Environmental Health PerspectivesVol. 40, pp. 227-232, 1981

Chelating Agents in Biological Systemsby Jack Schubert*

Chelation enables metals to be transported to or from vulnerable target sites, and to hinder orfacilitate their carcinogenic potential. In the reverse sense, metals are capable of ligandscavenging via complexation or mixed complex formation-the latter being the result ofinteraction with binary complexes. Consequently, metal complexes can be utilized for thetransport of selected organic chemotherapeutic drugs to target organs, or for the decorporationof those toxic organic compounds which are able, before or after metabolic activation, ofreacting with metals or 1:1 metal complexes. It is emphasized that the degree to which metal ionsinteract in vivo should employ the conditional constants which take into account competitionfrom other ions, especially Ca2 +, H +, and OH-. The genotoxic consequences ofthe various chemicalfactors involved in chelation, along with examples: kinetics, stabilization of oxidation states,lipophilicity, and mixed ligand formation, are discussed.

IntroductionThe transition metal ions inevitably exist as

metal complexes in biological systems by interac-tion with the numerous molecules possessing group-ings capable of complexation or chelation. Hencewe find essential metals such as Cu, Zn, Cr, Fe,Mn, and Co existing as binary and ternary chelatesof amino acids, carboxylic acids, and proteins.Without such interactions, life could not exist or bemaintained, or, as earlier expressed, "Life couldnot exist or even come into being without themediating action of metals" (1). Wood (2) made thecogent point when he wrote: ". . . metals areinvolved in every aspect of biosynthesis, biode-gradation, and in the assembly of macromolecularstructure. If you think that biochemistry is theorganic chemistry of living systems, then you aremisled: biochemistry is the coordination chemistryof living systems." Certainly not all biochemicalreactions involve metal compounds. On the otherhand metals are involved in such a large number ofessential biological reactions that life as we know itcould not exist without coordination compounds.The carcinogenic metals all have the ability-via

complexation or ternary complex formation-to

*Department of Chemistry, University of Maryland BaltimoreCounty, Catonsville, Maryland 21228.

August 1981

interact with DNA and other nuclear constituentsor to alter such cellular properties as membraneintegrity. Chelation phenomena enable metals to betransported to or away from vulnerable targetsites, and to facilitate or hinder those intracellularinteractions which may ultimately lead to cancer.In order to appreciate and apply the dual nature ofmetal chelation in carcinogenesis, both in its inductionand therapy, I shall review selected aspects of thechemistry of metal ions, chelate formation, and thestability of metal chelates. A more comprehensivereview of the chemistry of carcinogenic metals isgiven elsewhere by Martell (3). These chemicalproperties and resulting speciation all have a bearingon the ability of a metal ion to interact with targetmolecules. For example, while Cr(VI) as chromate,CrO42-, enters cells, the ultimate mutagen or car-cinogen from the chemical standpoint can only beCr(III) which is the state which chelates withbiological molecules as reference to the Sillen-Martell tables reveals (4). In fact, Cr(III) is notcarcinogenic because it cannot penetrate cell mem-branes, but Cr(VI) is reduced to Cr(III) within thecell. On the other hand, chelation with exogenouschelating agents can reduce or prevent itsmutagenicity (5). Similarly, results on the antitumoractivity of acid ruthenium amine complexes suggestthat reduction to Ru(II) occurs in vivo prior tometal coordination to nucleic acids (6).

227

I will emphasize the ability of chelants to combinewith and to transport metal ions-a process ofmetal scavenging. This property, especially whenwater-soluble, nonmetabolizable, and exeretablechelates are formed, is widely applied for thedecorporation of toxic metals, both stable andradioactive. A potentially useful "reverse" prop-erty is ligand scavenging in which the goal is toaccelerate the excretion of toxic organic substancescontaining groupings capable of binding to a non-toxic metal prior to or after metabolic activation.Binary complexes may also combine with an addi-tional ligand forming a mixed or ternary complex(7). This latter property appears to be the mecha-nism by which catecholamines (8, 9) are trans-ported as well as nucleotides (10) and oxygen. Inthis connection, therefore, it would appear thatmetal complexes could transport organic chem-otherapeutic drugs to target organs or removeexcessive levels of the drugs.

