a s m a r k e d l y i n h i b i t e d thereafter (Fig. 2). T h e i n h i b i t o r y effect o f S M - S O D c o n t i n u e d for 6 h a f t e r c a r r a g c c n i n injection. U n d e r identical c o n d i t i o n s , n e i t h e r S O D n o r H 2 0 2 in-

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Dose of SM 50D (m9fRg) Fig. 3. Dose-dependent inhibition by SM-SOD. A,timals were intravenously injected with varying dose of SM-SOD. After 5 ruth, 2 mg of c¢rrageenln solution (1(30 ~tl) was injected in the paw. 3 h afler carrageenin injeclion, the paw volume was measured as described in Fig. 2. Values show the increased paw volume i% of initial volume). Data were expressed as meall±S.D, derived from six to eight paws.

activated S M - S O D s h o w e d s u c h i n h i b i t o r y a c t i o n at a n y t i m e p o i n t tested. I n h i b i t i o n o f p a w e d e m a b y S M - S O D o c c u r r e d d o s e d e p e n d e n t l y (Fig, 3).

Effect of SM-SOD on the accumulation of Ecan's blue S i n c e E v a n ' s b l u e circulates b o u n d to a l b u m i n , t h e d y e h a s b e e n used t o s t u d y c h a n g e s in vascular p e r m e a bifity f o r a l b u m i n [18]. S i n c e E v a n ' s blue m a r k e d l y a c c u m u l a t e d in t h e c a r r a g e e n i n - t r e a t e d p a w , vascular p e r m e a b i l i t y f o r a l b u m i n w o u l d be increased at t h e site of injection. T o test w h e t h e r S M - S O D i n h i b i t s t h e earr a g e e n i n - i n d u c e d increase i n vascular p e r m e a b i l i t y , t h e a m o u n t s o f t h e d y e a c c u m u l a t e d i n the p a w were de-

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Fig. 4. Eff~t of SM-SOD on earrageenin induced paw edema. Under light diethyl ether anesthesia, rats were injected with 0.2 ml of saline or SM-SOD (10 mg/kg) into the tall vein ] rain before giving carrageenin (10 mg/kgi. 2 h after earrageenin treatment, animals were intravenously injected with 0.5 ml of 1% Evan's blue. At I h after giving Evan's blue. animals were killed by bleeding. Arrows indicate the Evan's hlue-a~umulated lesion, A, control group; B, SOD treated ~oup; C, SM-SOD treated group.

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Fig. 5. Effect of SM-SOD on vascular permeability of the paw. Under light diethyl ether anesthesia, rats were injected with 0.2 ml of saline or $M-SOD (10 mg/kg) into the tail vein 1 rain before being given car rageenin. At O, 1 and 2 h after carrageenin treatment, animals were intravenously injected with 0.5 ml of 1% Evan's blue. I h after Bran's blue injection, animals w¢~ killed by bleeding. The paw was excised and the tissue-associated dye was extracted in 2 ml of dimethyl formemide as described in the text. After 48 h of extraction, the amounts of the extracled dye were detarralned spect rophotometrically al 600 nm. Data show the difference in the amoums of the dye found between the control and carrageenin-treated paw~ Open columns, soline-treated group; closed columns. SM-SOD treated group. Data show mean ± S.D. derived from eight 1o ten paws r e t r a i n e d in a n i m a l s a f t e r i n t r a v e n o u s a d m i n i s t r a t i o n o f e i t h e r saline o r S M - S O D , A c c u m u l a t i o n o f the d y e w a s i n h i b i t e d significantly b y S M - S O D , b u t n u t b y saline (Fig. 4). T h e i n h i b i t o r y effect o f S M - S O D w a s m u s t a p p a r e n t at 2 h a f t e r a d m i - . i s t r n t i o n o f c a r r a g e c n i n (Fig. 5). T o s t u d y t h e m e c h a n i s m for t h e c h a n g e in vascular p e r m e a b i l i t y at t h e initial stage o f t h e e d e m a , d i a m i n e oxidase, a n e n z y m e t h a t d e g r a d e s h i s t a m i n e a n d o t h e r d i a m i n e s , w a s injected w i t h c a r r a g e e n i n . I n t e r e s t i n g l y ,

