Free Radical Biology& Medicine, Vol. 13, pp. 533-541, 1992 Printed in the USA.All rightsreserved.

0891-5849/92 $5.00 + .00 Copyright © 1992PergamonPress Ltd.

Original Contribution T H E I N H I B I T O R Y E F F E C T S O F 21 M I M I C S O F SUPEROXIDE DISMUTASE ON LUMINOL-MEDIATED CHEMILUMINESCENCE EMITTED FROM PMA-STIMULATED POLYMORPHONUCLEAR LEUKOCYTE

TIAN YAPING,* FANG YUNZHONG,* LUO QINHUI,t SHEN MENGCHANG,t LtJ QIN, t and SHEN WENMEI* *Beijing Institute of Radiation Medicine, Beijing, China 100850; *Coordinate Chemistry Institute, Nanjing University, Nanjing, China 210008; and *BiochemistryDepartment, Great Wall Hospital, Beijing, China 100853

(Received 20 December 1991; Revised 10 March 1992; Re-revised 27 May 1992; Accepted 28 May 1992) Abstract--Four groups comprising 21 superoxide dismutase (SOD) mimics synthesized by us were comparatively studied for their inhibitory effects on luminol-mediated chemiluminescence emitted from phorbol myristate acetate (PMA)-stimulated polymorphonuclear leukocyte (PMNL). Among these groups, 20-membered macrocyclic bicopper(II) complexes and 13-membered macrocyclic dioxotetramine copper(If) complexes exhibited relatively higher activities of scavenging reactive oxygen species (ROS) produced by PMA-stimulated PMNL as compared with polyamine Cu(II)-Zn(II) complexes and copper(II) complexes of bis-shiff-base. Moreover, distinctly different effects of SOD mimics in the biologicalsystem have been found even in the same group. It is suggested that the biological effects of some SOD mimics are related to their structures. Keywords--SOD mimics, Luminol, Chemiluminescence, PMA-stimulated PMN, Free radicals

logical systems, the phorbol myristate acetate (PMA)-stimulated polymorphonuclear leucocyte (PMNL) was selected as an experimental model. Since PMNL as one kind of phagocytic cell has the ability to recognize infecting microbes and antigenic substances, it can cause phagocytosis, activation of the respiratory burst metabolism and generation of ROS. s-16 In such cases, O2 is primarily generated from the reduced nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase system; and then H202, "OH, HOC1, and ~O2are derived subsequently from it. 17'18 Cellular production and etilux of ROS may be detected by luminol-mediated chemiluminescence. Although this system is nonspecific for 02 measurement, it is an excellent one for comparatively screening SOD mimics by their effects of scavenging ROS in terms of chemiluminescence emitted from PMA-stimulated PMNL. A few reports have been found on the comparative investigation of the activities of various SOD mimics in living cells. In this study, through the application of PMA-stimulated PMNL and luminol-mediated chemiluminescence, the effects of SOD mimics on the biological system are found to be closely related to their structures. Phosphate-buffered saline (PBS)-glucose-albumin

INTRODUCTION

Superoxide dismutase (SOD) has the ability of catalytically dismutating superoxide radical (02) to hydrogen peroxide and oxygen) This provides a forefront defense against the injurious effect of endogenously generated 02 and other reactive oxygen species (ROS) derived subsequently from 07. It is very reasonable to study the effectiveness of SOD in preventing and curing ROS-involved diseases. 2-7 However, in the course of many years' studies, some practical problems have been encountered in clinical trials. One of these is that the enzyme having molecular weight of about 33,000 daltons can scarcely penetrate biomembrane. To find SOD-like compounds with low molecular weight, high biological activity, and nontoxicity, various Cu(II) complexes have been extensively synthesized as potential SOD mimics by many researchers, but the appropriate ones have not been obtained as yet. MATERIALS AND METHODS

Twenty-one SOD mimics were synthesized by us. For studying their effects on scavenging ROS in bioAddress correspondence to: Fang Yunzhong. 533

534

l . YAPIN6 e; a /

solution (PBS-G-A) was prepared with PBS containing 1 g/L of D-glucose and I g/L of bovine albumin. Dextran solution (6 g ofdextran T70) was dissolved in 100 mL of saline. All SOD mimics were dissolved in distilled water to a concentration of I raM.

