CANCER BIOTHERAPY & RADIOPHARMACEUTICALSVolume 18, Number 3, 2003© Mary Ann Liebert, Inc.

Radiotoxicity of Iodine-125 and Other Auger-Electron-Emitting Radionuclides: Background toTherapy

S. James Adelstein,1 Amin I. Kassis,1 Lisa Bodei,2 and Giuliano Mariani31Experimental Nuclear Medicine, Department of Radiology, Harvard Medical School, Boston,Massachusetts; 2Nuclear Medicine Division, European Institute of Oncology, Milan, Italy; 3RegionalCenter of Nuclear Medicine, University of Pisa Medical School, Pisa, Italy

ABSTRACT

Auger-electron cascades with their ability to deposit energy in extremely small volumes, typically in therange of cubic nanometers, have served as valuable probes of radiobiologic phenomena. Results from theirexperimental use form part of the evidence that nuclear DNA is the most radiosensitive cell element; thatchromosomal aberrations and large scale double-strand breaks are correlated with reproductive survival;that neoplastic transformation and also mutagenesis are greatest at low doses with high specific ionization;and that, like high linear-energy-transfer radiation, Auger-electron cascades can lead to bystander effects.We have also learned that radiobiologic responses to Auger-electron emission are particularly sensitive tothe site of decay, not only within the cell but also in the nucleus within the fine structure of chromatin.

Key Words: Auger-electron emitters, radionuclide therapy, biologic effects, DNA damage

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INTRODUCTION

The effects elicited by Auger-electron cascadesconstitute one of the most dramatic radiobiologicevents, with intriguing implications for the radiotherapy of tumors. From the early 1960s,Auger-electron-emitting radioisotopes have beenexplored for one purpose or another, primarily inexperimental biomedical research settings. At aninfluential conference held in 1975 at the Nu-

clear Research Center in Jülich (organized by L. E. Feinendegen and G. Stöcklin), the majorgroups working in this field met to compare theirfindings and discuss directions for future re-search.1

Peculiar to these radionuclides is their abilityto deposit energy in extremely small volumes,typically in the range of cubic nanometers (Fig.1). Neglected initially for therapeutic purposesbecause of their low energy and consequent shortrange, Auger-electron cascades are now being se-riously considered.

In this personal historical review, we will sum-marize the physics of Auger-electron production,the chemical consequences of Auger-electronemission, the biologic sequelae of this emission,

Address reprint requests to: Giuliano Mariani; Director,Regional Center of Nuclear Medicine, University of PisaMedical School; Via Roma 67, I-56126 Pisa, Italy; Tel.:139-050-993390; Fax: 39-050-5521100E-mail: g.mariani@do.med.unipi.it

and its application to the treatment of cancer inexperimental settings. A subsequent paper willpresent some of the latest findings and speculateon the application of these radionuclides for fu-ture therapeutic use in the clinic.

PHYSICS AND CHEMISTRY

Observed originally by Pierre Auger in 1925,Auger electrons are produced when a vacancy iscreated in the inner electron shell of an atomicspecies.2 This primary vacancy can be induced byelectron capture, internal conversion, or photo-electric interaction with external photons. The re-sidual atom (and, in the case of electron capture,the residual nucleus) is highly excited, and de-ex-citation occurs through a cascade of atomic va-cancies, with the emission of characteristic Augeror Coster–Kronig electrons or atomic x-rays and,in the instance of electron capture, of gamma pho-tons. In the case of iodine-125, for example, theinitial vacancy caused by electron capture from aninner shell with the ensuing Auger-electron cas-cade and the de-excitation of the residual telluriumnucleus with internal conversion lead to the depo-sition in a 20-nm sphere of approximately 2 keVfrom a cascade of about 20 Auger electrons.3,4

The spectrum of emitted electrons is very com-plex and mainly involves electrons with a sub-cellular range of penetration. Detailed MonteCarlo calculations have been performed to deter-mine the spectra emitted by chromium-51, iron-55, selenium-75, bromine-80m, technetium-99m,indium-111, iodine-123, iodine-125, and thallium-201.5,6 Table 1 shows the electron emissions fromiodine-125. The residual atom is highly chargedand extremely electron avid. When incorporated

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Figure 1. Localized energy deposition around iodine-125decay site. (Top) Energy (eV) deposited in 1-nm thick, con-centric spherical shells. (Bottom) Differential profile of theaverage absorbed energy density (eV per nm3). The sharpdrop in the first 5 nm illustrates the highly localized natureof the low-energy electron emissions.

