- Email: [email protected]
Role of metal complexes in the formation-detoxication action of active oxygen species
21
Bioelectrochemistty and Bioenergetics, 18 (1987) 21-28 A section of J. Electroanal. Chem., and constituting Vol. 232 (1987) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
1008 - ROLE OF METAL COMPLEXES IN THE FORMATIONDETOXICATION ACTION OF ACTIYE OXYGEN SPECIES*
GIDON
CZAPSKI
and SARA GOLDSTEIN
Department of Physical Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904 (Israel)
SUMMARY A general mechanism for the action of metal compounds either in enhancing the biological damage induced by active oxygen species or in protecting the biological target against these species is proposed. The effect of several copper complexes and iron and copper bleomycin complexes on DNA cleavage is discussed in view of this mechanism.
INTRODUCTION
The role of metal ions in many biological systems has been well established [l-3]. Metal ions are involved in the action of many enzymes, such as superoxide dismutase (SOD), catalase and xanthine oxidase, and in the function of many other molecules of biological interest, such as a hemoglobin, cytochromes and chlorophylls. All these compounds lose their biological activity when the metal ion is removed or replaced by a different metal ion. In many systems, the role of the metal ions lies in their ability to participate in oxidation-reduction reactions, or in their ability to bind other molecules reversibly. Metal ions can also affect the conformation of a molecule or can bind substrates in a site-specific manner. All of these features are crucial in the reaction mechanisms of these enzymes and biologically important compounds. Metal ions also play an important role in the action of drugs and toxic compounds [4-91. In some cases, the metal ions are essential for the action of these compounds, while in others, they just enhance the effect [4-81. There are other systems where the metal compounds provide protection by inhibiting the activity of various toxic compounds and drugs [4,10].
Presented at the Bioelectrochemical Biological Systems”, Obemai, 22-24
l
0302-4598/87/$03.50
Society Meeting October 1986.
0 1987 Elsevier Sequoia
S.A.
on “Formation
and Reactions
of Peroxides
in
22
In this article, we will try to draw out a general reaction mechanism for the action of metal compounds either in enhancing DNA damage induced by active oxygen species or in protecting DNA against these species. This will be demonstrated through a few well-studied systems. THE HABER-WEISS MECHANISM
REACTION
CATALYSED
BY METAL
IONS AND
THE SITE-SPECIFIC
in many biological processes, such as The expression of the toxicity of 0, phagocytosis [ll] and eschemia [12], the action of drugs such as adriamycin [6], as well as in the action of herbicides such as paraquat [8,9], demands the presence of copper or iron compounds. In many of these systems it is assumed that 0; is a precursor for OH’ radical, which is formed through reduction of the metal by 0, and subsequent reoxidation of the reduced metal by H,02. This is called the Haber-Weiss reaction or the Fenton reaction driven by 0, [13-191: M”+ + 0;
+ MC”-iI+ + O2
MC”-I)+ + H,O, net:
0,
-+ M”+ + OH-
(1) + OH’
(2)
+ H,Oz + 0, + OH- + OH’
(3)
In many of the systems studied, OH’ scavengers did not prevent the damage and rather high concentrations of scavengers were necessary, especially in in vivo studies to achieve protection [20]. Moreover, it was demonstrated that OH; formed through reaction (3), was more harmful than that generated directly by radiolysis [21]. These observations led to the assumption of the site-specific mechanism [17-221. According to this mechanism, the metal compound is bound to the biological target. OH-is generated in the vicinity of the target site and therefore it is more harmful than an OH-that is generated in the bulk of the solution. Biol - M”+ + 0;
--) Biol - MC”-‘)+ + 0,
Biol-M(“-‘)++HzOz-+
Biol-M”+ (
(la) . ..OH’ L damage
+OH-
(2a)
i
In cells or in very concentrated solutions of biological components, OH; being formed homogeneously, would react almost at the point where it was generated. Since in cells the concentrations of the various cell components exceed 1 M, and as the rate constant of OH’ with these components is > lo9 M-i s-l, it is easy to show that the OH’ would react within about lOPi s with one of its nearest-neighbour molecules, which are at most 5-10 A away from that OH: Therefore, in order to scavenge such an OH’ in the cell, one needs scavenger concentrations of - 1 M so that the scavenger would compete efficiently with the cell components. The homogeneous generation of OH’ radicals inside the cell would be quite inefficient in causing DNA damage since most of the OH’ radicals would react in the cell with their nearest neighbours and thus would not reach DNA. If, however,
23
instead of generating a very reactive OH; one introduces 0;) which is an unreactive species towards most biological molecules [23,24], this 0; would be capable of diffusing around the cell until it would meet a target with which it could react. If Cu(I1) or Fe(II1) ions or their complexes are bound to DNA or to any other target, 0; would reduce the metal and subsequently the reduced metal would react with mechanism for the H,O, to yield OH’ at the target site. Such a site-specific production of OH’ would be more efficient than the homogenous formation of OH’ in the cell, and hence it would be much more difficult to scavenge such an OH’ [17-221. THE PRODUCT OF THE REACTION OF H,O, WITH THE REDUCED METAL
It is generally believed that H,O, yields OH’radicals when it reacts with Cu(1) or Fe(I1) compounds. However, it is possible that reaction (2) or (2a) would generate species other than OH’ such as Fe(W) [25,26], Cu(II1) [27,28] or CuH,Oc [28]. These alternative entities for OH-are highly oxidizing species which are capable of damaging the biological target directly or of ultimately generating OH: As these species may have different reactivities towards both the target and OH’scavengers, they might show a very different toxicity as compared to OH; and it might be more difficult to scavenge some of these species with OH’scavengers. METAL-CATALYSED DISMUTATION DELETERIOUS EFFECT OF 0;
OF
0;
AND
ITS
COMPETITION
WITH
THE
Reactions (1) and (2) or (la) and (2a) explain the enhancement of the toxicity of 0; by metal compounds. On the other hand, because many copper compounds catalyse the dismutation of 0, very efficiently [29-341, it is not clear why these compounds enhance biological damage by 0, instead of protecting against its toxicity. The mechanism of the dismutation of 0; by SOD [35,36] and by many metal compounds [29-341 is generally assumed to be through the ping-pong mechanism: cu2+ + 0, Cu+ + 0, net :
+ cu+ + 0, + 2 H+ + Cu2+ + H20,
20;+2H++H,O,+O,
(I) (4) (5)
If reactions (1) and (2) or (la) and (2a) were to occur instead of reactions (1) and (4) or (la) and (4a), then the toxicity of 0; would be enhanced by the metal compound. In the case where reactions (4) or (4a) predominated over reaction (2) or (2a), the metal compound would protect the biological system from 0; toxicity. However, if the reoxidation of the cuprous complex by O2 uia reaction (- 1) were to compete with reaction (4) the catalytic activity of the compound would be lower. In the case of native SOD, protection is observed because the rate of reaction (2) is considerably lower than that of reaction (4). In this particular case, reaction (- 1)
24
kz
Hz’Jz
I CuL,H,O,
/I\
CuL2”+
OH’
l
Liqand
degradation
Scheme 1.