\j',

~

--~z - - - d . . . . . . --~ ,'

'

Isolation (ff PMNL Fresh, heparinized human blood (90 mE) was prepared and then 30 mL of 6% dextran T70 added and shaked gently. It was placed at room temperature for 1 h until all red blood cells sedimented. The upper buffy-coat layer containing PMNL was carefully transferred into a centrifuge tube and spun at 200 × g for 5 min. The supernatant was rejected and the sediment treated with hypotonic solution to hemolyze the contaminating red blood cells. After mixing well for 30 s, it was centrifuged at 200 × g for 3 rain. The sedimented cells were resuspended in 5.0 m L of PBSG-A solution and carefully layered on 50 mE of lymphocyte separating medium. The mixture was centrifuged at 1500 × g for 5 rain at 4°C. The pellet containing PMNL was washed with 50 mE of PBS-G-A solution and resuspended in it, counted, and adjusted to 106/mE.

Measurement of hlminol-mediated chemiluminescence emilted b.l, PMA-stimulated PMNL Prepared PMNE (0.5 mL), I mE of 0.1 mM luminol, and several #L of SOD mimics solution were mixed, and then PBS-G-A solution was added to make a total volume of 1.55 mL. PMA solution (50 /aL) was added to start the emission of luminescence, and the chemiluminescence was measured with a WDD-I luminometer (Beijing Second Optical Instrument Factory, Beijing, China) at definite time intervals. The ability of scavenging ROS in terms of inhibitory effect of SOD mimics on luminol-mediated chemiluminescence emitted from PMA-stimulated PMNE is expressed as inhibitory rate (%), which is calculated by the formula given in the section entitled

Reagents. SOD mimics Synthesis of 21 copper(II) complexes for mimicing SOD activities were accomplished as characterized to be acceptable by spectra and elemental analysis as well as by their activity of scavenging O~- (Refs. 1921 ). These complexes may be divided into four groups as follows:

/ ~ .......

~3 - - - z

,~

/\--/

6,

Fig. 1. F h e s t r u c t u r e of 2 0 - m c m b e r e d m a c r o c y c l i c b i c o p p e r ( l l ) complexes. R

H,

11-2,

,¥

CI , ('u2L'(CI:)(CIO4)2"CH3OH: No. 1

n

X

Br , Cu2L'(Br)(CIO4)3-CH3OH: No. 2

1,

X - O H . ('u21,'(OH)(C104)3 - CH~OH: No. 3

R - CH3,

.k"

I . ('u,L'(I)(CIO4) 3" CH3OH: No. 4

X

Na.('u2L'(N3)(C104)3"Ctt3OH: No. 5

;t V : O H , Cu2L(OH)(CIO4)3" 2H20: No. 6 X - Br , ('u2L(Br)(CI04)3.4H20:

No. 7

L' a n d L d e n o t e m a c r o c y c l i c ligand with R - H a n d R respectively.

('H3.

Group I. Twenty-membered macrocyclic bicopper(II) complexes with bridging ligands C1, B r , OH-, I , and N 3 were synthesized by template condensation of 2,6-diformylpyridine (DFP) or diacetylpyridine (DAP) with 1,3-diaminopropane. Their structural tbrmulas are shown in Fig. l.

Group 2. Thirteen-membered dioxotetraamine macrocyclic copper(I1) complexes and related linear copper(II) complexes (Fig. 2). Group 3. Polyamine Cu(lI)-Zn(II) complexes with imidazolate bridge (Fig. 3).

Group 4. Copper(ll) complexes of bis-sh~ff:base synthesized by condensation of 2-acetylpyridine or pyridine-2-aldehyde with polyamine (Fig. 4). Reagents Luminol(5-amino- 1,2,3,4-tetrahydrophthalazin1,4-dion), PMA, and dextran T70 were purchased from Merck-Schuchardt (Hohenbrunn bei Mtinchen, Germany), Sigma Chemical Co. (St. Louis, MO), and Pharmacia Co. (Sweden), respectively. Other chemicals bought from Beijing Chemical Factory were of analytical grade. Bovine serum albumin was obtained from Shanghai Biological Products Factory. Cu,

Inhibitoryeffectsof SOD

535

NO2 NH

FL-N'

o~o

I--N,; , N " I [ ~ N , /

o

,N

cu

L N ~,/

H2NJ "~,.