Table 1. Auger,a Coster-Kronig (CK),* and Internal Conversion (IC)† Electrons from the Decay of Iodine-125

Average energy Range(MeV) Yield/Decay (mm)

IC 1 K 3.65E-03 1.91E-01 4.98E-01IC 1 L 3.06E-02 1.10E-01 1.86E101IC M,N 3.41E-02 2.84E-02 2.31E101Auger KLL 2.24E-02 1.38E-01 1.08E101Auger KLX 2.64E-02 5.90E-02 1.43E101Auger KXY 3.02E-02 6.50E-03 1.82E101CK LLX 2.19E-04 2.64E-01 1.02E-02Auger LMM 3.05E-03 1.25E100 3.73E-01Auger LMX 3.67E-03 3.40E-01 5.04E-01Auger LXY 4.34E-03 2.11E-02 6.62E-01CK MMX 1.27E-04 1.44E100 6.43E-03Auger MXY 4.61E-04 3.28E100 2.25E-02CK NNX 2.99E-05 3.51E100 1.15E-03Auger NXY 3.24E-05 1.09E101 1.97E-03CK OOX 6.00E-06 3.66E100 1.50E-04

*Total yield of Auger and CK electrons per decay 5 24.9; Auger and CK energy released per decay 5 12,241 eV.†Total yield of IC electrons per decay 5 0.94; IC energy released per decay 5 7,242 eV.

into simple organic molecules, charge transferleads to molecular disruption and fragmentation.This has been observed with CH3

125I and C2H5125I7 as well as with 5-[125I]iodouracil.8

When iodine-125 affixed to a base is incorporatedinto duplex deoxyribonucleic oligonucleotides, theAuger cascade produces disruption of ,5 base pairson both sides of the decaying atom, in the con-tralateral as well as the ipsilateral strand.9 This de-structive action is probably due to the energy trans-fer of low-energy electrons with a range , 25 nmas well as to the residual electronic excitation of thedecayed atom. Although atomic recoil could play arole in some cases of Auger-electron emission, foriodine-125 the recoil energy is insufficient to breakthe carbon–iodine bond.

In examining the effects of Auger-electronemission on macromolecular DNA, most atten-tion has been focused on the production of sin-gle-strand breaks (SSB) and double-strand breaks(DSB). The earliest observations were made withiodine-125 incorporated into bacteriophage andbacterial DNA.10,11 In these systems, frozen un-der glycerol, it was demonstrated that one iodine-125 decay produced one DSB.

More recent studies have dealt with iodine-125brought into approximation with plasmid DNAthrough a minor-groove-binding dye, Hoechst33342,12,13 or by a triplex-forming oligonucleo-

tide.14 Iodine-125-labeled Hoechst 33342 is acurved, aromatic, dye molecule that docks into theminor groove of duplex DNA bringing the iodine-125 atom within several angstroms of the nucleicacid surface (Fig. 2). The effects of both this dyeand 125I-labeled antipyrine, which has no affinityfor DNA and is distributed freely in solution, havebeen studied in the production of SSB and DSB inpUC19 plasmids, a supercoiled form of DNA. Bothmolecules are equally efficient in producing SSB,but 125I-iodoHoechst 33342 is ,7 times more effi-cient per decay than 125I-iodoantipyrine in produc-ing DSB. When the radical scavenging agent DMSOis added to the solution, it protects against the pro-duction of SSB by both agents, but less so for 125I-iodoHoechst 33342 than for 125I-iodoantipyrine. Onthe other hand, DMSO provides no protectionagainst the production of DSB by 125I-iodoHoechst33342, while it provides ,25-fold protection againstthe production of DSB by 125I-labeled antipyrine(Fig. 3). From these experiments, it appears that io-dine-125 in close proximity to DNA produces oneDSB per decay by a direct mechanism, while radi-cals from the decay of iodine-125 at a distance fromDNA produce DSB indirectly through a SSB inter-mediary, as do gamma photons, and this latter ac-tion is susceptible to radical scavenging.

In the case of DNA packaged as chromatin inmammalian cells, the effect of radical scavengers onthe production of DSB is dramatically different.When iodine-125 is incorporated into cellular DNAby incubating cells with 5-[125I]iodo-29-deoxyuri-dine (125IUdR) and this incubation occurs in the ab-sence or presence of DMSO, there is a striking de-crease in the number of DSB produced in the latterinstance. This is observed both at 3°C and in thefrozen state.15,16 The dose modification factor(DMF) decreases with an increasing number of de-cays and is greatest at low doses. These observa-tions are best explained by postulating two mech-anisms for DSB production: (i) a direct effect at thesite of decay not modulated by DMSO, and (ii) anindirect effect at sites many base pairs distant fromthe site of decay, but sufficiently close because ofthe packaging of DNA in the cellular chromatin tobe within the range of the watery radicals producedby the Auger electrons. Such dual mechanismswould account for the scavenging effect of DMSOon reducing the effectiveness of the watery radi-cals and for the decrease in DMF with increasingnumber of decays as the direct effect predominates.Recent experiments (Walicka, Adelstein and Kas-sis, unpublished results) suggest that whatever themechanisms of DSB production, the DSB result-

