[CANCER RESEARCH 33, 2588-2595, November 1973]

Paramagnetic Changes during Development of a TransplantedAKR/J Leukemia in Mice as Measured by Electron SpinResonancel

Harold M. Swartz, Colin Mailer, Sunandini Ambegaonkar, William E. Antholine,Donald R. McNellis, and Stuart J. Schneller

Radiation Biology and Biophysics Laboratory of the Department of Radiology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226

SUMMARY

Paramagnetic changes occurring during tumor development were studied in the mouse AKR leukemia (transplanted) model system using electron spin resonance. Paramagnetic changes were found only in blood and in organsdirectly involved by the leukemic cells. The change inblood consisted of an increase in a paramagnetic transitionelement complex, and occurred prior to the tissue changes.In the liver, both free radicals and normally occurring transition elements decreased when the liver became extensively infiltrated by the leukemic cells. Similar results werefound in the spleen, except that in the spleen there also wasan increase of a paramagnetic transition element or elements, with an apparent g factor near g = 2.003, plus thedevelopment of additional hyperfine structure in proportion to tumor involvement. An early rise in free radicals,which has been reported as characteristic of tumor development, was not observed in these studies.

INTRODUCTION

ESR2 studies in cancer research were initially inspiredby theoretical speculations regarding the carcinogenic potential of free radicals (2, 5) and by the fact that someprocesses that induce free radicals are carcinogenic (8).Other related theories which involve free radicals are thatthe rate of cell division is inversely related to the level ofintracellular free radicals (3) and that the active formsof some carcinogens are free radicals (27). The first experimental ESR results revealed that most neoplastic tissues had decreased free radical concentrations (6, 10, 15,28). Subsequent studies of human tumors and of spontaneous and experimental animal tumors confirmed thatmost tumor tissues have few free radicals (7, 11, 12, 22,30).

A few exceptions to the general finding of decreased freeradicals in tumors have been reported. Some of these aredue to increased melanin, which is a stable free radical

'These studies were supported by research funds from the JohnHartford Foundation and Oshkosh Area United Fund.

2The abbreviations used are: ESR, electron spin resonance; DPPH.2,2-diphenylpicrylhydrazyl.

Received February 15, 1973; accepted July 11, 1973.

(14). In some other studies a true increase in metabolicallyactive free radicals have been reported. Pavlova andLivenson (17) noted increased ESR signals in leukocytes ofpatients with chronic lymphoid leukemia, but noted normal levels in leukocytes of patients with acute leukemia orchronic myeloid leukemia. Saprin et al. (19-21) reportthat a significant increase in free radicals is characteristicof early tumor growth, with free radical levels decreasingto below normal as the tumor matures. They have observedthis pattern in a number of tumor systems, including amouse La cell leukemia. Chumakov et al. (4) also reported early free radical increases in livers and kidneys ofmice with AKR leukemia. This pattern has not been observed by most other workers, but the latter studies wereprimarily on chemically induced hepatomas (7, 11, 30). Anadditional difference has been the use of lybphilized specimens by Saprin et al. (19-21) and Chumakov et al. (4). Itis now well established (see Ref. 9 for a recent review) thatfree radical signals are produced by interaction of lyophi-lized tissues with oxygen.

Although hypothesized free radical changes providedthe initial experimental impetus, it soon became apparentthat non-free radical ESR signals (from paramagnetic traceelements) also change in carcinogenesis. One of the earliestfindings was that the amount and type of paramagnetictrace elements in mature tumors differed from corresponding normal tissues (15, 16). In studies of chemicallyinduced hepatomas, a new ESR signal was noted prior tothe onset of other biochemical or histological indicationsof carcinogenesis (11, 29, 30).

To provide additional data on paramagnetic changesduring tumor development, we report here a study of thetime dependence of ESR signals from an experimentalAKR mouse leukemia using nonlyophilized, quickly frozenwhole tissues.

MATERIALS AND METHODS

Mice. Six-week-old AKR mice (The Jackson Laboratory, Bar Harbor, Maine) were given i.v. injections of IO3AKR/J leukemia cells from spleens of leukemic mice onDay 0. In a subsidiary experiment (see "Discussion"), the

number of cells injected into groups of 6 mice varied fromIO2 to IO6. Chart 1 summarizes the time course of the

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Paramagnetic Changes in A KR Leukemia by ES R

LIVE fi WEIGHTS

20

03

{02

SPLEEN WEIGHTS

0 5 IO ISDAYS

THYMUS WEIGHTS

i 10DAYS

30 Q AKR/J (H w* 1 mie«rtctivtttt io' BW SH7 Diasialls IV

30

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Chart I. Organ weights and mortality data for experimental AKRleukemia; IO3 blast cells injected i.v. at Day 0. Mortality data from 30

disease and weight changes in liver, spleen, and thymus.The spleen enlarges as the disease develops, and its weightis considered the most reliable indicator of the extent ofthe disease (1). The leukemic cells are found primarily inthe spleen and do not appear in the peripheral blood atleast until the last 2 days before death. The leukemic animals were sacrificed periodically throughout the experiment, 6/day. A control group of mice given infections ofnonleukemic spleen cells was also sacrificed, 4 to 6 animals/day.

