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Manganese Dipyridoxyl Diphosphate: MRI Contrast Agent with Antioxidative and Cardioprotective Properties?
Biochemical and Biophysical Research Communications 254, 768 –772 (1999) Article ID bbrc.1998.0131, available online at http://www.idealibrary.com on
Manganese Dipyridoxyl Diphosphate: MRI Contrast Agent with Antioxidative and Cardioprotective Properties? In Vitro and ex Vivo Assessments 1 Heidi Brurok,* Jan Henrik Ardenkjær-Larsen,† Georg Hansson,† Sissel Skarra,* Kirsti Berg,* Jan O. G. Karlsson,‡ Ib Laursen,§ and Per Jynge* *Department of Physiology and Biomedical Engineering, Faculty of Medicine, Norwegian University of Science and Technology, Medisinsk-Teknisk Senter, N-7005 Trondheim, Norway; †Nycomed Innovation AB, Ideon-Malmo¨, S-20512 Malmo¨, Sweden; ‡Department of Pharmacology, Linko¨ping University, S-581 85 Linko¨ping, Sweden; and §Department of Automation, Denmark University of Technology, DK-2800 Lyngby, Denmark
Received December 21, 1998
Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent for magnetic resonance imaging (MRI) of the liver. Aims of the study were to examine if MnDPDP possesses superoxide dismutase (SOD) mimetic activity in vitro, and if antioxidant protection can be demonstrated in an ex vivo rat heart model. Superoxide ( •O 22) and hydroxyl radicals ( •OH 2) were generated in xanthine oxidase and Fenton reactions. Spin adducts with 5,5-dimethyl-1-pyrroline-N-oxide were detected by electron spin resonance spectroscopy. Contractile function and enzyme release were monitored in rat hearts during hypoxia-reoxygenation. Low mM concentrations of MnDPDP and its metabolite Mn dipyridoxyl ethylene-diamine (MnPLED) dismutated •O 22, but showed no activity in Fenton or catalase reactions. MnDPDP 30 mM improved contractile function and reduced enzyme release in rat hearts during reoxygenation. It is concluded that MnDPDP and MnPLED possess SOD mimetic activities and may thereby protect the heart in oxidative stress. © 1999 Academic Press
Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent (Teslascan, Nycomed Imaging AS) for magnetic resonance imaging (MRI) of the liver (1–3). MnDPDP (Fig. 1) is metabolized in biological systems (4, 5) by a process which includes transmetallation of Mn with zinc (Zn), dephosphorylation of the ligand with formation of dipyridoxyl ethylene-diamine (PLED), and the relase of divalent Mn ions (Mn 21). It is the uptake and retention of paramagnetic Mn 21 in 1 This study was supported by grants from the Research Council of Norway, the Norwegian University of Science and Technology and Nycomed Imaging AS.
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
normal hepatocytes that shortens proton longitudinal spin relaxation time (T1) thereby providing contrast between normal and pathological liver tissue. Experimental studies have also shown that MnDPDP may be an effective MRI marker in acute myocardial infarction by discriminating between occluded or reperfused infarcts (6) and by revealing graded information about myocardial function and metabolism (Brurok, nonpublished data). Reactive oxygen species (ROS) include radicals like superoxide ( •O 22) and hydroxyl ( •OH 2) and nonradicals such as hydrogen peroxide (H 2O 2) (7). Excess production of ROS together with a weakened endogenous defence are major factors behind myocardial ischemia-reperfusion injuries (8). Consequently, there is a need for exogenous strategies. One approach would be to mimic (mitochondrial) Mn-based superoxide dismutase (Mn-SOD) which converts •O 22 to H 2O 2 and O 2 (9). Recently several Mn-complexes have been shown to possess SOD-mimetic activity (10 –14). The aims of the present study were to examine whether MnDPDP and/or main metabolites possess antioxidant properties when tested in vitro and ex vivo in rat hearts subjected to an oxidative stress. MATERIALS AND METHODS Test substances. MnDPDP, MnPLED, ZnPLED and the sodiumliganded chelator (Na 2DPDP) were examined in vitro and MnDPDP ex vivo. Electron spin resonance (ESR) spectroscopy. Spin adducts of •O 22 and •OH 2 were detected with the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) (14, 15), which was protected from spontanous oxidation. Deionized water (conductivity less than 0.056 mS) was used to reduce contamination by trace metals. The experiments were run on a Bruker ESP 300 E spectrometer (Bruker Spectrospin SA, Wissembourg, France) using a glass capillary (20 ml) or a flat quarts cell (200 ml). Spectra were obtained at room temprature (21-23°C)
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (Yellow Springs Instrument Co., Ohio, USA). Oxygen saturation in 5 mM H 2O 2 in the presence and absence of test substance was measured after 3 min of reaction. Catalase was used as a positive control.