303

Figure 2. Pictorial representation of 125I-iodoHoechst33342–DNA complex. The small molecule has its benzimi-dazole (Bz1 and Bz2) rings perpendicular to the plane of theDNA minor groove. The arc-like shape of the iodoHoechst33342 molecule allows it to follow the minor groove wheninteracting with the arched DNA double helix.

ing from DNA-bound iodine-125 are much moredifficult to repair than those produced by low lin-ear-energy-transfer (LET) radiation and a higherfraction is unrepaired in the former case (Fig. 4).

For radioiodine incorporated into DNA, two iso-topes of iodine have been compared by labeling theDNA of V79 cells with 123IUdR or 125IUdR. Therelative number of DSB produced from the decayof these radioisotopes is related to the energy de-posited in 5-nm to 20-nm spheres. Iodine-125 de-

posits approximately twice the energy of iodine-123 in these volumes, while the production of DSBis between 1.3- and 2.2-fold greater, depending onthe degree of DNA repair assumed.17,18

RADIOBIOLOGY

Although the first biologic observations madewith Auger-electron-emitting radionuclides took

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Figure 3. DNA strand breaks produced in plasmid DNA by bound and unbound iodine-125 in the presence and absence of DMSO.Damage produced by iodine-125 decay in pUC19 plasmid DNA from 125I-iodoHoechst 33342 (125IH) (left) and 125I-iodoantipyrine(125IAP) (right) with and without DMSO. (Top) Disappearance of supercoiled DNA. A greater protection with DMSO is observed onirradiation by 125IAP than by 125IH. (Middle) Appearance and disappearance of single-strand breaks (SSB). DMSO reduces the pro-duction of SSB in both instances. The initial slope reduction, however, is greater for irradiation by 125IAP than by 125IH. (Bottom)Production of double-strand breaks (DSB). In the case of irradiation by 125IAP, DMSO markedly curtails the appearance of DSB,which are produced from two proximate SSB. In the case of irradiation by 125IH, DMSO has no influence on DSB production andthe disappearance of DSB is due to fragmentation of DNA. With 125IH, the DSB are produced by direct, nonscavengeable action.

advantage of their low-energy electrons to pro-duce microautoradiographs,19 principal interestwas aroused by their extreme toxicity demon-strated in bacteriophage and bacteria.11,20 Atabout the same time, a number of investigatorsobserved the potent cytotoxic effects of iodine-125 when incorporated into the DNA of mam-malian cells.21–24 Moreover, two striking differ-ences were observed when survival curves foriodine-125 were compared with those for thebeta-particle emitters, iodine-131 and tritium. Forrodent cells, the shoulder on clonogenic survivalcurves observed with low-LET radiation is seenwith iodine-131 and hydrogen-3, while an expo-nential decline, characteristic of high-LET radia-tion, is seen with iodine-125. Moreover, the slopeof the survival curve obtained with iodine-125 ismuch steeper than the final slopes seen with thetwo beta-particle emitters (Fig. 5). In these stud-ies, the mean radioactivity content at 37% sur-vival (D37) for 125IUdR, 131IUdR and tritiatedthymidine (3HTdR) is 3.7, 43.0, and 60.7mBq/cell, respectively.

Three other biologic endpoints have also beenexamined: the production of chromosomal aber-rations, cell transformation, and gene muta-tion.24–26 Chromosomal aberrations are inducedmore effectively by iodine-125 than by iodine-131 or hydrogen-3 (Fig. 6). These include singleor multiple chromatid breaks and chromatid ex-change figures of variable complexity including

those resulting from SSB (fragments, chromatidbreaks) and DSB (dicentrics and rings). Cyto-toxicity is directly related to chromosomal dam-age in all cases with plots of survival versusbreaks per cell (Fig. 7) superimposable for thethree radionuclides.

When the same radionuclides incorporated intoDNA are compared for their ability to induce celltransformation, significant differences are ob-served. At low doses, where cell survival is high,the transformative action of iodine-125 exceedsthat of iodine-131, which, in turn, exceeds that oftritium (Fig. 8). At higher doses and lower cellsurvival, the relations of transformation to cyto-toxicity are parallel. The results suggest that thelarge DNA deletions caused by the decay of in-corporated iodine-125, even at the lowest doses,have a greater initial effect on transformation thando small absorbed doses from the low-LET betaemitters, presumably because the low-LET radi-ation causes more repairable injury (SSB andbase damage).