can be neglected compared to reaction (4) (k_, = (0.44 f 0.12) M-’ s-l [36]), and thus SOD catalyses the dismutation of 0, very efficiently. In the absence of DNA, a copper compound (CULT) may react with 0;) 0, and H202 according to Scheme 1. For many copper compounds, k, = k, = lo* - lo9 M-’ s-l [29-361, k_, = 103-lo5 M-i s-i [34,37-391, k,= 103-lo4 M-i s-l [34,39], and under typical biological conditions [0,] = 0.24 mM, [O;] = lo-” M and [H20Z] = low8 M [40,41]. Therefore, in contrast to the case of SOD, with these substances reaction (- 1) will compete efficiently with reaction (4). In this case, the real ~rnover rate constant will be lower than k,,, = 2 klk4/(kl + k4), which is obtained only if reactions (1) and (4) proceed, and kreo, will be given by 2
klk4/{(k-1P21/PJ)
+ kl + k4), and hence L/keal
= 1+ [k-IP21/{(kI +
k4)[0;]}]. If k_,[O,] -=z (k, + k4)[0;], as in the case of SOD, k,,, = krear, otherwise k,,, > kreal. When krea, becomes very small, 0, will disappear through
different pathways. Thus, under physiological conditions, a copper complex which has a similar k,,, to that of SOD but its reduced form is reoxidized quickly by 0,, would exhibit lower protection as compared to SOD, and a relatively high concentration of this compound would be necessary to protect the system from 0; toxicity. THE MECHANISM COMPOUNDS
OF DNA
DAMAGE
INDUCED
BY 0;
IN THE
PRESENCE
OF METAL
If a metal compound binds to a biological target, reactions (l), (2) and (4) are replaced by reactions (la), (2a) and (4a) and the rate constants of the relevant reactions might change. It has been shown that Cu(II)-phenanthroline, (Op),Cu2+, enhances DNA damage induced by 0; [42,43]. As this complex is almost as efficient as SOD in catalysing the dismutation of 0; through reactions (1) and (4) (k,,, = (5.1 f 0.5) x lo8 M-’ s-* [34]), and taking into account k_, = (5.0 f 0.5) X lo4 M-’ s-l [34] and k, = (1100 f 150) M-’ s-l [34] and the physiological concentrations of 0;) H202 and 0,, one would expect that this complex would either protect DNA against 0; toxicity or that it would be inefficient in causing DNA damage in the
25
I _______L____-_
imechanism
l
Scheme 2.
I L__
_DNA
degradation
_________-,
I I I
presence of 0;. This paradox has been resolved by demonstrating that (Op),Cu*+ and (Op)*Cu+ bind to DNA to form ternary complexes, which react very slowly with 0, and 0, as compared to the free complexes [44]. For the various copper complexes studied in the presence of DNA, k,, = k, and reactions (la) and (4a) were too slow to be measured [44]. However, one can reduce and oxidize the ternary copper complexes by 0, through the free copper complexes, which are in rapid equilibrium with the ternary complexes. The mechanism of DNA damage induced by 0; in the presence of a copper complex and H,O, can be simplified by neglecting reactions (la), (-la) and (4a), and it is given in Scheme 2. When we studied the kinetic parameters which are included in Scheme 2, we were able to explain why and how (Op)2Cu2+ enhances DNA damage induced by 0; instead of providing protection. In the presence of DNA, k,, x==k,, and k, = k2n, while in its absence, k, -=xk, [34,44]. Therefore, one expects effective production of OH’radicals at the binding site. The Cu(II)-bipyridyl complex, (Bpy),Cu*+, does not degrade DNA under similar conditions to those of the Op system [42,43]. This is surprising as we have shown that with the Bpy system k,, B k,, and k, = k,,. However, we found that the binding constant of the cuprous complex to DNA is much lower with the Bpy system than with the Op system. Therefore, one observes only slight DNA damage with the Bpy system as most of the oxidizing species are formed through reaction (2) and not reaction (2a), and we expect less DNA damage when the oxidizing species is formed non-site specifically as compared to that formed site specifically. An efficient drug of this kind is one which forms a ternary complex with the metal ion and the biological target for which K, is not too high. If K, is too high, will be very low, and as a result the rate of the the concentration of free CuLy reduction by 0, will be too low to compete with the self-dismutation of 0; or with its reactions with other substrates.