FIG. 1. Molecular structure of MnDPDP (3).
with frequency 9.736 GHz; sweep width 60 and 30 Gauss; resolution 8192 and 1024 points and sweep time 45 sec. Recording started 20 sec after onset of reactions. 4-6 spectra were collected in sequence, and measurements were made in the 4 min. •
2 2
Xanthine oxidase reaction. O was generated in the xanthine oxidase reaction (14, 15). The reaction mixture contained: 2.5 mM hypoxanthine; 18.5 U/ml xanthine oxidase; 100 mM DMPO; and test substance. No buffering or chelating agents were included (14), and pH was 6.0. •O 22 produces the DMPO-perhydroxyl adduct (DMPOOOH) which rapidly decays into a DMPO-hydroxyl adduct (DMPOOH) with longer halflife. Corresponding ESR spectra shows a shortlived sextet replaced by a quartet. Free radical production was expressed as the signal intensity (amplitude) of the peak appearing at 3470 Gauss in the quartet, and inhibition by test substances as a per cent decrease in the amplitude. Inhibition of the xanthine oxidase enzyme by test substances was investigated by measurements of the rate of urate formation by spectrophotometry at 290 nm (16). Fenton reaction. •OH 2 was produced by Fe 21 catalyzed degradation of hydrogen peroxide (H 2O 2) (14, 15). The reaction mixture contained: 5 mM H 2O 2; 20 mM FeSO 4/NaEDTA; 100 mM DMPO; and test substance. In parallel experiments the •OH 2 scavenger 1,3dimethyl-2-thiourea (DMTU) was tested. The pH in the control reaction was 3.5. ESR revealed DMPO-OH as the sole adduct, appearing as a quartet. Data processing was as described for the xanthine oxidase reaction. Catalase reaction. Catalase-like effects of test substances were examined by measuring oxygen production (14) by a Clark electrode
FIG. 2.
Ex vivo heart experiments. Isolated rat hearts (Wistar) were perfused in the Langendorff mode (16) with Krebs-Henseleit bicarbonate buffer (17) at 37°C and at a flow rate of 10 ml/min. The perfusate was equlibrated with 95% O 2 and 5% CO 2 during normoxic perfusion and with 95% N 2 and 5% CO 2 during hypoxic perfusion. Left ventricular (LV) developed pressure (LVDP) and lactic dehydrogenase (LDH) (18) in the coronary effluate were measured. After a control period of 20 min, hearts were subjected to hypoxic perfusion for 120 min followed by normoxic perfusion for 30 min. MnDPDP was either absent (control) or present (30 mM) in the perfusates during hypoxia and reoxygenation. Chemicals. MnDPDP, MnPLED, ZnPLED and Na 2DPDP were supplied by Nycomed Imaging AS, Oslo, Norway. Other chemicals used were: hypoxanthine, xanthine oxidase, catalase, DMPO and DMTU (Sigma Chemical Co., St. Louis, MO, USA); and FeSO 4, H 2O 2, NaEDTA, and MnCl 2 (Merck AG, Darmstadt, Germany). Statistics. Results (n 5 3–5) are expressed as the mean 6 standard error of the mean (SEM). Comparison between groups were made by use of one-way ANOVA and subseqently by Fischer9s Protected Least Significant Difference (PLSD) test.
RESULTS Xanthine oxidase reaction. Control reaction revealed that DMPO-OOH evolved rapidly (within 60 sec) towards DMPO-OH, the latter with hyperfine splitting constants of a N 5 14.71 G and a H/ B 5 14.91 G (Fig. 2). Without substrate no spin adducts were observed. Adding MnDPDP or MnPLED did not influence the timing of DMPO-OOH and DMPO-OH formation, but the substances markedly reduced the signal intensity of both adducts. Per cent reductions (Fig. 3) by MnDPDP (MnPLED) were: 5 mM 25% (25%); 10 mM 73% (49%); 25 mM 83% (71%); 50 mM 81% (83%); 100 mM 90% (81%); and 500 mM 92% (85%).
ESR spectra in the 1 min and the 4 min of the xanthine oxidase reaction: upper panel, control; lower panel, 50 mM of MnDPDP. 769
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FIG. 4. Contractile function (LVDP) during hypoxia and reoxygenation without (solid symbols) and with (open symbols) 30 mM of MnDPDP present.
FIG. 3. Concentration-responses of MnDPDP and MnPLED in the xanthine oxidase reaction. Data are derived from signal intensities appearing at 3470 Gauss in the spectrum of DMPO-OH in the 4 min. Results are expressed in per cent of control values obtained in the absence of test substance, and are presented as mean 6 SEM.
ZnPLED 500 mM neither influenced the timing of spin adduct formation nor led to any significant supression in signal intensity compared to control. In contrast, Na 2DPDP 500 mM prolonged the lifetime of DMPO-OOH, delayed the appearance of the DMPO-OH spin adduct, and reduced signal intensities by 73% compared to control. MnDPDP and MnPLED induced a concentration dependent rise in the rate of urate formation (to 163% and 132% at 100 mM). No such rise was observed for ZnPLED. Na 2DPDP showed a concentration dependent reduction (by 79% at 100 mM).
Ex vivo heart experiments. LVDP was maintained at a higher level with MnDPDP in the perfusate (Fig. 4), and on reoxygenation LVDP was 30-45% higher than control. LDH-release (Fig. 5) was low during hypoxia in both groups, but rose instantly on reoxygenation. The accumulated LDH release during reoxygenation was reduced by 70% with MnDPDP. DISCUSSION MnDPDP and MnPLED in low mM concentrations showed true SOD-mimetic activities in vitro, as evi-
Fenton reaction. Only the DMPO-OH adduct with its characteristic quartet (15) appeared in the ESR spectra. 500 mM of MnDPDP, MnPLED and ZnPLED revealed small increases in signal intensity (by 22%, 18% and 18%; significant for MnDPDP) when compared to control. Na 2DPDP, on the other hand, showed a concentration-dependent marked inhibition of spin adducts by 3%, 58% and 78% at respectively 50 mM, 250 mM and 500 mM. The •OH 2 scavenger DMTU 10 mM and 100 mM reduced spin adduct formation by 20% and 74%, respectively. Catalase reaction. MnDPDP, MnPLED, ZnPLED and Na 2DPDP (500 mM) had no effect on H 2O 2 dismutation in contrast to the catalase control.