Sample Preparation. Samples were taken of whole blood,spleen, liver, thymus, muscle, and brain from both control and leukemic animals. The technique used for thesolid tissues was to remove the organ, weigh it, insert thetissue into a 4-mm (inside diameter) glass tube by gentlepressure, and then immediately freeze the tube and tissuein liquid nitrogen. Blood samples were taken by syringefrom the heart; a 4-mm glass tube was filled and thenfrozen. The total time between removal of the organ andfreezing was about 5 min. To run the samples in the ESRspectrometer, the frozen tissue or blood icicles were removed from the glass tubes by warming the surface justsufficiently to allow the now-cylindrical sample to bepushed into a narrow-tailed Dewar flask full of liquidnitrogen. The Dewar flask was then placed in the spectrometer resonant cavity.

ESR Spectrometer. We used a Varian Associates ModelE-9 ESR spectrometer operating at 9.29 GHz. The resonant cavity was a Varian TE104 dual-sample cavity. Cavity1 of the dual cavity contained a spin and g-factor standard,DPPH in benzene, and Cavity 2 contained the narrow-tailed Dewar flask and the sample. Once the sample wasin Cavity 2, the variable coupler matched the cavity tothe waveguide and the spectrometer was set to operate inthe absorption mode.

Each sample of tissue was examined at several different microwave power levels to determine the power-saturation characteristics of its paramagnetic components. Saturation occurs when the observed ESR signal height doesnot increase in proportion to the square root of incidentmicrowave power. This takes place when the paramagneticspecies absorb energy faster than they can release it, resulting in an increased number of spins in the upper level.Each paramagnetic species has a characteristic rate atwhich it releases energy to its surroundings (the spin-lattice relaxation time), and this can be helpful in identifying classes or specific types of paramagnetic species.Frozen tissues have 2 main types of paramagnetic species:transition metal ions (with very short spin-lattice relaxation times) and free radicals (with relatively long relaxation times). Power-saturation studies, combined with g-factor determinations, provide an excellent means ofdifferentiating between these 2 types of spin species (25).For our tissues, from normal and leukemic mice, we foundon the basis of complete power-saturation studies of representative samples that we could characterize most samplesby obtaining ESR spectra in the following order: (a) a high-power (200-mW) spectrum primarily for observation ofthe transition metal ion signals [most tissue free radicalsignals are saturated at 77°Kwith such a high power,

while transition ions are not (25)]; (b) the DPPH standardto give a check on the sensitivity of the spectrometer fromsample to sample and to provide a g-factor standard(2.0036) for calculation of the experimentally observed gfactor; (c) a low-power spectrum, taken at 0.1 mW to observe any free radical signals. At such a power, the transition metal ion signals have decreased to about one-thirtiethof their high-power amplitude, while that of the now-un-saturated free radical signal has increased from its 200-mW value. Complete power studies of the ESR signalsshowed that, at 0.1 mW, the free radical signal at g =2.0036 was unsaturated.

In most cases the samples were large enough that theESR-sensitive volume in Cavity 2 (~2.0-cm long by 0.4cm in diameter) was completely filled with sample. Whenit was not filled, a correction factor, determined with standard samples of known length, was applied to adjust theobserved signal amplitude to its "100% filled" value.

Analysis. The spectra (microwave power absorbed versusmagnetic field) were recorded as 1st derivatives of the ESRabsorption lines. Estimates of the amount of absorptionwere obtained by measuring the height of the extremes ofthe 1st derivative display (maximum positive deflectionto maximum negative deflection). This is a valid measureof relative absorption intensities of different samples provided the compared line widths and line shapes are thesame (18).

In most instances, true g-factor determinations could notbe made because of the complex shape of the spectra(mainly due to overlapping signals). In this paper we usethe convention of "apparent" g-factor i.e., the empiricallyobserved cross-over (zero) point of the derivative curves.On all of the spectra, the positions where g-factors andsignal heights were measured are marked.