(No. 8)

N¢ "~N HI I~

N~M

°o~°

Clta

o~o

Ha N'--"I

"~" |

N

•

f

j" ~ N

N

'

N

!•- - N H /2

(No. 9)

(No. 10)

N

i

"H a, N. I- - - -

(No. 11)

Fig. 2. The structureof 13-membereddioxotetraaminemacrocycliccopper(ll) complexesand related linearcopper(ll) complexes. Zn-SOD purified by us from bovine erythrocyte proved to be homogeneously pure. Phosphate-buffered saline (PBS, pH 7.4, 50 mM) consisted of 40 mM Na2HPO4, 10 mM KHEPO4, and 145 mM NaC1. Luminol stock solution (1 mM) was prepared with dimethyl sulfoxide (DMSO) and diluted with PBS to the concentration of 0.1 mM on use. PMA stock solution (5 mg/mL) was prepared with acetone and diluted with PBS to 1.25/zg/mL just before use. Hypotonic solution for hemolysis consisted of 3 g NaC1 and 372 mg of disodium ethylenediaminetetraacetic acid (EDTA) dissolved in l L of distilled water. The inhibitory rate of SOD mimics (%) was

I

Br- > I- > OH- > N~. Among them the N3 bridged complex has the lowest inhibitory rate for scavenging ROS, which is similar to the results reported previously.19'2°

537

of their ability for scavenging ROS. By comparing No. 8 and No. 9, it is found that both have the same coordination atoms and similar structures; the only difference between the features of the two complexes is that the ligand of No. 8 is linear, while that of No. 9 is macrocyclic. Perhaps the coordination center of the former is more easily accessible for small molecules (such as Oj) than that of the latter. When one H atom of methylene located between two amide groups is substituted by 4-nitrobenzyl, the activity for scavenging ROS increases. The mononuclear linear polyamine complexes have higher activity for scavenging ROS than the corresponding binuclear complexes. Kimura et al. 22 reported that these kinds of macrocyclic polyamine complexes have definite abilities for dismutating O~- in aqueous solution. The present article further shows that the dioxotetraamine copper(II) complexes have relatively strong abilities for scavenging ROS in physiological conditions.

Inhibitory effects of group 2 SOD mimics The mimics of No. 8-11 are macrocyclic dioxotetraamine copper(II) complexes. Their inhibitory rates are listed in Table 2, and Fig. 7 shows the luminescent curves in the presence of No. 8. The results shown in Table 2 indicate that, except for No. 9, all the complexes have activities for scavenging ROS similar to No. 1-4, and among them No. 8 is a notable complex with the highest activity. Extra modification of complexes may lead to the decrease

Inhibitory effects of group 3 SOD mimics Except for No. 12, four other complexes in the third group contain imidazolate-bridged Cu(II) and Zn(II) similar to the coordinated structure in the active site of CuZn-SOD. Recently, this kind of complex has attracted great interest from coordination chemists trying to synthesize complexes with similar active site structures of CuZn-SOD. The abilities of five SOD mimics at three different concentrations for

ol ()

t.¢h

10

2O

30

Time interval (rain) Fig. 6. The luminescent curve in the presence of SOD mimic No. 2. Luminescent curves ofPMA-stimulated PMNL respiratory burst system have been estimated in the presence of three different concentrations of SOD mimic No. 2. • denotes the control system; whereas O, A, and • represent 6.4 uM, 15.9 uM, and 31.3 uM of SOD mimic No. 2 in the testing systems, respectively.

538

1-. YAPING el al.