FIG. 5. Effluent LDH release during hypoxia and reoxygenation without (solid symbols) and with (open symbols) 30 mM of MnDPDP present.
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denced by reduced spin adduct formation with ESR/ DMPO, and by maintained urate production, which excluded direct enzyme inhibition. MnDPDP and MnPLED revealed no antioxidant actions in the Fenton or catalase reactions. MnDPDP 30 mM protected rat hearts during hypoxia and rexogenation as shown by an improved contractile function and by a reduction in enzyme release. ZnPLED did not reduce spin adduct formation or urate production in the xanthine oxidase reaction. This is in analogy with the Cu, Zn SOD enzyme in which Zn plays a structural role only (19). Na 2DPDP delayed occurrence and reduced signal intensity of spin adducts and reduced urate production, most probably due to metal binding in the molybdenum plus Fe containing xanthine oxidase (20). This finding may be in accordance with Blasig (15), who showed a similar effect with diethylene-triamine-pentaacetic acid (DETAPAC) present in the xanthine oxidase reaction. Some other findings deserve comment. Firstly, MnDPDP and MnPLED increased the rate of urate production, probably due to removal of •O 22 as one end product in the xanthine oxidase reaction. Secondly, in our xanthine oxidase assay MnDPDP and MnPLED did not provide complete inhibition of spin adduct formation. This may be explained by trace metal contamination and by an element of Fenton reaction in which MnDPDP and MnPLED were ineffective. Contributing may also be a rise in H 2O 2 due to dismutation of •O 22 (7, 9). Thirdly, MnDPDP, MnPLED and ZnPLED induced a minor elevation of spin adducts in the Fenton reaction. This is difficult to explain since it concerns both investigated metals (Mn and Zn) and chelates (DPDP and PLED), and since a catalytic role of Mn in formation of •OH 2 has been held as unlikely (21) in studies of Mn-EDTA, Mn-pyrophosphates and Mn-polyphosphates. Archibald and Fridovich (9) have shown that Mn 21 when combined with common anions, may form complexes with SOD activity. To avoid false positive effects with Mn 21 released from MnDPDP or MnPLED (4, 5), we like investigators of Mn-containing compounds (14) omitted both buffering and chelating agents in the reaction mixtures. A few experiments (n 5 2) were undertaken in order to examine the effect of Mn 21 (MnCl 2) in the xanthine oxidase reaction. They confirmed that MnCl 2 alone produced the characteristic ESR spectrum of Mn 21 (22), but was without SOD activity. On the other hand, MnCl 2 in the presence of lactate or phosphate produced no specific Mn 21 spectrum, but greatly reduced the formation of spin adducts. Furthermore, recent experiments (nonpublished data) in our laboratory using nitro blue tetrazolium (NBT) and spectrophotometry as detection tools (23), demonstrated SOD activity of MnCl 2 as well as of MnDPDP and MnPLED when present in phosphate buffer at pH 7.8. Concerning the catalase reaction, in
which we did not demonstrate any effect by our test substances, Stadtman et al. (24) have reported activity of Mn 21-bicarbonate complexes. Together with the observations described by Archibald and Fridovich (9), this strengthens the proposition that Mn 21, when present in an optimal chemical environment, possesses catalytic and antioxidant activities. Cardiac hypoxia followed by reoxygenation, as applied in the present rat heart experiments, represents a situation with an increased production of ROS, with • O 22 being particularly important in initiating a deleterious ROS cascade (8, 25). •O 22 converts beneficial nitric oxide (NO) to highly toxic peroxynitrite, inititates release of Fe from Fe-sulfur centers in proteins (26) and produces H 2O 2 available for generation of hyperreactive •OH 2. SOD mimetics may play a therapeutic role in acute myocardial infarction by lowering • O 22 on its own, and by conserving NO. The end result will be improved microvascular function (27–29) and prevention of postischemic reperfusion injuries (29) following standard fibrinolytic treatment. Our results are in good correspondence with those of the de Leiris group (14, 30) studying Mn-salen complexes (10) in vitro and ex vivo. Accordingly, the SOD mimetic EUK-8 closely resembles MnDPDP and MnPLED in the xanthine oxidase reaction, as less than 50 mM completely inhibited spin adduct generation measured by ESR/DMPO after 20 sec. EUK-8, however, differed from MnDPDP and MnPLED by also displaying catalase-like activity (14). In ex vivo heart experiments EUK-8 attenuated reperfusion-induced arrhythmias, preserved contractile function and improved cardiac ultrastructure (14, 30). This resembles the action of MnDPDP which has shown protective properties in cardiac hypoxia-reoxygenation in our study and against anthracycline-induced injury in a recent mice left atrium study (31). In conclusion, we have shown that the MRI contrast agent MnDPDP and its metabolite MnPLED, have SOD-mimetic properties with a potential to reduce oxidative injuries in the heart. ACKNOWLEDGMENTS The advice and support from Professor Klaes Golman and senior researchers Go¨ran Pettersson and Robertson Towart, Nycomed Innovation AB, Malmo¨, Sweden is gratefully acknowledged.