Means, standard deviations, and standard errors of the

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Swartz el al.

means are tabulated or plotted out in "Results." Student's/-test was applied to test the significance of the controland experimental means, the limits of significance beingat the 5% level. All the data sets were also tested statistically to determine whether the data points were distributednormally about their mean values. It was found that noneof the distributions differed significantly (at the 5% level)from a normal distribution.

RESULTS

The experimental results can be divided into 2 groups,those organs that had no statistically significant difference between ESR spectra from control and leukemicmice, and those that did. Brain, thymus, and muscle arein the 1st class; liver, spleen, and blood are in the 2nd.

Brain, Thymus, and Muscle

Representative ESR spectra obtained from brain, thymus, and skeletal muscle are shown in Charts 2 and 3.Qualitatively and quantitatively, the spectra did not change

during the course of the leukemia. The apparent gfactors, mean signal heights (calculated at the pointsindicated in Charts 2 and 3), and standard deviations forcontrol and experimental peaks are summarized in Table1. A r test between the mean signal heights of the controland leukemic samples showed no significant difference atthe 5% level.

Spleen, Liver, and Blood

Spleen. The ESR changes observed in the spleen werecomplex, with overlapping signals that could not be resolved into pure lorentzian or guassian line shapes.

Representative spectra are shown in Chart 4. Although

IIUKÃŒMIA tHYMUi

A CONTICI »AIN».07« r053 2jq035 1.95t I »33

1.105 2.079 1.053 1.011 1.0035 1.979 1956 I.9I3 1991 Ili«

Chart 3. Representative ESR spectra from thymus and muscle ofleukemic mice. Conditions as for Chart 2, except thymus modulation,5 gauss.

Table 1Peak-lo-peak ESR signal heights for brain, thymus, and muscle

Chart 2. ESR spectra from brains of mice on Day 9. Experimentalconditions: The microwave frequency was 9.29 GH¿;incident power levelas stated; sample temperature, 77°K;sweep rate, 400 gauss in 16 min;

100 kHz modulation, 8 gauss; time constant, 3 sec. The DPPH in benzene is a Cavity 1 and the sample is in a Varian finger Dewar flask inCavity 2 of a Varian TE104 dual-sample cavity. The g-factor scale appears above the DPPH signal in B. The gains are the same in both spectra; the larger noise in B is due to liquid nitrogen bubbling in the fingerDewar flask, t, g-factor positions: , peak-to-peak heights.

BrainControlExperimentalThymusControlExperimentalMuscleControlExperimental303038304653g5253,9,8=

2.003°±±±±i±3.3.7C82.55.109.271(35)"(33)(24)(ID(30)(30)86815646g=3

±.3±.2

±.7±1.94"98127.4.2.1.6(37)(33)(30)(30)

" Measured at O.I mW.6Measured at 200 mW.'Mean ±S.D." Numbers in parentheses, number of samples.

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Paramagnetic Changes in AKR Leukemia by ESR

A. CONTIOl SHEEN

Chart 4. ESR spectra from control and leukemic :lions as for Chart 2.

ilcens. ESR condi-

spleen does have multiple resonances, only those centered near g = 2 appeared to change significantly duringthe course of development of the disease.

The basic spectrum at g = 2 consisted of at least 2 different peaks. One, with an apparent g factor of 2.003, hada peak-to-peak line width of 20 gauss, was seen best atlow-microwave power levels, and was readily power saturated. The g factor and derivative maxima were the samein leukemic animals and in controls, but the line shapeobserved at low-power levels appeared to change, withsome broadening of the wings in the animals with leukemia.This broadening may have been due to an increase in the2nd component described in the following paragraph. Thelow-power signal has the classical ESR features of a tissue-free radical (25), and it is our assumption that it is

'one and, here, it is referred to as the free radical signal.

We estimated its change during the course of the diseaseby measuring the peak-to-peak height at low microwavepower (0.1 mW). Because the line shape did change, sucha quantitative estimation can be only an approximation.The presence of a significant amount of a 2nd signal in thelow-power spectra, such as that described below, shouldlead to an overestimation of the free radical signal. Thevalues of the peak-to-peak measurements of the spleenfree radical for the control and leukemic mice are shownin the upper half of Chart 5. In leukemic mice, the amountof the free radical appears to remain the same as or lessthan that in the controls.