26
The equilibrium constants K, and K, determine whether most of the reaction with H,O, will yield target or ligand damage. If K, is small and the equilibrium is shifted mainly towards CuLl, H,O, will react with CuLl in a non-site-specific mechanism. This would lead either to drug destruction or at most to the formation of an oxidizing entity, which would only partially damage the target. If K, is high so that most of the CuL: is present as a ternary complex, the reaction with H,O, will yield oxidizing entities at the target site. However, the oxidizing species might attack the drug itself, causing its degradation. Whether the oxidizing species will attack the drug or DNA depends on the size of the drug used and on the way the drug binds to the biological target. If K, is high and the equilibrium is shifted towards ligand dissociation, no damage will be expected, as we have shown that DNA=CuL+ does not react with H,O, [44]. The rate of the reoxidation of the various ternary cuprous complexes by 0, decreased as the concentration of DNA increased, indicating that the rate of reaction ( - la) is lower than that of reaction (- 1) [44]. Therefore, we can conclude that as the rate of the oxidation of the cuprous complex by 0, decreases, DNA cleavage increases since the oxidation of the ternary cuprous complex by 0, does not compete with that by H,O,. In general, knowing the kinetic parameters, which include the relevant rate constants and equilibria constants in Scheme 2, one can predict whether a given compound will protect against 0; toxicity or enhance it. The measurements and the knowledge of these parameters can be used to design an efficient drug. This is not limited to DNA damage, as in Scheme 2 DNA can be replaced by other biological targets. DNA CLEAVAGE INDUCED BY BLEOMYCIN
The mechanism by which the drug bleomycin (Blm) degrades DNA is different from that suggested in Scheme 2. Blm is a much larger molecule than Op and its action in vitro demands iron in order to express its cytotoxic effect [4,5,45]. Nevertheless, in some respects the action of Blm is similar to that of Op. In both cases, a metal ion, a reducing agent and 0, are essential for causing DNA degradation. DNA damage in the case of Blm is caused through the reaction of the ferrous complex with 0, forming an activated complex, which degrades DNA [46]. This process does not involve the Haber-Weiss reaction, and most probably OH’is not formed. The copper complex of Blm does not degrade DNA in vitro [4,47]. This complex is not reduced by 0, because of its high redox potential [47]. However, with other reductants such as thiols or CO; it is possible to reduce it [47-491. We have demonstrated that when Blm-Cu(1) reacts with 0, it forms Blm-Cu(I1) reversibly, while when it reacts with H,O, drug degradation occurs in both the absence and presence of DNA [49]. This is possibly the reason why BLM-Cu(I1) does not degrade DNA in the presence of a reducing agent and H,O,; on the contrary, the addition of Cu(I1) ions inhibits DNA cleavage [4,47].
21
In the case of Op, catalase inhibits the damage [42,43], while in the case of Blm it enhances it [50]. The reason for this is that the reaction of Blm-Fe(I1) in the absence and presence of DNA with H,O, causes degradation of the drug [46,51], while with the Op system it does not, and only in the presence of DNA does it cause DNA cleavage [42,43]. The difference between (Op),Cu(I), on the one hand, and Blm-Cu(1) and Blm-Fe(II), on the other hand, is in their reaction with H,O,. In the former case, the ligand is not degraded either in the absence or in presence of DNA. In the presence of DNA, degradation of DNA takes place [42,43], while with the latter ones, drug degradation occurs efficiently in both the absence and presence of DNA [46,51]. CONCLUSIONS
We believe that the described mechanism, given in Scheme 2, is quite general for the role of metal compounds in the action of various drugs, toxic compounds and normal metabolic processes. It seems that the mechanism of DNA cleavage by Blm-Fe(I1) is an exception. The general features of the mechanism of biological damage in the presence of metal complexes are as follows: (1) The oxidized and reduced metal complexes form ternary complexes with the biological target. (2) There is a cycle of oxidation-reduction reactions of the metal complex. In can be replaced by other reducing agents such as some cases, 0;) as a reductant, vitamin c, glutathione and NAD(P)H. Reoxidation of the metal can take place through reaction with 0;) O2 or H,O,. (3) The reaction of the reduced form with H,O, yields an oxidizing species, which can be either OH’or a metal peroxo complex or a higher valency state of the metal, which causes damage. Reoxidation by 0, or 0, does not contribute to any biological damage. The efficiency of a compound to cause damage can be predicted to some extent. It depends on the equilibrium and rate constants mentioned in Scheme 2, and by choosing a compound with appropriate equilibrium and rate constants, one can design an efficient drug. ACKNOWLEDGEMENTS
This work was supported by grant No. 1409 of the Council GSF Neuherberg, F.R.G. and Israel Academy of Science.
of Tobacco
Research,
REFERENCES 1 S.D. Aust, L.A. Morehouse and C.E. Thomas, 2 A.V. Xavier (Editor), Frontiers in Bioinorganic
J. Free Rad. Biol. Med., 1 (1985) 1. Chemistry, VCH Publishers, U.S.A.,
1986.