REFERENCES
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1. Elizondo, G., Fretz, C. J., Stark, D. D., Rocklage, S. M., Quay, S. C., Worah, D., Tsang, Y. M., Chen, M. C. M., and Ferrucci, J. T. (1991) Radiology 178, 73–78. 2. Rocklage, S. M., Watson, A. D., and Carvlin, M. J. (1992) in Magnetic Resonance Imaging (Stark, D. D., and Bradley, W. G., Eds.), Mosby Year Book Inc, St. Louis. 3. Tirkkonen, B., Aukrust, E., Couture, E., Grace, D., Haile, Y.,
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4. 5.
6.
7.
8. 9. 10.
11. 12. 13. 14. 15. 16. 17.
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Holm, K. M., Hope, H., Larsen, Å., Sivertesen Lunde, H., and Sjøgren, C. E. (1997) Acta Radiol. 38, 780 –789. Grant, D., Zech, K., and Holtz, E. (1994) Invest. Radiol. 29, S249-S250. Toft, K. G., Hustvedt, S. O., Grant, D., Martinsen, I., Gordon, P. B., Friisk, G. A., Korsmo, Å. J., and Skotland, T. (1997) Acta Radiol. 38, 677– 689. Pomeroy, O. H., Wendland, M., Wagner, S., Derugin, N., Holt, W. W., Rocklage, S. M., Quay, S., and Higgins, C. B. (1989) Invest. Radiol. 24, 531–536. Halliwell B. (1997) in Antioxidants: The Basics—What They Are and How to Evaluate Them (Sies. H., Ed.), pp. 3–20, Academic Press, San Diego. Garlick, P. B., Davies, M. J., Hearse, D. J., and Slater, T. F. (1987) Circ. Res. 61, 757–760. Archibald, F. S., and Fridovich, I. (1982) Arch. Biochem. Biophys. 214, 452– 463. Baudry, M., Etienne, S., Bruce, A., Palucki, M., Jacobsen, E., and Malfroy, B. (1993) Biochem. Biophys. Res. Commun. 192, 964 – 968. Darr, D., Zarilla, K. A., and Fridovich, I. (1987) Arch. Biochem. Biophys. 258, 351–355. Faulkner, K. M., Liochev, S. I., and Fridovich, I. (1994) J. Biol. Chem. 269, 23471–23476. Riley, D. P., and Weiss, R. H. (1994) J. Amer. Chem. Soc. 116, 387–388. Tanguy, S., Boucher, F. R., Malfroy, B., and de Leiris, J. G. (1996) Free Radical Biol. Med. 21, 945–954. Blasig, I. E., Ebert, B., Wallukat, G., and Loewe, H. (1989) Free Radical Res. Commun. 6, 303–310. Spitz, D. R., and Oberley, L. W. (1989) Anal. Biochem. 179, 8 –18. Langendorff, O. (1895) Pflu¨gers Arch. Physiol. 61, 291–332.
18. Krebs, H. A., and Henseleit, K. (1932) Hoppe Seyler’s Z. Physiol. Chem. 210, 33– 66. 19. Moss, D. W. (1979) in Enzymes in Cardiology (Hearse, D. J., de Leiris, J., and Loisance, D., Eds.), pp. 145–198, John Wiley & Sons, New York. 20. Cowan, J. A. (1993) Inorganic Biochemistry, pp. 254 –260, VCH Publishers, New York. 21. Stryer, L. (1995) Biochemistry, 4th ed., p. 756, W. H Freeman and Company, New York. 22. Cheton, P. L., and Archibald, F. S. (1988) Free Radical Biol. Med. 5, 325–333. 23. Laursen, I., and Larsen, J. C. (1988) Fysisk Tidsskrift 86, 9. 24. Stadtman, E. R., Berlett, B. S., and Chock, P. B. (1990) Biochemistry 87, 384 –388. 25. Hearse, D. J., Humphrey, S. M., and Chain, E. B. (1973) J. Mol. Cell. Cardiol. 5, 395– 407. 26. Liochev, S. I., and Fridovich I. (1997) Free Radical Biol. Med. 23, 668 – 671. 27. Asplund, A., Grant, D., Karlsson, J. O. G. (1994) J. Pharmacol. Exp. Ther. 271, 609 – 618. 28. Matsanuga, K., and Furchgott, R. F. (1991) J. Pharmacol. Exp. Ther. 259, 1140 –1146. 29. Hearse, D. J., and Saldanha, C. (1997) in Coronary Microcirculation during Ischaemia and Reperfusion (Haunsø, S., Aldershvile, J., and Hastrup Svendsen, J., Eds.), pp. 195–210, Munksgaard, Copenhagen. 30. Pucheu, S., Boucher, T., Sulpice, T., Tresallet, N., Bonhomme, Y., Malfroy, B., and de Leiris, J. (1996) Cardiovasc. Drugs Ther. 10, 331–339. 31. Towart, R., Jynge, P., Refsum, H., and Karlsson, J. O. G. (1998) Arch. Pharmacol. 358(Suppl. 2), R626.