Chelate Formation and In VivoStability

In biological systems important factors in theactions of metal chelates include: (1) intrinsic stabil-ity; (2) chelant-metal ratios; (3) stability as afunction of pH; (4) competition from other metalsand ligands, both endogenous and exogenous; (5)overall charge and lipophilicity; and (6) rates ofhydrolysis and chelate formation. An excellentreview of the chemistry of chelation and factorsgoverning stability is provided by Mellor (11).

Chelating molecules possess at least two func-tional groups containing donor atoms capable ofcombining with the metal ion and so situated that aring is formed with the metal ion. Since mostchelants contain protons, these compete with themetal ion for the chelant:

M + HnL = ML + nH+ (1)

where L represents the ligand and M the metal ion.Generally we will focus on the binary 1:1 chelatesand occasionally on 1:2 chelates, i.e., ML2. Thefraction of the metal present as a chelate, ML,derives from mass action:

ML/M = K/L) (2)

The equation shows that the fraction ofM in chelateform, (ML)/[(M) + (ML)], is enhanced either byincreasing the concentration of the ligand, or by aligand produeing a higher formation constant, Kf.Ligand concentrations, whether endogenous or ex-

228

ogenous, do not or cannot be varied greatly, e.g.,most chelants such as EDTA or diethylenetri-aminepentaacetic acid (DPTA) used for therapy ofmetal poisoning, become toxic above peak plasmalevels of 10-3-104M. However, the values ofKfmayrange from 100 to > 1030. Obviously, in the choice ofchelants for biological use, the Kf values offergreater flexibility for the scavenging and release ofbound metals or ligands.When exogenous chelating agents are employed

in biological systems, the stability of the binarycomplex as expressed by simple mass action doesnot necessarily reflect actual in vivo behavior. Forexample, in in vivo systems, Hg(II) is not bound byEDTA, despite the fact that the Kf of HgEDTA isvery high,A028. However, when competition fromsome of the common ligands in the plasma isconsidered, namely OH- and serum albumin for Hgand H+ and Ca2' for EDTA, we find that there ispractically no net binding by EDTA for the mercu-ry. When calculations commonly employed in ana-lytical and coordination chemistry are adapted tobiological systems (12-14), they give the "condi-tional constant" which provides a more directmeasure of metal chelate stability under biologicalconditions.One example (12) of the usefulness of the condi-

tional constant is the following: Fe(III)EDTA isabout 106 times stronger then Cu(II)EDTA inconventional terms. Yet at pH 7, the Cu(II)EDTAchelate is more than 102 times stronger thanFe(III)EDTA (Fig. 1). This result, for example,has a bearing on the ease of dissociation in vivo ofthese chelates since they could result in the releaseof free toxic metal ions in vulnerable tissue or cells.

Rate EffectsThe rates at which chelate or complex ion forma-

tion take place have important biological conse-quences, as, for example, in the manifestation orreversal of metal ion mutagenicity in microbial testsystems (5). The rapid or labile reactions areusually completed by the time of mixing while theinert or robust systems may take minutes to days.While most metal chelates, at least of the divalenttransition metals, Co, Cd, Mn, Zn, and Cu arelabile, those involving Cr(III), Fe(III), Co(III),Mo(III), W(III), Mn(IV), Ru(II), Ni(II), and Pt(IV),are often inert (11). Outer orbital complexes(sp2d3)are usually labile and inner orbital (d2sp3) complexesare either labile or inert, the former when at leastone d orbital is vacant, otherwise they are inert.