28 3 K.B. KarIin and J. Zubieta (Editors), Biological and Inorganic Copper Chemistry, Adenine Press, New York, 1984, Vols. 1 and 2. 4 E.A. Sausville, J. Peisach and S.B. Horwitz, Biochemistry, 17 (1978) 2740. 5 J.W. Lown and S.K. Sim, Biochemistry, 77 (1977) 1150. 6 J.R.F. Muidini, B.K. Sinha, L. Gianni and C.E. Myers, FEBS Lett., 172 (1984) 226. 7 R. Cone, S.K. Hason, J.W. Lown and A.M. Morgan, Can. J. Biochem., 54 (1976) 219. 8 C.C. Winterbourn and H.C. Sutton, Arch. Biochem. Biophys., 235 (1984) 116. 9 R. Kohen and M. Chevion, Free Rad. Res. Commun., 1 (1985) 71. 10 A.C.M. FiIho, M.E. Hoffman and R. Meneghibi, Biochem. J., 18 (1984) 273. 11 B.M. Babior, N. EngI. J. Med., 298 (1978) 659. 12 B. Hammond, H.A. Kontos and M.L. Hess, Can. J. Physiol. Pharmacol., 63 (1985) 173. 13 F. Haber and J. Weiss, Proc. R. Sot. London, Ser. A, 147 (1934) 332. 14 W.H. Koppenol, J. Butler and J.W. van Leeuwen, Photochem. Photobiol., 28 (1978) 665. 15 G. Czapski and Y.A. Ilan, Photochem. Photobiol., 28 (1978) 651. 16 E.W. Kellog and I. Fridovich, J. Biol. Chem., 250 (1975) 8812. 17 R.C. Lynch and I. Fridovich, Biochim. Biophys. Acta, 571 (1979) 195. 18 K.L. Fong, P.B. McCay, J.L. Poyer, H.P. Misra and B.B. Kcele, Chem. Biol. Inter., 15 (1976) 77. 19 J.J. Van Hemmen and W.J.A. Meuling, B&him. Biophys. Acta, 402 (1975) 131. 20 G. Czapski, Isr. J. Chem., 24 (1984) 29. 21 A. Samuni, M. Chevion and G. Czapski, Radiat. Res., 99 (1984) 562. 22 E.K. Hodgson and I. Fridovich, Biochemistry, 14 (1975) 5294. 23 G. Czapski, Annu. Rev. Phys. Chem., (1971) 171. 24 B.H.J. Bielski and G.G. Shiue, Oxygen Free Radicals and Tissue Damage, Ciba Foundation Series 65 (New Series), 1979, p. 43. 25 W.H. Koppenol and J.F. Liebman, J. Phys. Chem., 88 (1984) 99. 26 W. Bors, C. Michel and M. Saran, Eur. J. B&hem., 95 (1979) 621. 27 G.R.A. Johnson, N.B. Nazhat and R.A. Saadalla-Nazhat, J. Chem. Sot., Chem. Commun., (1985) 407. 28 S. Goldstein and G. Czapski, J. Free Rad. Biol. Med., 1 (1985) 373. 29 J. Rabani, D. Klug-Roth and J. LiIie, J. Phys. Chem., 77 (1973) 1169. 30 D. Khtg-Roth and J. Rabat-ii, J. Phys. Chem., 80 (1976) 587. 31 J. Weinstein and B.H.J. Bielski, J. Am. Chem. Sot., 102 (1980) 4916. 32 R. Brigelius, R. Spottl, W. Bors, E. Lengfelder, M. Saran and U. Weser, FEBS Lett., 47 (1974) 72. 33 M. Younes, E. Lengfelder, S. Zienau and U. Weser, B&hem. Biophys. Res. Commun., 81 (1978) 576. 34 S. Goldstein and G. Czapski, J. Inorg. Chem., 24 (1985) 1087. 35 E.M. Fielden, P.B. Roberts, R.C. Bray, D.J. Lowe, G.N. Mautner, G. Rotillo and L. Calabrese, B&hem. J., 139 (1974) 49. 36 D. KIug, I. Fridovich and J. Rabani, J. Am. Chem. Sot., 95 (1973) 2786. 37 A. ZuberbuhIer, Helv. Chim. Acta, 53 (1970) 473. 38 A.L. Crumbliss and A.T. Poulos, Inorg. Chem., 14 (1975) 1529. 39 I. Pecht and M. Anbar, J. Chem. Sot. A, (1968) 1902. 40 A. Boveris, Adv. Exp. Med. Biol., 78 (1977) 67. 41 A. Boveris and E. Cadenas, Superoxide Dismutases, CRC Press, Boca Raton, FL, 1982, Vol. 2, p. 15. 42 L.E. Marshall, D.R. Graham, K.A. Reich and D.S. Sigman, Biochemistry, 20 (1981) 244. 43 K.A. Reich, L.E. Marshall, D.R. Graham and D.S. S&man, J. Am. Chem. Sot., 103 (1981) 3582. 44 S. Goldstein and G. Czapski, J. Am. Chem. Sot., 108 (1986) 2244. 45 E.A. Sausville, J. Peisach and S.B. Horwitz, B&hem. Biophys. Res. Commun., 173 (1976) 814. 46 R.M. Burger, J. Peisach and S.B. Horwitz, J. Biol. Chem., 256 (1981) 11636. 47 T. Suzuki, J. Kuwahara and Y. Sugiura, Biochemistry, 24 (1985) 4719. 48 J.H. Freedman, S.B. Horwitz and J. Peisach, Biochemistry, 21 (1982) 2203. 49 S. Goldstein and G. Czapski, Int. J. Rad. Biol., 51 (1987) 693. 50 J.M.C. Gutteridge, A.P.C. Beard and G.J. QuinIan, Biochem. Inter., 10 (1985) 441. 51 S. Goldstein and G. Czapski, Free Rad. Res. Commun., 2 (1987) 259.