Much of our information about the mutageniceffects of Auger-electron emitters comes from thelaboratory of John B. Little. Using 125IUdR and3HTdR, he and his colleagues26 examined cyto-toxicity, the production of DSB, and the appear-ance of mutant 6-thioguanine resistance (6TGR)in human lymphoblastoma cells. Table 2 sum-marizes their results. At low doses, 125IUdR ismuch more mutagenic than 3HTdR, with signif-icant increases in mutant fraction occurring withlittle change in survival for the former compound.At higher doses the mutation-fraction versus sur-

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Figure 4. Repair of DNA double-strand breaks. Unrejoineddouble-strand breaks (DSB) produced in V79 cells after 6 hof incubation with 125IUdR for strand scission factors (SSF)reflecting various degrees of damage. For damage caused byDNA-bound iodine-125, ,20% of DSB are unrepaired, whilefor that caused by x-rays, only ,5% are unrepaired (Walicka,Adelstein and Kassis, unpublished data).

Figure 5. Clonogenic survival of V79 cells with DNA-in-corporated iodine-125, iodine-131, and hydrogen-3.

vival-fraction curve has about the same slope asthe x-ray data, reminiscent of the observationswith cell transformation. In the same cell line,125IUdR is also more mutagenic than x-rays and

131IUdR in producing hprt mutants. In a syn-chronized population of CHO cells, it has beenshown that the most susceptible period for the in-duction of hprt mutants occurs while the genomeis replicating in early S phase. Compared with x-rays, the relative biologic effectiveness (RBE)values for iodine-125 incorporated into DNA forcell killing and induction of hprt mutations aremuch higher than those for tritium. With both ra-dionuclides, ,50% of the mutant clones exhibitpartial gene deletions and ,25% are missing atotal gene.27

For all these radiobiologic experiments, the ra-diohalogens were introduced into DNA by incu-bating the cells with 5-radioiodo-29-deoxyuridine(*IUdR), a thymidine analog in which iodine sub-stitutes for the methyl group in position 5 of thepyrimidine ring (Fig. 9). Because the van der Waals’radius of the halogen approximates that of themethyl group, the cellular enzymes incorporate theanalog at the same site and rate as a thymine base.

With cytotoxicity as an endpoint, two other ra-diohalogens, themselves Auger-electron emitters,have been compared with iodine-125, namely,bromine-77, and iodine-123, when they replaceiodine-125 by isomorphous substitution into thethymidine analog.17,18,28–30 Clonogenic survival

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Figure 6. Chromosomal aberrations in V79 cells inducedby DNA-incorporated iodine-125, iodine-131, and hydro-gen-3. Total chromosome breaks (chromatid plus chromo-some type) produced immediately (0 h) or 24 h after re-moval of cells from radioactive medium.

Figure 7. Clonogenic survival of V79 cells as function oftotal chromosome breaks induced by DNA-incorporated io-dine-125, iodine-131, and hydrogen-3. Chromosomal breaksare scored 24 h after removal of cells from radioactive medium.

Surviving fraction

Tran

sfor

man

ts p

er d

ish

1.5

1.2

0.9

0.6

0.3

01.0 0.8 0.6 0.4 0.2

Figure 8. Malignant transformation in BALB/3T3 cells byiodine-125, hydrogen-3, and x- rays. Transformants per dishinduced by 125I-iododeoxyuridine ( d ), 3H-thymidine ( s ),and (x) x-rays.

curves remain exponential but the slopes are shal-lower with D37 (mean lethal dose) of 1.5, 2.6, and4.8 mBq/cell, respectively, for 125IUdR, 123IUdR,and 77BrUdR. However, the energy deposited perdecay in 5-nm spheres is in the ratio of 1000 to560 to 300 eV. Consequently, the energy de-posited per nucleus is approximately the same at37% survival. This correlation suggests that nu-clear recoil, electronic excitation, and chemicaltransmutation are probably of minor importanceto the biologic toxicity observed, as these physi-cal properties differ in all three radionuclides.

The position of iodine-125 in the cell is, how-ever, of critical importance to its radiotoxicity. Itwas recognized early that extracellular iodine-125 is of low toxicity despite its 30 keV photonemission.23 When the mitochondrial stain iodo-rhodamine is labeled with iodine-125 and incor-

porated into the cytoplasm of cultured rodentcells, a clonogenic survival curve with a distinctshoulder and a D37 of 4.6 Gy to the nucleus isfound (Fig. 10).31 While this value compares wellwith the 5.8 Gy x-ray D37, it contrasts with thatof DNA-incorporated 125IUdR, which has a D37of 0.8 Gy to the cell nucleus and demonstratesthe high RBE associated with Auger-electron cas-cades.