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Manganese Dipyridoxyl Diphosphate: MRI Contrast Agent with Antioxidative and Cardioprotective Properties? In Vitro and ex Vivo Assessments 1 Heidi Brurok,* Jan Henrik Ardenkjær-Larsen,† Georg Hansson,† Sissel Skarra,* Kirsti Berg,* Jan O. G. Karlsson,‡ Ib Laursen,§ and Per Jynge* *Department of Physiology and Biomedical Engineering, Faculty of Medicine, Norwegian University of Science and Technology, Medisinsk-Teknisk Senter, N-7005 Trondheim, Norway; †Nycomed Innovation AB, Ideon-Malmo¨, S-20512 Malmo¨, Sweden; ‡Department of Pharmacology, Linko¨ping University, S-581 85 Linko¨ping, Sweden; and §Department of Automation, Denmark University of Technology, DK-2800 Lyngby, Denmark
Received December 21, 1998
Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent for magnetic resonance imaging (MRI) of the liver. Aims of the study were to examine if MnDPDP possesses superoxide dismutase (SOD) mimetic activity in vitro, and if antioxidant protection can be demonstrated in an ex vivo rat heart model. Superoxide ( •O 22) and hydroxyl radicals ( •OH 2) were generated in xanthine oxidase and Fenton reactions. Spin adducts with 5,5-dimethyl-1-pyrroline-N-oxide were detected by electron spin resonance spectroscopy. Contractile function and enzyme release were monitored in rat hearts during hypoxia-reoxygenation. Low mM concentrations of MnDPDP and its metabolite Mn dipyridoxyl ethylene-diamine (MnPLED) dismutated •O 22, but showed no activity in Fenton or catalase reactions. MnDPDP 30 mM improved contractile function and reduced enzyme release in rat hearts during reoxygenation. It is concluded that MnDPDP and MnPLED possess SOD mimetic activities and may thereby protect the heart in oxidative stress. © 1999 Academic Press
Manganese dipyridoxyl diphosphate (MnDPDP) is a contrast agent (Teslascan, Nycomed Imaging AS) for magnetic resonance imaging (MRI) of the liver (1–3). MnDPDP (Fig. 1) is metabolized in biological systems (4, 5) by a process which includes transmetallation of Mn with zinc (Zn), dephosphorylation of the ligand with formation of dipyridoxyl ethylene-diamine (PLED), and the relase of divalent Mn ions (Mn 21). It is the uptake and retention of paramagnetic Mn 21 in 1 This study was supported by grants from the Research Council of Norway, the Norwegian University of Science and Technology and Nycomed Imaging AS.
0006-291X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
normal hepatocytes that shortens proton longitudinal spin relaxation time (T1) thereby providing contrast between normal and pathological liver tissue. Experimental studies have also shown that MnDPDP may be an effective MRI marker in acute myocardial infarction by discriminating between occluded or reperfused infarcts (6) and by revealing graded information about myocardial function and metabolism (Brurok, nonpublished data). Reactive oxygen species (ROS) include radicals like superoxide ( •O 22) and hydroxyl ( •OH 2) and nonradicals such as hydrogen peroxide (H 2O 2) (7). Excess production of ROS together with a weakened endogenous defence are major factors behind myocardial ischemia-reperfusion injuries (8). Consequently, there is a need for exogenous strategies. One approach would be to mimic (mitochondrial) Mn-based superoxide dismutase (Mn-SOD) which converts •O 22 to H 2O 2 and O 2 (9). Recently several Mn-complexes have been shown to possess SOD-mimetic activity (10 –14). The aims of the present study were to examine whether MnDPDP and/or main metabolites possess antioxidant properties when tested in vitro and ex vivo in rat hearts subjected to an oxidative stress. MATERIALS AND METHODS Test substances. MnDPDP, MnPLED, ZnPLED and the sodiumliganded chelator (Na 2DPDP) were examined in vitro and MnDPDP ex vivo. Electron spin resonance (ESR) spectroscopy. Spin adducts of •O 22 and •OH 2 were detected with the spin trap 5,5-dimethyl-1-pyrrolineN-oxide (DMPO) (14, 15), which was protected from spontanous oxidation. Deionized water (conductivity less than 0.056 mS) was used to reduce contamination by trace metals. The experiments were run on a Bruker ESP 300 E spectrometer (Bruker Spectrospin SA, Wissembourg, France) using a glass capillary (20 ml) or a flat quarts cell (200 ml). Spectra were obtained at room temprature (21-23°C)
768
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS (Yellow Springs Instrument Co., Ohio, USA). Oxygen saturation in 5 mM H 2O 2 in the presence and absence of test substance was measured after 3 min of reaction. Catalase was used as a positive control.