Bioelectrochemistty and Bioenergetics, 18 (1987) 21-28 A section of J. Electroanal. Chem., and constituting Vol. 232 (1987) Elsevier Sequoia S.A., Lausanne - Printed in The Netherlands
1008 - ROLE OF METAL COMPLEXES IN THE FORMATIONDETOXICATION ACTION OF ACTIYE OXYGEN SPECIES*
GIDON
CZAPSKI
and SARA GOLDSTEIN
Department of Physical Chemistry, The Hebrew University of Jerusalem, Jerusalem 91904 (Israel)
SUMMARY A general mechanism for the action of metal compounds either in enhancing the biological damage induced by active oxygen species or in protecting the biological target against these species is proposed. The effect of several copper complexes and iron and copper bleomycin complexes on DNA cleavage is discussed in view of this mechanism.
INTRODUCTION
The role of metal ions in many biological systems has been well established [l-3]. Metal ions are involved in the action of many enzymes, such as superoxide dismutase (SOD), catalase and xanthine oxidase, and in the function of many other molecules of biological interest, such as a hemoglobin, cytochromes and chlorophylls. All these compounds lose their biological activity when the metal ion is removed or replaced by a different metal ion. In many systems, the role of the metal ions lies in their ability to participate in oxidation-reduction reactions, or in their ability to bind other molecules reversibly. Metal ions can also affect the conformation of a molecule or can bind substrates in a site-specific manner. All of these features are crucial in the reaction mechanisms of these enzymes and biologically important compounds. Metal ions also play an important role in the action of drugs and toxic compounds [4-91. In some cases, the metal ions are essential for the action of these compounds, while in others, they just enhance the effect [4-81. There are other systems where the metal compounds provide protection by inhibiting the activity of various toxic compounds and drugs [4,10].
Presented at the Bioelectrochemical Biological Systems”, Obemai, 22-24
l
0302-4598/87/$03.50
Society Meeting October 1986.
0 1987 Elsevier Sequoia
S.A.
on “Formation
and Reactions
of Peroxides
in
22
In this article, we will try to draw out a general reaction mechanism for the action of metal compounds either in enhancing DNA damage induced by active oxygen species or in protecting DNA against these species. This will be demonstrated through a few well-studied systems. THE HABER-WEISS MECHANISM
REACTION
CATALYSED
BY METAL
IONS AND
THE SITE-SPECIFIC
in many biological processes, such as The expression of the toxicity of 0, phagocytosis [ll] and eschemia [12], the action of drugs such as adriamycin [6], as well as in the action of herbicides such as paraquat [8,9], demands the presence of copper or iron compounds. In many of these systems it is assumed that 0; is a precursor for OH’ radical, which is formed through reduction of the metal by 0, and subsequent reoxidation of the reduced metal by H,02. This is called the Haber-Weiss reaction or the Fenton reaction driven by 0, [13-191: M”+ + 0;
+ MC”-iI+ + O2
MC”-I)+ + H,O, net:
0,
-+ M”+ + OH-
(1) + OH’
(2)
+ H,Oz + 0, + OH- + OH’
(3)
In many of the systems studied, OH’ scavengers did not prevent the damage and rather high concentrations of scavengers were necessary, especially in in vivo studies to achieve protection [20]. Moreover, it was demonstrated that OH; formed through reaction (3), was more harmful than that generated directly by radiolysis [21]. These observations led to the assumption of the site-specific mechanism [17-221. According to this mechanism, the metal compound is bound to the biological target. OH-is generated in the vicinity of the target site and therefore it is more harmful than an OH-that is generated in the bulk of the solution. Biol - M”+ + 0;
--) Biol - MC”-‘)+ + 0,
Biol-M(“-‘)++HzOz-+
Biol-M”+ (
(la) . ..OH’ L damage
+OH-
(2a)
i
In cells or in very concentrated solutions of biological components, OH; being formed homogeneously, would react almost at the point where it was generated. Since in cells the concentrations of the various cell components exceed 1 M, and as the rate constant of OH’ with these components is > lo9 M-i s-l, it is easy to show that the OH’ would react within about lOPi s with one of its nearest-neighbour molecules, which are at most 5-10 A away from that OH: Therefore, in order to scavenge such an OH’ in the cell, one needs scavenger concentrations of - 1 M so that the scavenger would compete efficiently with the cell components. The homogeneous generation of OH’ radicals inside the cell would be quite inefficient in causing DNA damage since most of the OH’ radicals would react in the cell with their nearest neighbours and thus would not reach DNA. If, however,
23
instead of generating a very reactive OH; one introduces 0;) which is an unreactive species towards most biological molecules [23,24], this 0; would be capable of diffusing around the cell until it would meet a target with which it could react. If Cu(I1) or Fe(II1) ions or their complexes are bound to DNA or to any other target, 0; would reduce the metal and subsequently the reduced metal would react with mechanism for the H,O, to yield OH’ at the target site. Such a site-specific production of OH’ would be more efficient than the homogenous formation of OH’ in the cell, and hence it would be much more difficult to scavenge such an OH’ [17-221. THE PRODUCT OF THE REACTION OF H,O, WITH THE REDUCED METAL
It is generally believed that H,O, yields OH’radicals when it reacts with Cu(1) or Fe(I1) compounds. However, it is possible that reaction (2) or (2a) would generate species other than OH’ such as Fe(W) [25,26], Cu(II1) [27,28] or CuH,Oc [28]. These alternative entities for OH-are highly oxidizing species which are capable of damaging the biological target directly or of ultimately generating OH: As these species may have different reactivities towards both the target and OH’scavengers, they might show a very different toxicity as compared to OH; and it might be more difficult to scavenge some of these species with OH’scavengers. METAL-CATALYSED DISMUTATION DELETERIOUS EFFECT OF 0;
OF
0;
AND
ITS
COMPETITION
WITH
THE
Reactions (1) and (2) or (la) and (2a) explain the enhancement of the toxicity of 0; by metal compounds. On the other hand, because many copper compounds catalyse the dismutation of 0, very efficiently [29-341, it is not clear why these compounds enhance biological damage by 0, instead of protecting against its toxicity. The mechanism of the dismutation of 0; by SOD [35,36] and by many metal compounds [29-341 is generally assumed to be through the ping-pong mechanism: cu2+ + 0, Cu+ + 0, net :
+ cu+ + 0, + 2 H+ + Cu2+ + H20,
20;+2H++H,O,+O,
(I) (4) (5)
If reactions (1) and (2) or (la) and (2a) were to occur instead of reactions (1) and (4) or (la) and (4a), then the toxicity of 0; would be enhanced by the metal compound. In the case where reactions (4) or (4a) predominated over reaction (2) or (2a), the metal compound would protect the biological system from 0; toxicity. However, if the reoxidation of the cuprous complex by O2 uia reaction (- 1) were to compete with reaction (4) the catalytic activity of the compound would be lower. In the case of native SOD, protection is observed because the rate of reaction (2) is considerably lower than that of reaction (4). In this particular case, reaction (- 1)
24
kz
Hz’Jz
I CuL,H,O,
/I\
CuL2”+
OH’
l
Liqand
degradation
Scheme 1.