When iodine-125 is brought into the nucleus andnot incorporated into DNA, clonogenic survivalcurves may remain exponential, but the cytotoxi-city may be significantly reduced. This can be seendramatically in the case of 125I-labeled tamoxifenwhen it is compared with 125IUdR and the ubiq-uitous 125I-antipyrine (distributed as is cell water)(Fig. 11).32 The iodotamoxifen is bound to estro-gen receptors in the cell nucleus of MCF7 cells,certainly positioning its radioiodine atom differ-ently than that of IUdR. Similar observations have been made with the DNA intercalating agent3-acetamido-5-iodoproflavine (A125IP), where theRBE is approximately two-thirds that of 125IUdR.33

On the other hand, Yasui and her colleagues34 haveshown in MCF7 cells that an iodine-125-labeledestrogen, E-17a[125I]iodovinyl-11bmethoxyestra-diol (125IVME2), is equally as cytotoxic per decayas 125IUdR. 125IVME2 is more efficient at pro-

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Table 2. Mutagenic Effects of 125IUdR Compared withThose of 3HTdR in Human Lymphoblastoma Cells

125IUdR 3HTdR

D0 (decays per cell) 28 3856TGR mutations per decay 1 3 1026 2 3 1028

DSB per decay 6 0.6

Figure 9. Structures of (A) 5–iodo-29-deoxyuridine and (B) thymidine.

A

B

ducing DSB than is 125IUdR by a factor of eight.It is unclear whether this difference is due to thepositioning of the radioiodine atom with regard tothe nuclear matrix or whether the degree of dam-age caused by the decay of iodine-125 in 125IUdR

results in a degree of fragmentation in DNA notseen with 125IVME2.

The mutagenic effects of A125IP at the hprt lo-cus have also been studied.35 In the lymphoblas-tic line examined, compared with 125IUdR, muta-genicity and cytotoxicity due to the intercalatingagent are 20 to 25%. Both 125IUdR and A125IP in-duce large-scale genetic effects (partial or com-plete deletions) at the hprt locus, as would be ex-pected from the high-LET-like properties of theAuger-electron cascades.

Other nonhalogen, Auger-electron-emitting ra-dionuclides exhibit the same effects.36–39 When

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Figure 10. Clonogenic survival of V79 cells as functionof intracellular concentration of mitochondrial 125I-iododi-hydrorhodamine (——) and nuclear 125I-iododeoxyuridine(– – –).

Figure 11. Clonogenic survival of MCF7 cells exposed to125I-iodotamoxifen (125ITAM), 125I-iodoantipyrine (125IAP),and 125I-iododeoxyuridine (125IUdR). 125ITAM cytotoxic-ity is differentiated between that associated with the wholecell (125ITAMwc) and that precipitable with DNA (125ITA-Mppt).

Figure 13. Clonogenic survival of V79 cells exposed to 125I-iododeoxyuridine and various concentrations of DMSO. Cellswere labeled with 125IUdR and accumulated decays at 2135°C.

Figure 12. Clonogenic survival of V79 cells exposed to various agents labeled with Auger-electron-emittingradionuclides (125I-DR 5 iodorhodamine, A125IP 5 3–ac-etamido-5–iodoproflavine, 123I/125IUdR 5 iododeoxyuri-dine, 77BrUdR 5 bromodeoxyuridine). The biologic effec-tiveness relative to x-ray is given.

located in the nucleus, they show a high degreeof toxicity, while in the cytoplasm or extracellu-lar, their action is like other low-LET radiation.Of particular interest is the DNA binding agentplatinum-195m as trans-platinum(II). Decay ofthe radionuclide produces an exponential survivalcurve whose RBE approximates that of A125IP.40

A comparison of the various survival curves isshown in Figure 12.

Recent experiments have shed light on thephysical mechanisms responsible for iodine-125toxicity when the radionuclide is incorporatedinto DNA.41–44 At 4°C, the radical scavengerDMSO (10%) reduces the D0 of 125IUdR in V79cells fivefold. When experiments are conductedat 2135°C so that a full range of DMSO con-centrations can be employed, the contributions ofdirect and indirect (radical-mediated) effects canbe quantified. At extrapolated, infinite DMSOconcentration (direct effects only) the D0 is 54 65 decays per cell; at zero DMSO concentration(both direct plus indirect effects), the D0 is 411 636 decays per cell (Fig. 13). From these results({1 2 (54/411) 5 0.87}), it can be inferred that,90% of the cytotoxicity is due to indirect ef-fects. As in the case of DSB production (seePhysics and Chemistry above), we interpret thisto imply that radicals produced at the iodine de-cay site can diffuse to base pairs at some distancealong the chromosome, but close to the site ofdecay because of chromatin packaging.