Hydrolyzable, polyvalent cations such as those ofCr, V, Ti, Zr, and Pu, undergo a complicated seriesof reactions with water and hydroxide ions, even-

Environmental Health Perspectives

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13

1I-NC

9

7

2 4 6pH

FIGURE 1. Variation of the log of the effeconstants with pH for the chelates of I

Cu(II)EDTA. The calculations includedCa++, OH-, and H+. The curves are talpublished elsewhere (12).

tually leading to inorganic polymers. I

ions, such as Cr2O72- which cannot e:pH > 5 will begin to change when sirin water. Thus, Cr2072- becomes chr(and HCrO4 . In fact, the genotoxicitnmeasured in the rec-assay, decreaseafter solution in water with half-lifeThe biological consequences of sloimean that different investigatorEapparently confficting results, depetime elapsed between the preparati(tion and the biological testing.

Mixed (Ternary) Comple.In biological systems metal ions u;

with two different ligands. For exaning of copper in serum by the normamino acids is greater than expectindividual binding properties. It timixed complexes are fonned in whicis bound simultaneously to two diacids. The biological significance of miis becoming more and more recognizeAugust 1981

in the excellent review by Martin and Scharff (15).Perrin (16) employed a simulated plasma solutioncontaining 22 amino acids and, by means of acomputer program, showed that at pH 7.4 most ofthe copper was present as a mixed complex ofCu-histidine-cysteine, and that an appreciable frac-tion of zinc was found as a mixed complex ofZn-cysteine-histidine (Fig. 2).In the presence of two chelants, the metal ion

may formn two independent binary complexes:

L + A +2M = ML + MA (3)

Frequently the number of donor atoms or groups ina multidentate ligand is less than the coordinationnumber of the metal ion. This permits anotherligand to occupy these sites which would otherwisecontain solvent molecules. Formation of the mixedcomplex usually occurs stepwise as follows:

L + A + M = L + A = MLA (4)

I I where ligand L has a higher denticity than A, which8 10 is usually bidentate. However, both ML and MA

are present to some extent, the relative fractions ofeach would depend on Idnetics and relative stabilities.

etive (conditional) Large, highly charged anions such as EDTA4-,Fe(III)EDTA and cannot accommodate two such molecules to givecomretition from ML2. However, even though the hexadentate EDTAken from a figure may occupy all of the coordination sites, another,

lower dentate ligand can displace one or more of thecoordinating carboxyl groups. With Cr(III), the

Further, many EDTA chelate is quinquedentate with a coordinatedxist as such at H20 in the sixth position (18). The coordinated H20mply dissolved is labilized by the free carboxylate of EDTA and)mate, CrO42-, easily replaced by acetate or azide to form they of Cr2O72- as mixed complex Cr(EDTA)Xn+ 1). The displacementd immediately of ligand groups during mixed complex formationof 30 min (5). can lead to important new properties not normnallyw conversions possible in the parent binary complex, e.g., forma-3 may obtain tion of protonated species and ligand-ligand inter-mnding on the actions within the coordination sphere (19).On of the solu- When a second ligand, A, occupies uncoordinated

sites in a binary complex, an important correlationensues, namely that the stability of the mixed

xes ligand chelate is greater, the larger the differencebetween the log K1/K2 values [the Kf in Eq. (2)] ofsually combine the binary complexes (20).nple, the bind- Statistically, a metal ion prefers or favors combi-Lally occurring nation with different ligands rather than two of the;ed from their same (21, 22). However, the stability of some mixedurns out that complexes exceed that expected from the statisticalh a copper ion factor by many orders of magnitude.ifferent amino It is interesting to note that in biological systemsixed complexes the imidazole moiety of histidine enhances the)d as described stability of mixed complexes when an -0- ligand

229

Copper Complexes.)

V.,

Zinc Complexes

In-z

U)

u 4. 6

I>Ic* _

XnU,

zm xa

NJ4 tN

yU)

C-1

NJ

3

NJj.