can be neglected compared to reaction (4) (k_, = (0.44 f 0.12) M-’ s-l [36]), and thus SOD catalyses the dismutation of 0, very efficiently. In the absence of DNA, a copper compound (CULT) may react with 0;) 0, and H202 according to Scheme 1. For many copper compounds, k, = k, = lo* - lo9 M-’ s-l [29-361, k_, = 103-lo5 M-i s-i [34,37-391, k,= 103-lo4 M-i s-l [34,39], and under typical biological conditions [0,] = 0.24 mM, [O;] = lo-” M and [H20Z] = low8 M [40,41]. Therefore, in contrast to the case of SOD, with these substances reaction (- 1) will compete efficiently with reaction (4). In this case, the real ~rnover rate constant will be lower than k,,, = 2 klk4/(kl + k4), which is obtained only if reactions (1) and (4) proceed, and kreo, will be given by 2
klk4/{(k-1P21/PJ)
+ kl + k4), and hence L/keal
= 1+ [k-IP21/{(kI +
k4)[0;]}]. If k_,[O,] -=z (k, + k4)[0;], as in the case of SOD, k,,, = krear, otherwise k,,, > kreal. When krea, becomes very small, 0, will disappear through
different pathways. Thus, under physiological conditions, a copper complex which has a similar k,,, to that of SOD but its reduced form is reoxidized quickly by 0,, would exhibit lower protection as compared to SOD, and a relatively high concentration of this compound would be necessary to protect the system from 0; toxicity. THE MECHANISM COMPOUNDS
OF DNA
DAMAGE
INDUCED
BY 0;
IN THE
PRESENCE
OF METAL
If a metal compound binds to a biological target, reactions (l), (2) and (4) are replaced by reactions (la), (2a) and (4a) and the rate constants of the relevant reactions might change. It has been shown that Cu(II)-phenanthroline, (Op),Cu2+, enhances DNA damage induced by 0; [42,43]. As this complex is almost as efficient as SOD in catalysing the dismutation of 0; through reactions (1) and (4) (k,,, = (5.1 f 0.5) x lo8 M-’ s-* [34]), and taking into account k_, = (5.0 f 0.5) X lo4 M-’ s-l [34] and k, = (1100 f 150) M-’ s-l [34] and the physiological concentrations of 0;) H202 and 0,, one would expect that this complex would either protect DNA against 0; toxicity or that it would be inefficient in causing DNA damage in the
25
I _______L____-_
imechanism
l
Scheme 2.
I L__
_DNA
degradation
_________-,
I I I
presence of 0;. This paradox has been resolved by demonstrating that (Op),Cu*+ and (Op)*Cu+ bind to DNA to form ternary complexes, which react very slowly with 0, and 0, as compared to the free complexes [44]. For the various copper complexes studied in the presence of DNA, k,, = k, and reactions (la) and (4a) were too slow to be measured [44]. However, one can reduce and oxidize the ternary copper complexes by 0, through the free copper complexes, which are in rapid equilibrium with the ternary complexes. The mechanism of DNA damage induced by 0; in the presence of a copper complex and H,O, can be simplified by neglecting reactions (la), (-la) and (4a), and it is given in Scheme 2. When we studied the kinetic parameters which are included in Scheme 2, we were able to explain why and how (Op)2Cu2+ enhances DNA damage induced by 0; instead of providing protection. In the presence of DNA, k,, x==k,, and k, = k2n, while in its absence, k, -=xk, [34,44]. Therefore, one expects effective production of OH’radicals at the binding site. The Cu(II)-bipyridyl complex, (Bpy),Cu*+, does not degrade DNA under similar conditions to those of the Op system [42,43]. This is surprising as we have shown that with the Bpy system k,, B k,, and k, = k,,. However, we found that the binding constant of the cuprous complex to DNA is much lower with the Bpy system than with the Op system. Therefore, one observes only slight DNA damage with the Bpy system as most of the oxidizing species are formed through reaction (2) and not reaction (2a), and we expect less DNA damage when the oxidizing species is formed non-site specifically as compared to that formed site specifically. An efficient drug of this kind is one which forms a ternary complex with the metal ion and the biological target for which K, is not too high. If K, is too high, will be very low, and as a result the rate of the the concentration of free CuLy reduction by 0, will be too low to compete with the self-dismutation of 0; or with its reactions with other substrates.