Table 2. l-he Inhibitory Rate of Macrocyclic Dioxotetraamine Copper(If) Complexes (%) The No. of SOD Mimics 8 9 10 I1

6.4 #M

15.9 uM

31.3 uM

34.2 25.4 36.9 33.2

75.2 29.5 52.3 62.7

84.4 38.6 61.5 79.5

scavenging ROS in terms of inhibitory rates are shown in Table 3. The luminescent curve of complex No. 14 is plotted in Fig. 8. Although five SOD mimics have different structures, as shown in Fig. 3, they have almost similar inhibitory rates at the same concentration, and the inhibitory rates change little when varying the concentrations. This may be attributable to the probable break of their imidazolate bridges under biological conditions and subsequent formation of mononuclear complexes without such bridge structures.

Inhibitor), effects of group 4 SOD mimic.s In the fourth group of SOD mimics, five members having the structure of copper(II) complexes of bischfffbase are listed in Fig. 3, and their abilities of

scavenging ROS expressed by inhibitory rates are shown in Table 4. The typical luminescent curve of No. 17 is given in Fig. 9. The results in Table 4 indicate that complexes No. 17-19 have little activity for scavenging ROS, which is proposed to be the unstable and easily destroyed structure of SOD mimics. Thus, they seem to be unsuitable as SOD mimics, whereas No. 20-21 have better scavenging ability. In our previous communications, ~9-2~,23the evaluation of 21 SOD mimics is dependent on their activities estimated by pulse radiolysis as well as the riboflavin illumination method. The results expressed as abilities for dismutating O5 showed that the activities of SOD mimics were much lower than that of native SOD (e.g., the activity of SOD mimic No. I was found to be about 400-fold lower). It is very, interesting to find that in this study, the inhibitory effect of this SOD mimic on the luminol-mediated chemiluminescence emitted from PMA-stimulated PMNL is strikingly similar to that of native SOD. Moreover, the activities of other SOD mimics for scavenging ROS are quite different from that of SOD mimic No. 1. Two groups of 20-membered macrocyclic bicopper complexes and 13-membered macrocyclic dioxotetraamine copper(II) complexes have higher abilities than two other groups of SOD mimics, polyamine Cu(II)-

g g

i

-Io

2o

3o

Time interval (min) Fig. 7. The luminescence curve in the presence of SOD mimic No. 8. Luminescent curves of PMA-stimulated PMN L respiratory burst system in the presence of three different concentration of SOD mimic No. 8 have been estimated. • denotes the control system; whereas O, A, and * represent 6.4 uM, 15.9 uM, and 31.3 uM of SOD mimic No. 8 in the testing systems, respectively.

Inhibitory effects of SOD Table 4. The Inhibitory Rates of Cu(ll) Complexes of Bi-SchiffBase (%)

Table 3. The Inhibitory Rates of Indiazolate-Bridged Copper(ll) and Zinc(lI) Complexes (%) The No. of SOD Mimics 12 13 14 15 16

6.4 taM

15.9 taM

31.3 taM

I 1.4 10.0 13.3 12.9 15.4

18.7 18.8 15.6 20.4 18.5

16.3 22.6 18.7 17.5 19.7

Zn(II) complexes and Cu(II) complexes of bi-shiffbase. Even in the same group, the activities of the complexes change with the variation of their structures. These facts indicate that the structure of SOD mimics is closely related to their biological function, and a minor change of the structure will lead to obvious change of SOD mimic's activity for scavenging ROS. Although the activity of CuZn-SOD is higher than that of SOD mimics, the macromolecular structure prevent the enzyme from penetrating biomembrane, and it can only scavenge ROS outside the PMAstimulated PMNL, whereas SOD mimics can scavenge ROS intracellularly as well as extracellularly. This suggests that the ability of CuZn-SOD for scavenging ROS produced by the PMA-stimulated PMNL is much less than that of the enzyme in noncellular systems. This may be due to its poor ability to penetrate biomembrane. Regarding the relationship between the inhibitory effects of SOD mimics on luminol-mediated chemilu-