Cu(ll) and Zn(ll) Complexes of Amino AcidsFIGURE 2. Calculated distribution of Cu(II) and Zn(II) among 22 amino acids; Cu2+ = 2.7 X 10-7M and Zn2+ = 3.3 x 10-6 M. The

amino acid concentrations were those known to exist in blood plasma. SAA is a composite representing alanine + arginine +isoleucine + leucine + valine. The bar graphs were constructed from data tabulated by Perrin and Agarwal (17). Altogether 19copper species were estimated to exist, accounting for 98% of the total copper, while 20 zinc species accounted for 93% of the totalzinc. In the case of copper, about 86% of all the species were mixed ligand complexes, while about 40% of the zinc species weremixed ligand complexes.

atom is involved (19). With synthetic chelants suchas nitrilotriacetic acid (NTA) and imidazole, transi-tion metals form strong mixed complexes. In natu-rally occurring mixed complexes with proteins, theimidazole moeity from histidine together with -0-

donor atoms is ubiquitous (e.g., hemoproteins andthe non-heme hemocyanins).

Oxidation-ReductionChelation serves to stabilize that oxidation state

of a metal ion forming the most stable chelate withthe particular chelant. For example, an unusualoxidation state of Ag2+ can be stabilized by chela-tion with 2,2'-dipyridyl and that ofNi3 + by chelationwith o-phenylenedimethylarsine (11). The stabilityof a metal complex to oxidation-reduction is afunction of the stability of the metal complex. Theseare important considerations in biological systemswhen it is desired to use or maintain a givenoxidation state.

Biological Consequences andApplications

I have already alluded to several biological roles230

of chelation. In this section I will cite severaladditional ones useful for biomedical purposes.

Lipophilicity and IntracellularTransport

In the classical work of Albert on the anti-microbial activity of the Fe(II) and Cu(II) chelatesof 8-hydroxyquinoline and their derivatives (23),only the neutral 1:2 chelate was able to passthrough the cell membrane. Within the cell the 1:2chelate dissociated into the 1:1 chelate which wasthe actual antibacterial agent, presumably by forminga ternary complex with essential enzymes. Similarly,with Fe(III), only the neutral 1:3 chelate couldpenetrate the cell, where again dissociation tookplace. It is possible, as Gershon and co-workershave shown (24), to replace one of the 8-hydro-xyquinolines in the 1:2 chelate of copper withbidentate arylhydroxy acids such as salicylate. Theresulting mixed ligand complex is also neutral andantibacterial. The cobalt(II)-EDTA chelate can com-bine with cyanide, and, in fact, the mixed complexhas been recommended and used as an antidotecyanide poisoning (16).As a means of enhancing intracellular penetra-

Environmental Health Perspectives

I

-)

° 200

-

G)Q 1

o

I .-

tion by synthetic chelating agents, esterification ofthe functional groupings, such as the carboxyls inEDTA, have been accomplished on the assumptionthat the active ligand groupings within the cell arereleased by hydrolysis of the ester. In fact, suchcompounds have been effective in removing heavymetals such as Ce, Y, and Pu from the liver.Unfortunately, these esterified compounds have alow therapeutic index (14).

It is feasible to use metabolic inhibitors to jammetabolic reactions such as the tricarboxylic acidcycle. For example, Pb reacts with citrate to form amoderately strong chelate but parenteral adminis-tration of citrate has little effect because of therapid metabolism of the citrate. When sublethaldoses of sodium fluoroacetate are given to rats, thecitrate levels increase 20-fold or more within thespleen, kidney, and liver. In fact, fluoroacetateadministration caused a small but significant (15%)decrease in the acute toxicity of Pb (25).

Catalase-like ActivityThe decomposition ofH202 is catalyzed by chelated

metal ions though the rate is not as efficient as theiron-containing enzyme, catalase, in terms of thenumber of molecules of H202 decomposed per unittime. The mechanism of catalysis by syntheticchelates, which is far greater than that due to theuncomplexed metal ion, utilize the chelating prop-erties of peroxide which acts as a bidentate ligand,HOO-, as described by Wang (26). Copper chelatessuch as Cu - histamine and Cu - histidine are goodcatalysts at pH 7. The mechanism involves againthe chelating property of HOO-. It was shown (27)that two adjacent coordination sites of the Cu(II)must be available. Hence, if the quadridentatecopper is chelated by a tridentate or tetradentatechelant, no catalytic property is observed.