26
The equilibrium constants K, and K, determine whether most of the reaction with H,O, will yield target or ligand damage. If K, is small and the equilibrium is shifted mainly towards CuLl, H,O, will react with CuLl in a non-site-specific mechanism. This would lead either to drug destruction or at most to the formation of an oxidizing entity, which would only partially damage the target. If K, is high so that most of the CuL: is present as a ternary complex, the reaction with H,O, will yield oxidizing entities at the target site. However, the oxidizing species might attack the drug itself, causing its degradation. Whether the oxidizing species will attack the drug or DNA depends on the size of the drug used and on the way the drug binds to the biological target. If K, is high and the equilibrium is shifted towards ligand dissociation, no damage will be expected, as we have shown that DNA=CuL+ does not react with H,O, [44]. The rate of the reoxidation of the various ternary cuprous complexes by 0, decreased as the concentration of DNA increased, indicating that the rate of reaction ( - la) is lower than that of reaction (- 1) [44]. Therefore, we can conclude that as the rate of the oxidation of the cuprous complex by 0, decreases, DNA cleavage increases since the oxidation of the ternary cuprous complex by 0, does not compete with that by H,O,. In general, knowing the kinetic parameters, which include the relevant rate constants and equilibria constants in Scheme 2, one can predict whether a given compound will protect against 0; toxicity or enhance it. The measurements and the knowledge of these parameters can be used to design an efficient drug. This is not limited to DNA damage, as in Scheme 2 DNA can be replaced by other biological targets. DNA CLEAVAGE INDUCED BY BLEOMYCIN
The mechanism by which the drug bleomycin (Blm) degrades DNA is different from that suggested in Scheme 2. Blm is a much larger molecule than Op and its action in vitro demands iron in order to express its cytotoxic effect [4,5,45]. Nevertheless, in some respects the action of Blm is similar to that of Op. In both cases, a metal ion, a reducing agent and 0, are essential for causing DNA degradation. DNA damage in the case of Blm is caused through the reaction of the ferrous complex with 0, forming an activated complex, which degrades DNA [46]. This process does not involve the Haber-Weiss reaction, and most probably OH’is not formed. The copper complex of Blm does not degrade DNA in vitro [4,47]. This complex is not reduced by 0, because of its high redox potential [47]. However, with other reductants such as thiols or CO; it is possible to reduce it [47-491. We have demonstrated that when Blm-Cu(1) reacts with 0, it forms Blm-Cu(I1) reversibly, while when it reacts with H,O, drug degradation occurs in both the absence and presence of DNA [49]. This is possibly the reason why BLM-Cu(I1) does not degrade DNA in the presence of a reducing agent and H,O,; on the contrary, the addition of Cu(I1) ions inhibits DNA cleavage [4,47].
21
In the case of Op, catalase inhibits the damage [42,43], while in the case of Blm it enhances it [50]. The reason for this is that the reaction of Blm-Fe(I1) in the absence and presence of DNA with H,O, causes degradation of the drug [46,51], while with the Op system it does not, and only in the presence of DNA does it cause DNA cleavage [42,43]. The difference between (Op),Cu(I), on the one hand, and Blm-Cu(1) and Blm-Fe(II), on the other hand, is in their reaction with H,O,. In the former case, the ligand is not degraded either in the absence or in presence of DNA. In the presence of DNA, degradation of DNA takes place [42,43], while with the latter ones, drug degradation occurs efficiently in both the absence and presence of DNA [46,51]. CONCLUSIONS
We believe that the described mechanism, given in Scheme 2, is quite general for the role of metal compounds in the action of various drugs, toxic compounds and normal metabolic processes. It seems that the mechanism of DNA cleavage by Blm-Fe(I1) is an exception. The general features of the mechanism of biological damage in the presence of metal complexes are as follows: (1) The oxidized and reduced metal complexes form ternary complexes with the biological target. (2) There is a cycle of oxidation-reduction reactions of the metal complex. In can be replaced by other reducing agents such as some cases, 0;) as a reductant, vitamin c, glutathione and NAD(P)H. Reoxidation of the metal can take place through reaction with 0;) O2 or H,O,. (3) The reaction of the reduced form with H,O, yields an oxidizing species, which can be either OH’or a metal peroxo complex or a higher valency state of the metal, which causes damage. Reoxidation by 0, or 0, does not contribute to any biological damage. The efficiency of a compound to cause damage can be predicted to some extent. It depends on the equilibrium and rate constants mentioned in Scheme 2, and by choosing a compound with appropriate equilibrium and rate constants, one can design an efficient drug. ACKNOWLEDGEMENTS
This work was supported by grant No. 1409 of the Council GSF Neuherberg, F.R.G. and Israel Academy of Science.
of Tobacco
Research,
REFERENCES 1 S.D. Aust, L.A. Morehouse and C.E. Thomas, 2 A.V. Xavier (Editor), Frontiers in Bioinorganic
J. Free Rad. Biol. Med., 1 (1985) 1. Chemistry, VCH Publishers, U.S.A.,
1986.