Fig 6. Effect of diamine oxidas¢ on paw edema. Under light diethyI elher anesthesia, animals were subcutaneously injected with carrageeain (10 mg/kg) and diamine oaidase (500/Lg,/kg) and time-dependent changes in paw volume were delem'dned. Other conditions were ihe same ~ in Fig. g. Data were e0~pressod as m~n_+S,D, dell,led from six paws. o, conllol group; O, diamine oxidase treated groups,

the effect o f c a r r a g e e n n i o n t h e incre-as¢ i n vascular p e r m e a b i l i t y w a s m a r k e d l y i n h i b i t e d b y d i a m i n e oxidase particularly at its early stage (Fig. 6).

Histological examination V a s c u l a r p e r m e a b i l i t y of e n d o t h e l i a l cells m a r k e d l y c h a n g e s d u r i n g t h e initial step o f i n f l a m m a t i o n [3,4]. L e u k o c y t e s also p l a y a n i m p o r t a m role in t h e p r o p a g a t i o n o f i n f l a m p a a t i o n a t its d i f f e r e n t stages. T o u n d e r s t a n d the cellular m e c h a n i s m b y w h i c h S M - S O D inh i b i t e d t h e increase i n vascular permeability, histological e x a m i n a t i o n o f c a r r a g e e n i n - t r e a t e d p a w w a s carried out. A t 3 h a f t e r c a r r a g e e n i n t r e a t m e n t , the p a w w a s excised, fixed w i t h f o r m a l i n , a n d stained w i t h h e m a t o x / l i n a n d cosin. Histological e x a m i n a t i o n o f t h e p a w rcve.aled t h a t a large n u m b e r of p o l y m o r p h o n u c l e a r leukocytes ( P M N ) w e r e f o u n d to m i g r a t e to t h e earta-

Fig 7. Effectof S M - S O D on P M N inFdlmfion.Under lightdiethy}ether ~esthesia, a~mals were intravenouslyinjectedvdth O ~ ml of salineor

SM-SOD (10 mg/kg). After 5 min, tbey wel'¢ given carragee~in to the paw. At 3 h after ~rrageenin treatrcmnt, animals were killed by bleeding. The paw was excised and fu~ed in 10% formalin solution for overnight. Tissue specimens were stained by hematoxilin and cosin as described in the text, A, saline-treated group; B. SM-SOD treated group.

378 geenin-treated site of the paw (Fig. 7). Intravenous administration of SM-SOD markedly inhibited the migration of PMN. Again, no such inhibitory action was seen with SOD. Change in SOD actiuity of the paw after carrageenin treatment Since carrageenin increased the vascular permeability of the paw, surface membranes of a tissue, particularly those of endothelial cells, would be perturbed by this treatment. Hence, levels of intracelhilar scavenging enzymes, such as SOD, may possibly be decreased in earrageenin-treated tissues by plasma membrane destruction. Such a decrease in scavenging activity of a tissue might secondarily enhance oxidative injury of vascular endothelial cells. To test this possibility, SOD activity in both sides of the paw was determined 1 h after giving carrageenin. However, no significant difference in the enzyme activity was found between the control and carrageenin-treated paws (65 5:5 units/ paw). Accumulation of SM-SOD in the carrageenin treated paw To determine the amount of SM-SOD accumulated in the earrageenin-treated paw, 1251-labeled SM-SOD was administered intravenously and det trained for tissue-associated radioactivity. 1 h after administration of SM-SOD (10 mg/kg), 8 + 1.2 and 80 5:7 units of the enzyme were found to accumulate in the control and the carragceniu-treated side of the paw, respectively. Discusslan The present work demonstrates that the injured-sitedirected SM-SOD markedly inhibited the occurrence of the paw edema during 1-3 h after carrageenin treatment. This suggests that superoxide radical a n d / o r its metabolite(s) might play a critical role in the pathogenesis of carrageenin-indueed paw edema. It should be noted that, during 1 h after earrageeuln treatment, SM-SOD markedly inhibited the accumulation of the dye without inhibiting th