Viral InteractionsMetal chelates which contain vacant coordination

sites appear to combine with a sulfur bridge of theprotein coating of a virus. This, as proposed byEichhorn (28) changes cleavage, enabling the DNAto leave the virus (15). Virus T2 bacteriophageinfect E. coli by a similar mechanism. The DNApasses from the phage to the bacteria, an essentialstep in viral replication. Cd(CN)2 appears to act bythe formation of a bond between the metal complexand a sulfur bridge of the protein coating of thevirus. With virus T2 bacteriophage it is believedthat a zinc complex in the cell wall of the bacteriumexists and is bound to a sulfur-containing bond ofthe protein coating which is then destroyed. Note

that these proposed mechanisms involve ternarycomplex formation.

MiscellaneousComplexes and chelates of platinum(II) are prov-

ing to be effective anticancer agents. The cis-dichlorodiammineplatinum, a mixed complex, isfinding widespread clinical applications as describedby Rosenberg (29) in a significant review. Otheractive complexes of Pt include those in which one ofthe ligands is bidentate, such as dichloroethyl-enediammineplatinum. It is conceivable that thesePt compounds interact with DNA via ternarycomplex formation.Mixed complex formation may be utilized also as

a ligand scavenger to transport ligands or hastentheir elimination, as mentioned earlier. This phe-nomenon could, in principle, utilize nontoxic binarychelates for the transport of chemotherapeuticdrugs to target sites, or to detoxify poisonoussubstances including pesticides which possess func-tional groupings capable of chelation or, whichacquire such groupings during metabolism. Of in-terest is the role of metal and ligand scavenging inthe induction or reversal of mutagenic effects (5).

Metals and their complexed forms play importantroles in various diseases (30). For example, copperand copper complexes have been postulated to playa role in inflammatory processes (31). In a series ofpapers, Williams and coworkers (32) have reviewedand analyzed the role of metal complexes in rheu-matoid arthritis, especially those of ternary coppercomplexes.

It is tempting to conclude that the so-called roleof metals in carcinogenesis reflects the transport ofthe binary and ternary complexes of the metals toand from intracellular target sites.

This work was supported by U.S.D.O.E. Contract No.EE-77-S-02-4369.

REFERENCES

1. Schubert, J. Interaction of metals with small molecules inrelation to metal-protein complexes. In: Chemical Specificityin Biological Interactions, F. R. N. Gurd, Ed., AcademicPress, New York, 1954.

2. Wood, J. M. Biological cycles for elements in the environ-ment. Naturwiss. 62: 357 (1975).

3. Martell, A. E. Chemistry of carcinogenic metals. Environ.Health Perspect. 40: 207 (1981).

4. Sillen, L. G., and Martell, A. E., Eds. Stability Constantsof Metal-Ion Complexes. The Chemical Society, London,1971.

5. Gentile, J. M., Hyde, K., and Schubert, J. Chromium

August 1981 231

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6. Clarke, M. J., Bitler, S., Rennert, D., Buchbinder, M., andKelman, A. D. Reduction and subsequent binding of ruthe-nium ions catalyzed by subcellular components. J. Inorg.Biochem. 12: 79 (1980).

7. Schubert, J. Mixed complex formation-new therapeuticapproaches. Trends Pharmacol. Sci., 2: 6, 50 (1981).

8. Colburn, R. W., and Maas, J. W. Adenosine triphosphate-metal-norepinephrine ternary complexes and catecholaminetransport. Nature 208: 37 (1965).

9. Maas, J. W., and Colburn, R. W. Co-ordination chemistryand membrane function with particular reference to thesynapse and catecholamine transport. Nature 208: 41 (1965).

10. Sigel, H., and Amsler, P. E. Hydrolysis of nucleosidephosphates. 6. On the mechanism of the metal ion promoteddephosphorylation of purine nucleoside 5' -triphosphate. J.Am. Chem. Soc. 98: 7390 (1976).

11. Mellor, D. P. Chemistry of chelation and chelating agents.In: The Chelation of Heavy Metals, A. C. Sartorelle, Ed.,Pergamon Press, Oxford, 1975.