For the past several years, it has been knownthat high-LET radiation, in the form of alpha par-ticles, deposited in one cell could produce cyto-

toxicity, cell transformation, and mutations in aneighboring cell.45 Recently, using an in vitro–invivo system, we have demonstrated this bystandereffect in cells prelabeled with 125IUdR.46 WhenLS174T colon adenocarcinoma cells lethally la-beled with 125IUdR are injected subcutaneously

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Figure 14. Treatment of mouse ovarian ascites tumor with 125I-iododeoxyuridine. 1 3 104 tumor cells alone ( s ); tumor cellsplus 4.6 MBq 125IUdR at 24, 36, 48, and 60 h (j ); tumor cells plus 4.6 MBq 125IUdR at 24, 28, 32, and 36 h (m ). The lastschedule matches the cell cycle of the tumor cells and labels all the cells.

Figure 15. Survival of mouse ovarian tumor ascites treatedwith 125I/131I-iododeoxyuridine in vivo. Treatment was started24 h after intraperitoneal inoculation of 1 3 106 tumor cells.Four or seven doses were given at 4 h intervals. 131IUdRshows no antineoplastic activity (131I decays by beta–gammaemission with little yield of Auger or conversion electrons).Seven doses of 555 kBq 125IUdR reduce tumor cell survivalto 1 3 1025.

into mice, they do not grow into tumors. Whenunlabeled cells are injected they do. Whenlethally labeled cells are co-injected with live un-labeled cells, the latter are inhibited in theirgrowth. With an increasing fraction of labeledcells, the degree of inhibition reaches a limitingvalue at about 60% in 15 days. Dosimetric con-siderations have shown that this inhibition of tu-mor growth is not due to direct irradiation of un-labeled cells by the injected iodine-125. Instead,it appears to be due to inhibitory action of theneighboring labeled cells by an as yet to be de-termined mechanism. This phenomenon could beof significance for radionuclide therapy with io-dine-125 as indicated below.

EXPERIMENTAL THERAPY

In addition to their usefulness as biophysical probesof radiation mechanisms, the extreme radiotoxicityof Auger-electron-emitting radionuclides invitestheir application in radiotherapy for cancer. In thespectrum of particles used in experimental ra-dionuclide therapy, Auger-electron emitters withtheir ranges in nanometers are the most fastidiousin terms of placement within radiosensitive nucleartargets with little cross-fire (cell-to-cell) irradiation.Alpha particles have ranges in micrometers andbeta particles in millimeters so that, in their use,there is no requirement for all cells in a tumor tobe labeled. (On the other hand, if the bystander ef-fect described above contributes significantly to cy-totoxicity, this requirement for Auger-electronemitters may be dropped as well.)

Most experimental therapy with Auger-elec-

tron cascades has used 125IUdR. Unfortunately,the direct use of radiohalogenated pyrimidine nu-cleosides presents some formidable problems.The agents are cell-cycle dependent and incor-porated into DNA at the time of DNA synthesis(S phase) only. Moreover, the nucleosides are po-tentially incorporated into the DNA of all prolif-erating cells in addition to that of tumors. Fur-thermore, efficient hepatic dehalogenation limitsthe residence time of the intact compound in thecirculation. Thus, three conditions must be metfor 125IUdR to be practically useful. First, the tar-geted tumor cells should be in a region that is iso-lated and can be directly accessed. Second, oncewithin the vicinity of tumor cells, the agentshould be freely diffusible and available. Third,

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Figure 16. Survival of rats bearing 9L gliosarcoma brain tumors following treatment with 125I-iododeoxyuridine.

Figure 17. Photomicrograph of spinal cord from rat bear-ing implanted 9L gliosarcoma. The catheter is surrounded bytumor which is, in turn, surrounded by normal spinal cord.

when the agent has left the target region it shouldbe converted rapidly into an inactive form andthe radioactivity should be excreted from thebody. These requirements can be met theoreti-cally by locoregionally directed administration of125IUdR to accessible body spaces such as theperitoneum, brain, spinal fluid, and bladder. Allfour sites have been explored.

Our first studies were with an ovarian ascitestumor in the mouse.47–51 At an early stage, thesetransplantable cells are all in cycle and confinedto the peritoneum. After intraperitoneal injectionof 123I/125IUdR, the cells are bathed with the ra-dioactive agent; when the agent escapes from theperitoneum and enters the circulation, it is rapidlydeiodinated, and the radionuclide is either ex-creted or taken up by the thyroid gland (if the lat-ter is not blocked by cold iodide). Scintigramsobtained after the i.p. injection of 123IUdR show

that in a normal mouse within 24 h most of theradionuclide has left the peritoneum, whereas ina mouse with ascites the radionuclide is taken upby the tumor cells and retained in the peritoneum.From an analysis of the cell cycle, a treatmentschedule has been devised that would label all ofthe tumor cells. Survival curves obtained after125IUdR administration to mice with ascites showthat mean survival time is prolonged in the ap-propriately treated group and that about 35% ofthe animals have been cured (Fig. 14). Becausein this tumor there is a regular relation betweensurvival time and number of tumor cells inocu-lum injected, it is possible to transpose the datainto tumor-cell survival fractions as a function ofadministered dose (Fig. 15). With seven injec-tions at 4-hour intervals, tumor-cell survival is re-duced nearly five logs, sufficient to be in the cu-rative range. As shown, 131IUdR has little-to-no