FIG. 1. Molecular structure of MnDPDP (3).
with frequency 9.736 GHz; sweep width 60 and 30 Gauss; resolution 8192 and 1024 points and sweep time 45 sec. Recording started 20 sec after onset of reactions. 4-6 spectra were collected in sequence, and measurements were made in the 4 min. •
2 2
Xanthine oxidase reaction. O was generated in the xanthine oxidase reaction (14, 15). The reaction mixture contained: 2.5 mM hypoxanthine; 18.5 U/ml xanthine oxidase; 100 mM DMPO; and test substance. No buffering or chelating agents were included (14), and pH was 6.0. •O 22 produces the DMPO-perhydroxyl adduct (DMPOOOH) which rapidly decays into a DMPO-hydroxyl adduct (DMPOOH) with longer halflife. Corresponding ESR spectra shows a shortlived sextet replaced by a quartet. Free radical production was expressed as the signal intensity (amplitude) of the peak appearing at 3470 Gauss in the quartet, and inhibition by test substances as a per cent decrease in the amplitude. Inhibition of the xanthine oxidase enzyme by test substances was investigated by measurements of the rate of urate formation by spectrophotometry at 290 nm (16). Fenton reaction. •OH 2 was produced by Fe 21 catalyzed degradation of hydrogen peroxide (H 2O 2) (14, 15). The reaction mixture contained: 5 mM H 2O 2; 20 mM FeSO 4/NaEDTA; 100 mM DMPO; and test substance. In parallel experiments the •OH 2 scavenger 1,3dimethyl-2-thiourea (DMTU) was tested. The pH in the control reaction was 3.5. ESR revealed DMPO-OH as the sole adduct, appearing as a quartet. Data processing was as described for the xanthine oxidase reaction. Catalase reaction. Catalase-like effects of test substances were examined by measuring oxygen production (14) by a Clark electrode
FIG. 2.
Ex vivo heart experiments. Isolated rat hearts (Wistar) were perfused in the Langendorff mode (16) with Krebs-Henseleit bicarbonate buffer (17) at 37°C and at a flow rate of 10 ml/min. The perfusate was equlibrated with 95% O 2 and 5% CO 2 during normoxic perfusion and with 95% N 2 and 5% CO 2 during hypoxic perfusion. Left ventricular (LV) developed pressure (LVDP) and lactic dehydrogenase (LDH) (18) in the coronary effluate were measured. After a control period of 20 min, hearts were subjected to hypoxic perfusion for 120 min followed by normoxic perfusion for 30 min. MnDPDP was either absent (control) or present (30 mM) in the perfusates during hypoxia and reoxygenation. Chemicals. MnDPDP, MnPLED, ZnPLED and Na 2DPDP were supplied by Nycomed Imaging AS, Oslo, Norway. Other chemicals used were: hypoxanthine, xanthine oxidase, catalase, DMPO and DMTU (Sigma Chemical Co., St. Louis, MO, USA); and FeSO 4, H 2O 2, NaEDTA, and MnCl 2 (Merck AG, Darmstadt, Germany). Statistics. Results (n 5 3–5) are expressed as the mean 6 standard error of the mean (SEM). Comparison between groups were made by use of one-way ANOVA and subseqently by Fischer9s Protected Least Significant Difference (PLSD) test.
RESULTS Xanthine oxidase reaction. Control reaction revealed that DMPO-OOH evolved rapidly (within 60 sec) towards DMPO-OH, the latter with hyperfine splitting constants of a N 5 14.71 G and a H/ B 5 14.91 G (Fig. 2). Without substrate no spin adducts were observed. Adding MnDPDP or MnPLED did not influence the timing of DMPO-OOH and DMPO-OH formation, but the substances markedly reduced the signal intensity of both adducts. Per cent reductions (Fig. 3) by MnDPDP (MnPLED) were: 5 mM 25% (25%); 10 mM 73% (49%); 25 mM 83% (71%); 50 mM 81% (83%); 100 mM 90% (81%); and 500 mM 92% (85%).
ESR spectra in the 1 min and the 4 min of the xanthine oxidase reaction: upper panel, control; lower panel, 50 mM of MnDPDP. 769
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FIG. 4. Contractile function (LVDP) during hypoxia and reoxygenation without (solid symbols) and with (open symbols) 30 mM of MnDPDP present.
FIG. 3. Concentration-responses of MnDPDP and MnPLED in the xanthine oxidase reaction. Data are derived from signal intensities appearing at 3470 Gauss in the spectrum of DMPO-OH in the 4 min. Results are expressed in per cent of control values obtained in the absence of test substance, and are presented as mean 6 SEM.
ZnPLED 500 mM neither influenced the timing of spin adduct formation nor led to any significant supression in signal intensity compared to control. In contrast, Na 2DPDP 500 mM prolonged the lifetime of DMPO-OOH, delayed the appearance of the DMPO-OH spin adduct, and reduced signal intensities by 73% compared to control. MnDPDP and MnPLED induced a concentration dependent rise in the rate of urate formation (to 163% and 132% at 100 mM). No such rise was observed for ZnPLED. Na 2DPDP showed a concentration dependent reduction (by 79% at 100 mM).
Ex vivo heart experiments. LVDP was maintained at a higher level with MnDPDP in the perfusate (Fig. 4), and on reoxygenation LVDP was 30-45% higher than control. LDH-release (Fig. 5) was low during hypoxia in both groups, but rose instantly on reoxygenation. The accumulated LDH release during reoxygenation was reduced by 70% with MnDPDP. DISCUSSION MnDPDP and MnPLED in low mM concentrations showed true SOD-mimetic activities in vitro, as evi-
Fenton reaction. Only the DMPO-OH adduct with its characteristic quartet (15) appeared in the ESR spectra. 500 mM of MnDPDP, MnPLED and ZnPLED revealed small increases in signal intensity (by 22%, 18% and 18%; significant for MnDPDP) when compared to control. Na 2DPDP, on the other hand, showed a concentration-dependent marked inhibition of spin adducts by 3%, 58% and 78% at respectively 50 mM, 250 mM and 500 mM. The •OH 2 scavenger DMTU 10 mM and 100 mM reduced spin adduct formation by 20% and 74%, respectively. Catalase reaction. MnDPDP, MnPLED, ZnPLED and Na 2DPDP (500 mM) had no effect on H 2O 2 dismutation in contrast to the catalase control.