12. Schubert, J. The chemical basis of chelation. In: IronMetabolism, F. Gross, Ed., Springer-Verlag, Berlin, 1964.

13. Schubert, J. Heavy metals-toxicity and environmental pol-lution. In: Metal Ions in Biological Systems, S. K. Dhar,Ed., Plenum Press, New York, 1973.

14. Catsch, A., and Harmuth-Hoene, A. E. Pharmacology andtherapeutic applications of agents used in heavy metalpoisoning. In: The Chelation of Heavy Metals, A. C.Sartorelli, Ed., Pergamon Press, Oxford, 1979.

15. Martin, R. P., and Scharff, J. P. Mixed ligand complexesand their biological significance. In: An Introduction toBio-Inorganic Chemistry, D. R. Williams, Ed., C. C.Thomas, Springfield, Ill., 1976.

16. Perrin, D. D. Medicinal chemistry. In: Topics in CurrentChemistry, Vol. 64, Springer-Verlag, Berlin, 1976, pp.181-217.

17. Perrin, D. D., and Agarwal, R. P. Multimetal-multiligandequilibria: a model for biological systems. In: Metal Ions inBiological Systems, Vol. 2, Mixed Ligand Complexes, H.Sigel, Ed. Marcel Dekker, New York, 1973.

18. Sulfab, Y., Taylor, R. S., and Sykes, A. G. Rapidequilibration of the ethylenediamine-N,N,N',N'-tetraacetoaquochromate (III) complex with chromate

(VI), molybdate (VI), tungstate (VI), and azide.Labilization of the aquo ligand by the free carboxylateand substitution of chromium (III). Inorg. Chem. 15:2388 (1976).

19. Sigel, H., Fischer, B. E., and Prys, B. Biological implica-tions of the stability of ternary complexes in solution.Mixed-ligand complexes with manganese (II) and other 3dions. J. Am. Chem. Soc. 99: 4489 (1977).

20. Sovago, I., and Gergely, A. Factors influencing the forma-tion of mixed ligand complexes of nickel (II) and zinc (II).Inorg. Chim. Acta 37: 233 (1971).

21. Sharma, V. J., and Schubert, J. Statistical factor in theformation and stability of ternary and mixed complexes, J.Chem. Educ. 46: 506 (1969).

22. Beck, M. T. Chemistry of Complex Equilibria, VanNostrand-Reinhold Co., London, 1970.

23. Albert, A. Selective Toxicity, Chapman and Hall, London,1979.

24. Gershon, H., and Parmegiani, R. Antimicrobial activity of8-quinolinol, its salts with salicylic acid and 3-hydroxy-2-naphthoic acid and the respective copper (II) chelates inliquid culture. Appl. Microbiol. 11: 62 (1963).

25. Fried, J., Rosenthal, M., and Schubert, J. Induced accumu-lation of citrate in therapy of experimental lead poisoning.Proc. Soc. Exptl. Biol. Med. 92: 331 (1956).

26. Wang, J. H. On the detailed mechanism of a new type ofcatalase-like action. J. Am. Chem. Soc. 77: 4715 (1955).

27. Sharma, V. S., and Schubert, J. Catalase activity of metalchelates and mixed ligand complexes in the neutral pHregion, III. Inorg. Chem. 10: 251 (1971).

28. Eichhorn, G. L. Metal ion catalysis in biological systems. In:(Adv. Chem. Ser., Vo.. 37) American Chemical Society,Washington, D.C., 1963, pp. 37-55.

29. Rosenberg, B. Platinum complexes for the treatment ofcancer, Interdiscipl. Sci. Revs. 3: 134 (1978).

30. Kharasch, N., Ed. Trace Metals in Health and Disease,Raven Press, New York, 1979.

31. Schubert, J. The relationship of metal-binding to salicylateaction. In: Metal-Binding in Medicine, M. J. Seven, Ed., J.B. Lippincott, Philadelphia, 1960.

32. Jackson, G. E., May, P. M., and Williams, D. R. Metal-ligand complexes involved in rheumatoid arthritis-VI. J.Inorg. Nucl. Chem. 40: 1227 (1978).

232 Environmental Health Perspectives