539

The No. of SOD Mimics

6.4 uM

15.9 taM

31.3 taM

0 0 3.4 18.7 19.2

13.6 0 9.8 35.0 33.0

21.5 0.5 -57.6 --

17 18 19 20 21

minescence and their structure, the mechanism may be very complex. First, whether the Cu(II) ion in SOD mimics would be dissociated or not in the biological system must be considered. Although Cu(II) in an acidic medium is capable of dismutating O~- (Refs. 24-27), the toxicity of free Cu(II) ion prevents its use in biological systems. Various Cu(II)-containing complexes have been synthesized 28-32 in order to find a Cu(II) complex with effective biological activity, high ability for scavenging 02, and nontoxicity to biological systems. In another study (unpublished), we found that those Cu(II) complexes synthesized by us do not significantly interfere with the phagocytosis activity of PMNL at the concentration of SOD mimic used in this study. This may show that the coordinated Cu(II) in SOD mimics has little toxicity toward the PMAstimulated PMNL system. Most of these complexes retain their activities in aqueous solution, but the recent studies of Nagano and D a r r 33'34 showed that living cells and serum contained some factors which might be tightly bound

~.c ~g E

2°

•

"

2b

3b

Time interval (min) Fig. 8. The luminescent curve in the presence of SOD mimic No. 14. Luminescent curves of PMA-stimulated PMNL respiratory burst system have been estimated in the presence of three different concentrations of SOD mimic No. 14. • denotes the control system; whereas O, A, and • represent 6.4 taM, 15.9 taM, and 31.3 taM of SOD mimic No. 14 in the testing systems, respectively.

1

10

20 Time interval (rain)

I

30

Fig. 9. The luminescent curve in the presence of SOD mimic No. 17. Luminescent curves of PMA-stimulated PMNL respiratory burst system havebeen estimated in the presence of three different concentrations of SOD mimic No. 17. • denotes the control system; whereas O, A, and • represent 6.4 taM, 15.9 taM, and 31.3 taM of SOD mimic No. 17 in the testing systems, respectively.

540

T. YAPtNG el al.

with Cu(II) in SOD mimics and caused the release of Cu(II) from their active structure. Most of the copper complexes reported elsewhere were susceptible to inactivation by these factors. In our opinion, the estimation for activities of SOD mimics in aqueous solution may be suitable for research in vitro but not for research in vivo. The system most similar to the biological conditions should be used in screening SOD mimics. In our studies, living human PMNL cell was chosen as a first-used biological system for investigating SOD mimics not only because it was the living intact cell, but it also produced ROS when stimulated by PMA. If the tested complexes could keep their activities for dismutating 02 in such conditions, they must decrease the magnitude of the production of ROS. To keep the activity in biological conditions, the structure of SOD mimics should be stable enough. Four groups of Cu(II)-containing complexes synthesized by us as SOD mimics possess the activities of dismutating Oy. Two of these groups with the structure of macrocycle containing four amide coordinated with Cu(lI) as active site were found to be more stable and to have a high activity for scavenging ROS. On the other hand, the activities of the other two groups were much lower than the formers'. This seems to be contradictory to our previous communications, 19-21'23 in which the activities of SOD mimics were estimated by pulse radiolysis and the riboflavin illumination method. These results further demonstrated that the biological systems, such as living cells or protein-containing solutions, should be preferentially used in screening the SOD mimics. Since SOD mimics are proposed to be easily permeable to biomembrane, their biological effects may be superior to that of native SOD. It must be emphasized that there are more biological effects exerted by SOD mimics than those reported in this article. The protective effects of SOD mimics against the injurious actions of endogenous ROS on biomacromolecules, such as enzyme and DNA, have not been reported yet. Further studies of SOD mimics concerning these effects are being carried out, and the results will be reported later.

3.

4.

5,

6.

7.

8.

9. 10.

11.

12.

13.

14.

15. 16.

17.

18. Acknowledgements - - This study was supported by the National Natural Sciences Foundation of China. 19. REFERENCES

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ABBREVIATIONS

Cu--copper DAP--diacetylpyridine DFP--2,6-diformylpyridine DMSO--dimethyl sulfoxide H202--hydrogen peroxide HO2"--perhydroxyl radical O~---superoxide anion radical PBS-G-A--PBS-glucose-albumin solution PMA--phorbol myristate acetate PMNL~polymorphonuclear leukocyte ROS--reactive oxygen species SOD--superoxide dismutase Zn--zinc