FIG. 5. Effluent LDH release during hypoxia and reoxygenation without (solid symbols) and with (open symbols) 30 mM of MnDPDP present.
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denced by reduced spin adduct formation with ESR/ DMPO, and by maintained urate production, which excluded direct enzyme inhibition. MnDPDP and MnPLED revealed no antioxidant actions in the Fenton or catalase reactions. MnDPDP 30 mM protected rat hearts during hypoxia and rexogenation as shown by an improved contractile function and by a reduction in enzyme release. ZnPLED did not reduce spin adduct formation or urate production in the xanthine oxidase reaction. This is in analogy with the Cu, Zn SOD enzyme in which Zn plays a structural role only (19). Na 2DPDP delayed occurrence and reduced signal intensity of spin adducts and reduced urate production, most probably due to metal binding in the molybdenum plus Fe containing xanthine oxidase (20). This finding may be in accordance with Blasig (15), who showed a similar effect with diethylene-triamine-pentaacetic acid (DETAPAC) present in the xanthine oxidase reaction. Some other findings deserve comment. Firstly, MnDPDP and MnPLED increased the rate of urate production, probably due to removal of •O 22 as one end product in the xanthine oxidase reaction. Secondly, in our xanthine oxidase assay MnDPDP and MnPLED did not provide complete inhibition of spin adduct formation. This may be explained by trace metal contamination and by an element of Fenton reaction in which MnDPDP and MnPLED were ineffective. Contributing may also be a rise in H 2O 2 due to dismutation of •O 22 (7, 9). Thirdly, MnDPDP, MnPLED and ZnPLED induced a minor elevation of spin adducts in the Fenton reaction. This is difficult to explain since it concerns both investigated metals (Mn and Zn) and chelates (DPDP and PLED), and since a catalytic role of Mn in formation of •OH 2 has been held as unlikely (21) in studies of Mn-EDTA, Mn-pyrophosphates and Mn-polyphosphates. Archibald and Fridovich (9) have shown that Mn 21 when combined with common anions, may form complexes with SOD activity. To avoid false positive effects with Mn 21 released from MnDPDP or MnPLED (4, 5), we like investigators of Mn-containing compounds (14) omitted both buffering and chelating agents in the reaction mixtures. A few experiments (n 5 2) were undertaken in order to examine the effect of Mn 21 (MnCl 2) in the xanthine oxidase reaction. They confirmed that MnCl 2 alone produced the characteristic ESR spectrum of Mn 21 (22), but was without SOD activity. On the other hand, MnCl 2 in the presence of lactate or phosphate produced no specific Mn 21 spectrum, but greatly reduced the formation of spin adducts. Furthermore, recent experiments (nonpublished data) in our laboratory using nitro blue tetrazolium (NBT) and spectrophotometry as detection tools (23), demonstrated SOD activity of MnCl 2 as well as of MnDPDP and MnPLED when present in phosphate buffer at pH 7.8. Concerning the catalase reaction, in
which we did not demonstrate any effect by our test substances, Stadtman et al. (24) have reported activity of Mn 21-bicarbonate complexes. Together with the observations described by Archibald and Fridovich (9), this strengthens the proposition that Mn 21, when present in an optimal chemical environment, possesses catalytic and antioxidant activities. Cardiac hypoxia followed by reoxygenation, as applied in the present rat heart experiments, represents a situation with an increased production of ROS, with • O 22 being particularly important in initiating a deleterious ROS cascade (8, 25). •O 22 converts beneficial nitric oxide (NO) to highly toxic peroxynitrite, inititates release of Fe from Fe-sulfur centers in proteins (26) and produces H 2O 2 available for generation of hyperreactive •OH 2. SOD mimetics may play a therapeutic role in acute myocardial infarction by lowering • O 22 on its own, and by conserving NO. The end result will be improved microvascular function (27–29) and prevention of postischemic reperfusion injuries (29) following standard fibrinolytic treatment. Our results are in good correspondence with those of the de Leiris group (14, 30) studying Mn-salen complexes (10) in vitro and ex vivo. Accordingly, the SOD mimetic EUK-8 closely resembles MnDPDP and MnPLED in the xanthine oxidase reaction, as less than 50 mM completely inhibited spin adduct generation measured by ESR/DMPO after 20 sec. EUK-8, however, differed from MnDPDP and MnPLED by also displaying catalase-like activity (14). In ex vivo heart experiments EUK-8 attenuated reperfusion-induced arrhythmias, preserved contractile function and improved cardiac ultrastructure (14, 30). This resembles the action of MnDPDP which has shown protective properties in cardiac hypoxia-reoxygenation in our study and against anthracycline-induced injury in a recent mice left atrium study (31). In conclusion, we have shown that the MRI contrast agent MnDPDP and its metabolite MnPLED, have SOD-mimetic properties with a potential to reduce oxidative injuries in the heart. ACKNOWLEDGMENTS The advice and support from Professor Klaes Golman and senior researchers Go¨ran Pettersson and Robertson Towart, Nycomed Innovation AB, Malmo¨, Sweden is gratefully acknowledged.