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Table 3. Efficacy of 125IUdR Intrathecal Administration in Treatment of Leptomeningeal Metastases in Rats

Median paralysisTreatment (days) Survival fraction*

Saline 9.0 6 0.1 10.Single injection 11.2 6 0.1 0.6Five daily injections 12.3 6 0.1 0.4Five-day continuous infusion 15.2 6 0.4 0.1

*Survival fraction refers to fraction of tumor cells surviving, a calculation made possible because of a regular relationship be-tween number of cells injected and time of appearance of paralysis.

Figure 18. Biodistribution (left) and spinal cord activity (right) in rats bearing intrathecal 9L gliosarcoma and control rats fol-lowing intrathecal injection of 125I-iododeoxyuridine (370 kBq). On the right, distance is from the top of the cervical spine.

therapeutic effect. Results similar to 125IUdRhave been obtained with 123IUdR. As expected,the 123I-labeled compound is somewhat less ef-fective on a becquerel-per-becquerel basis, butthe short half-life of iodine-123 (13.2 h) com-pared with that of iodine-125 (60 days) couldmake the former more attractive for clinical pur-poses. In these experiments, little uptake of ra-dioiodine is found in normal proliferating tissues,

and no evidence is found for bone marrow tox-icity at therapeutically effective doses.

Locoregional therapy has also been attemptedinside the blood–brain barrier. In the first instance,intratumoral injection was made in a rat 9L gliosar-coma model.52,53 Scintigraphy demonstrated thatradioactivity is retained over a long period of timeafter the injection of 123IUdR into the tumor. Theamount of iodine-123 retained by the tumor cellsis so high that even 0.5-mm gliosarcomas can bevisualized. No radioactivity is retained by normalbrain. Autoradiographic studies with 125IUdRshow silver grains associated only with tumor cellsand none over normal brain tissue. Even clustersof tumor cells distant from the primary site con-tain radioactivity, and injection in one hemisphereof the rat can label tumor cells in the opposite lobe.With the thyroid gland blocked by potassium io-dide, radioactivity outside of the CNS is foundonly in urine and stomach contents, indicating thatlittle 125IUdR enters the circulation; radioiodine,mostly in the form of iodide, is excreted by thekidney and biliary systems. We conclude fromthese observations that 123I/125IUdR, injected di-rectly, diffuses freely throughout the rat brain andis taken up preferentially by dividing tumor cellsin the brain and not by normal cells in the brainand body.

Control animals given 127IUdR, i.e., nonra-dioactive agent, have been followed along with

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Figure 20. Onset of paralysis in rats with spinal cord tumors treated with 125I-iododeoxyuridine (74 MBq) 6 methotrexate. Sin-gle treatment (short dash) ; two treatments (long dash).

Figure 19. Radioactivity associated with spinal cord ofnormal (NT) and tumor-bearing rats (T) after intrathecal in-jection of 125I-iododeoxyuridine 6 methotrexate. TE671human rhabdomyosarcoma cells were implanted by cathe-ter in the spinal cords of immunodeficient rats. Animals wereinfused with 0.1 mM methotrexate (MTX) for 24 h, injectedwith ,37 kBq370 125IUdR, and infused again with MTX.

125IUdR-treated ones. All the control animals dieby day 19 with a median survival of 17 days,while the treated rats have a median survival of24 days with 20% living at 60 days (Fig. 16). Thisresult suggests a possible role for 123I/125IUdR insterilizing clusters of tumor cells left behind af-ter surgical removal of primary brain tumors.

The same 9L gliosarcoma has been implantedinto the spinal cord of rats (Fig. 17). Subsequentintrathecal administration of 125IUdR has led toconcentration of the radioactivity at the site of thetumor (Fig. 18). When 500,000 cells are im-planted, untreated animals developed hind-limbparalysis in 9 days and die 3 days later. At 3 daysafter gliosarcoma implantation, introduction of18.5 MBq 125IUdR by various treatment regi-mens produces the results in Table 3.