28 3 K.B. KarIin and J. Zubieta (Editors), Biological and Inorganic Copper Chemistry, Adenine Press, New York, 1984, Vols. 1 and 2. 4 E.A. Sausville, J. Peisach and S.B. Horwitz, Biochemistry, 17 (1978) 2740. 5 J.W. Lown and S.K. Sim, Biochemistry, 77 (1977) 1150. 6 J.R.F. Muidini, B.K. Sinha, L. Gianni and C.E. Myers, FEBS Lett., 172 (1984) 226. 7 R. Cone, S.K. Hason, J.W. Lown and A.M. Morgan, Can. J. Biochem., 54 (1976) 219. 8 C.C. Winterbourn and H.C. Sutton, Arch. Biochem. Biophys., 235 (1984) 116. 9 R. Kohen and M. Chevion, Free Rad. Res. Commun., 1 (1985) 71. 10 A.C.M. FiIho, M.E. Hoffman and R. Meneghibi, Biochem. J., 18 (1984) 273. 11 B.M. Babior, N. EngI. J. Med., 298 (1978) 659. 12 B. Hammond, H.A. Kontos and M.L. Hess, Can. J. Physiol. Pharmacol., 63 (1985) 173. 13 F. Haber and J. Weiss, Proc. R. Sot. London, Ser. A, 147 (1934) 332. 14 W.H. Koppenol, J. Butler and J.W. van Leeuwen, Photochem. Photobiol., 28 (1978) 665. 15 G. Czapski and Y.A. Ilan, Photochem. Photobiol., 28 (1978) 651. 16 E.W. Kellog and I. Fridovich, J. Biol. Chem., 250 (1975) 8812. 17 R.C. Lynch and I. Fridovich, Biochim. Biophys. Acta, 571 (1979) 195. 18 K.L. Fong, P.B. McCay, J.L. Poyer, H.P. Misra and B.B. Kcele, Chem. Biol. Inter., 15 (1976) 77. 19 J.J. Van Hemmen and W.J.A. Meuling, B&him. Biophys. Acta, 402 (1975) 131. 20 G. Czapski, Isr. J. Chem., 24 (1984) 29. 21 A. Samuni, M. Chevion and G. Czapski, Radiat. Res., 99 (1984) 562. 22 E.K. Hodgson and I. Fridovich, Biochemistry, 14 (1975) 5294. 23 G. Czapski, Annu. Rev. Phys. Chem., (1971) 171. 24 B.H.J. Bielski and G.G. Shiue, Oxygen Free Radicals and Tissue Damage, Ciba Foundation Series 65 (New Series), 1979, p. 43. 25 W.H. Koppenol and J.F. Liebman, J. Phys. Chem., 88 (1984) 99. 26 W. Bors, C. Michel and M. Saran, Eur. J. B&hem., 95 (1979) 621. 27 G.R.A. Johnson, N.B. Nazhat and R.A. Saadalla-Nazhat, J. Chem. Sot., Chem. Commun., (1985) 407. 28 S. Goldstein and G. Czapski, J. Free Rad. Biol. Med., 1 (1985) 373. 29 J. Rabani, D. Klug-Roth and J. LiIie, J. Phys. Chem., 77 (1973) 1169. 30 D. Khtg-Roth and J. Rabat-ii, J. Phys. Chem., 80 (1976) 587. 31 J. Weinstein and B.H.J. Bielski, J. Am. Chem. Sot., 102 (1980) 4916. 32 R. Brigelius, R. Spottl, W. Bors, E. Lengfelder, M. Saran and U. Weser, FEBS Lett., 47 (1974) 72. 33 M. Younes, E. Lengfelder, S. Zienau and U. Weser, B&hem. Biophys. Res. Commun., 81 (1978) 576. 34 S. Goldstein and G. Czapski, J. Inorg. Chem., 24 (1985) 1087. 35 E.M. Fielden, P.B. Roberts, R.C. Bray, D.J. Lowe, G.N. Mautner, G. Rotillo and L. Calabrese, B&hem. J., 139 (1974) 49. 36 D. KIug, I. Fridovich and J. Rabani, J. Am. Chem. Sot., 95 (1973) 2786. 37 A. ZuberbuhIer, Helv. Chim. Acta, 53 (1970) 473. 38 A.L. Crumbliss and A.T. Poulos, Inorg. Chem., 14 (1975) 1529. 39 I. Pecht and M. Anbar, J. Chem. Sot. A, (1968) 1902. 40 A. Boveris, Adv. Exp. Med. Biol., 78 (1977) 67. 41 A. Boveris and E. Cadenas, Superoxide Dismutases, CRC Press, Boca Raton, FL, 1982, Vol. 2, p. 15. 42 L.E. Marshall, D.R. Graham, K.A. Reich and D.S. Sigman, Biochemistry, 20 (1981) 244. 43 K.A. Reich, L.E. Marshall, D.R. Graham and D.S. S&man, J. Am. Chem. Sot., 103 (1981) 3582. 44 S. Goldstein and G. Czapski, J. Am. Chem. Sot., 108 (1986) 2244. 45 E.A. Sausville, J. Peisach and S.B. Horwitz, B&hem. Biophys. Res. Commun., 173 (1976) 814. 46 R.M. Burger, J. Peisach and S.B. Horwitz, J. Biol. Chem., 256 (1981) 11636. 47 T. Suzuki, J. Kuwahara and Y. Sugiura, Biochemistry, 24 (1985) 4719. 48 J.H. Freedman, S.B. Horwitz and J. Peisach, Biochemistry, 21 (1982) 2203. 49 S. Goldstein and G. Czapski, Int. J. Rad. Biol., 51 (1987) 693. 50 J.M.C. Gutteridge, A.P.C. Beard and G.J. QuinIan, Biochem. Inter., 10 (1985) 441. 51 S. Goldstein and G. Czapski, Free Rad. Res. Commun., 2 (1987) 259.