REFERENCES
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1. Elizondo, G., Fretz, C. J., Stark, D. D., Rocklage, S. M., Quay, S. C., Worah, D., Tsang, Y. M., Chen, M. C. M., and Ferrucci, J. T. (1991) Radiology 178, 73–78. 2. Rocklage, S. M., Watson, A. D., and Carvlin, M. J. (1992) in Magnetic Resonance Imaging (Stark, D. D., and Bradley, W. G., Eds.), Mosby Year Book Inc, St. Louis. 3. Tirkkonen, B., Aukrust, E., Couture, E., Grace, D., Haile, Y.,
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4. 5.
6.
7.
8. 9. 10.
11. 12. 13. 14. 15. 16. 17.
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Holm, K. M., Hope, H., Larsen, Å., Sivertesen Lunde, H., and Sjøgren, C. E. (1997) Acta Radiol. 38, 780 –789. Grant, D., Zech, K., and Holtz, E. (1994) Invest. Radiol. 29, S249-S250. Toft, K. G., Hustvedt, S. O., Grant, D., Martinsen, I., Gordon, P. B., Friisk, G. A., Korsmo, Å. J., and Skotland, T. (1997) Acta Radiol. 38, 677– 689. Pomeroy, O. H., Wendland, M., Wagner, S., Derugin, N., Holt, W. W., Rocklage, S. M., Quay, S., and Higgins, C. B. (1989) Invest. Radiol. 24, 531–536. Halliwell B. (1997) in Antioxidants: The Basics—What They Are and How to Evaluate Them (Sies. H., Ed.), pp. 3–20, Academic Press, San Diego. Garlick, P. B., Davies, M. J., Hearse, D. J., and Slater, T. F. (1987) Circ. Res. 61, 757–760. Archibald, F. S., and Fridovich, I. (1982) Arch. Biochem. Biophys. 214, 452– 463. Baudry, M., Etienne, S., Bruce, A., Palucki, M., Jacobsen, E., and Malfroy, B. (1993) Biochem. Biophys. Res. Commun. 192, 964 – 968. Darr, D., Zarilla, K. A., and Fridovich, I. (1987) Arch. Biochem. Biophys. 258, 351–355. Faulkner, K. M., Liochev, S. I., and Fridovich, I. (1994) J. Biol. Chem. 269, 23471–23476. Riley, D. P., and Weiss, R. H. (1994) J. Amer. Chem. Soc. 116, 387–388. Tanguy, S., Boucher, F. R., Malfroy, B., and de Leiris, J. G. (1996) Free Radical Biol. Med. 21, 945–954. Blasig, I. E., Ebert, B., Wallukat, G., and Loewe, H. (1989) Free Radical Res. Commun. 6, 303–310. Spitz, D. R., and Oberley, L. W. (1989) Anal. Biochem. 179, 8 –18. Langendorff, O. (1895) Pflu¨gers Arch. Physiol. 61, 291–332.
18. Krebs, H. A., and Henseleit, K. (1932) Hoppe Seyler’s Z. Physiol. Chem. 210, 33– 66. 19. Moss, D. W. (1979) in Enzymes in Cardiology (Hearse, D. J., de Leiris, J., and Loisance, D., Eds.), pp. 145–198, John Wiley & Sons, New York. 20. Cowan, J. A. (1993) Inorganic Biochemistry, pp. 254 –260, VCH Publishers, New York. 21. Stryer, L. (1995) Biochemistry, 4th ed., p. 756, W. H Freeman and Company, New York. 22. Cheton, P. L., and Archibald, F. S. (1988) Free Radical Biol. Med. 5, 325–333. 23. Laursen, I., and Larsen, J. C. (1988) Fysisk Tidsskrift 86, 9. 24. Stadtman, E. R., Berlett, B. S., and Chock, P. B. (1990) Biochemistry 87, 384 –388. 25. Hearse, D. J., Humphrey, S. M., and Chain, E. B. (1973) J. Mol. Cell. Cardiol. 5, 395– 407. 26. Liochev, S. I., and Fridovich I. (1997) Free Radical Biol. Med. 23, 668 – 671. 27. Asplund, A., Grant, D., Karlsson, J. O. G. (1994) J. Pharmacol. Exp. Ther. 271, 609 – 618. 28. Matsanuga, K., and Furchgott, R. F. (1991) J. Pharmacol. Exp. Ther. 259, 1140 –1146. 29. Hearse, D. J., and Saldanha, C. (1997) in Coronary Microcirculation during Ischaemia and Reperfusion (Haunsø, S., Aldershvile, J., and Hastrup Svendsen, J., Eds.), pp. 195–210, Munksgaard, Copenhagen. 30. Pucheu, S., Boucher, T., Sulpice, T., Tresallet, N., Bonhomme, Y., Malfroy, B., and de Leiris, J. (1996) Cardiovasc. Drugs Ther. 10, 331–339. 31. Towart, R., Jynge, P., Refsum, H., and Karlsson, J. O. G. (1998) Arch. Pharmacol. 358(Suppl. 2), R626.
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