Although positive results are obtained, it isclear that greater effectiveness would be re-quired, to be achieved either by dose escalation,prolongation of treatment, and/or pharmaceuti-cal manipulation. In pursuit of the latter, wehave turned to an alternative model of neoplas-tic meningitis using TE671 human rhab-domyosarcoma cells. These cells, when intro-duced into the spinal cavity of immunodeficientrats, form tumors along the leptomeninges. Af-ter pretreatment with methotrexate (MTX), in-cubation of these cells in vitro with 125IUdRleads to an increased uptake of 125IUdR per celland an increased percentage of cells taking upthe agent. Methotrexate, the standard for treat-ment of neoplastic meningitis in humans, in-creases the uptake of IUdR by blocking the in-trinsic (de novo) thymidine-phosphate pathwayas well as the dehalogenation of IUdMP (thefirst intermediate of the incorporation of IUdRinto DNA), and by interfering with cell divi-

sion, thereby increasing the fraction of cells inS phase. In the rat, when intrathecal MTX isadded to 125IUdR, the radioactivity found inspinal cord tumors is doubled (Fig. 19).

The combination of MTX–125IUdR (74 MBq)prolongs the onset of paralysis from 22 to 45days, while 125IUdR alone prolongs the onset to39 days (Fig. 20). Administration of two succes-sive MTX–125IUdR (74 MBq) cycles extends theparalysis-free period to 82 days with 30% cure(Fig. 20), while six successive cycles at 7.4 MBqextend the paralysis-free period to 90 days. Build-ing on standard therapy, these results promise anincremental improvement in what is now arapidly and uniformly fatal disease.

PROSPECTS

The major conclusions from studies included inthis paper are summarized in Table 4. Auger-electron cascades have served as valuable probesof radiobiologic phenomena. Results from theirexperimental use form part of the evidence thatnuclear DNA is the most radiosensitive cell ele-ment; that chromosomal aberrations and large-scale DSB production are correlated with repro-ductive survival; that neoplastic transformationand also mutagenesis are greatest at low doseswith high specific ionization; and that, like high-LET radiation, Auger-electron cascades can leadto bystander effects. We have also learned thatradiobiologic responses to Auger-electron emis-sion are particularly sensitive to the site of de-cay, not only within the cell but also within thenucleus in the fine structure of chromatin.

The exquisite and fastidious cytotoxicity re-sulting from properly positioned Auger-electron

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Table 4. Summary of Findings on DNA-Incorporated Iodine-125

Study Current view

Double-strand break formationNaked DNA Direct effectsCells Mostly indirect effects

Double-strand breaks per 125I decayNaked DNA 1Cells .1

Toxicity in mammalian cells High-LET clonogenic survival curveMostly scavengeable (indirect) effectNot limited to labeled cells (bystander effect)

Mutagenicity Greater than x-ray (at high survival)Transformation Greater than x-ray (at high survival)Therapeutic potential Very high

emitters makes the use of these radionuclides par-ticularly attractive for targeted therapy at the cel-lular level. This has been well demonstrated inanimal models. Translation of the promise intothe clinic remains a continuing challenge.

ACKNOWLEDGMENTS

The work reported herein has been supported inlarge part by NIH grant 5 R01 CA15523.

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Lisa Bodei, receivedher medical educationat the University of Pisaand completed her studythere for a postgraduatespecialty in nuclearmedicine in 1999. She iscurrently a staff physi-cian at the Division ofNuclear Medicine, Eu-

ropean Institute of Oncology, Milan.

Giuliano Mariani, re-ceived his medical de-gree from the CatholicUniversity of the Sa-cred Heart in Rome.He became affiliatedwith the Adelstein /Kassis group when heserved as the Italianattache for science and

technology in Boston between 1986 and 1990 andhas extended the findings of the laboratory to theclinic. He is the director of both the RegionalCenter of Nuclear Medicine and the SpecialtySchool in Nuclear Medicine at the University ofPisa Medical School.

S. James Adelstein com-pleted his medical edu-cation at Harvard Me-dical School and wenton to study biophysicsat the MassachusettsInstitute of Technol-ogy. He is a nuclearmedical specialist whohas had a long-stand-

ing interest in the biophysics and therapeutic ap-plications of Auger-electron cascades. Currently,he is a member of the Biological and Environ-mental Research Advisory Committee of the De-partment of Energy and Chairman of the Boardon Radiation Effects Research of the NationalAcademy of Sciences.

Amin I. Kassis, joinedDr. Adelstein’s labora-tory group at HarvardMedical School shortlyafter receiving his de-gree from McGill Uni-versity in 1976. His ma-jor focus has been theradiobiologic effects ofdiagnostic radiophar-

maceuticals, dosimetric estimations at the micro-scopic level, and the development of radiolabeledcompounds for cancer diagnosis and therapy. Heis the director of the Radiobiology and Experi-mental Radionuclide Therapy Section of the Lab-oratory for Experimental Nuclear Medicine.

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About the Authors