At high microwave powers (e.g., 200 mW) spleen usuallyhas a prominent peak centered at g = 2.003, with a peak-to-peak width of 35 gauss which is seen best at high microwave power levels and which does not power saturate below200 mW incident power. Its power-saturation characteristics indicate that it is due to a paramagnetic transitionelement rather than a free radical. The shape of this signalis complex, with indications of a plateau near the center ofthe resonance. During the course of the experiment, inseveral control spleens this plateau area became moreprominent (Chart 6). In the same region, leukemic micedeveloped a definite peak in rough proportionality to theextent of the leukemia, as is shown by histogram in Chart6. This line-shape change makes it difficult quantitativelyto estimate changes in intensity of the transition element orelements contributing to the main g = 2.003 signal. If oneassumes that the additional peak that developed duringthe course of the leukemia has a negligible contribution atthe position of the derivative maxima, then peak-to-peak measurements provide a measure of the change inintensity of this signal. Such a measurement is given inthe lower half of Chart 5. Double integration of this signal complex was not attempted because of the variablysloping base line and the inevitable inclusion of the 2nd peakin such a calculation.

Liver. The liver ESR signals contain a large number of

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Chart 5. Graph of spleen ESR g = 2.0 signal height at 200 and O.ImW incident microwave power versus time after injection of AKR leukemia cells. The control (O) and leukemic (•)mean ±S.E. are given foreach day.

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Swartz et al.

peaks, mainly from transition ions (13, 25), plus a g =2.003 "free radical" signal (Chart 7). As the leukemia pro

gressed all signal levels decreased, but the line shapes didnot change nor did new peaks appear. Results for a typical high-power peak (g = 1.94) and the low-power signalat g = 2.003 are given in Chart 8.

ESR SPECTRA (200mw) OF MICE SPLEENS DURING « ewesDEVELOPMENT OF AKR LEUKEMIA

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246 8 IO 12 14DAY CONTROL

Chart 6. Histogram of doublet appearance in high-power ESR spectra of spleen. Typical spectra are shown on the right. Note that only theleukemic mice between Days 9 and 13 show full development of thesplitting. The spectrum from the Day 14 experimental sample wasqualitatively different from all other spectra.

NORMAL MOUSE LIVER

MOUSE LIVERHill Dor """rfttivînçIO3

AKR/J Ituttmio cells

Chart 7. ESR spectra from normal and leukemic mouse livers. ESRconditions are as for Chart 2, except time constant, 1 sec; and sweep rate,1000gauss in 4 min for 200 mW power, and 8 min for 0. l mW power.

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O 2 4 6 8 10 12

Days after InjectionChart 8. Graph of liver signal height at g = 2.0 and g = 1.94. The

control and experimental mean ±S.E. are given.

Blood. Chart 9 shows ESR spectra of the blood of theleukemic mice, and Chart 10 gives the time course of thechange of the height of the high-power peak. This significantly increased in height above the control values. Thereare 2 important points to note. First, the increase occurs byDay 2. Second, this increase plateaus out very early andthen stays approximately constant until the animals startto die. This contrasts with the time sequence of changesfor other parameters of leukemia development (Charts1, 5, and 7). If we look at the best indicator of the development of the leukemia-spleen weight, this starts going upat Days 4 to 6 and reaches a peak at about Day 10. Fromthe mortality data mentioned previously, the animals donot start to die until about Day 11. The ESR changes inspleen and liver became significant about Day 6 and progressed until the animals were moribund. Thus the bloodchanges occur very early and peak much earlier than otherchanges associated with the development of the leukemia.We explored this problem further by giving 5 groups ofmice injections of different quantities of cells varying froma hundred to a million cells, sacrificing them at Days 1, 2,

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MOUSE WHOLE BLOODCONTROL

DPPH

MOUSE WHOLE BLOODDAY-II - Leukemia DPPH

Chart 9. ESR spectra from normal and leukemic mice blood. ESRconditions as for Chart 2, except scan rate, 720 gauss in 30 min; time constant, 10 sec.

WHOLE MOUSE BLOOD

32.

28.

. 24.

20

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16

12

8O 2 4 6 8 IO 12 14

EXPERIMENTAL DAY AFTER RECEIVING 10s AKR/J

LEUKEMIA CELLS

Chart 10. The mean height of the main peak in the blood of mice infected with AKR/J leukemia as a function of time after injection. Thevertical bars give the S.E. Each mean was an average of the spectra from4 to 8 mice.

and 4, and then doing ESR analyses of their blood. Theresults of this study are summarized in Chart 11. By the1st day after injection of the leukemic cells, the mice thathad received relatively high numbers of cells showed a

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Paramagnetic Changes in AKR Leukemia by ESR

MOUSE WHOLE BLOOD

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Day I

O IO1 IO2 IO3 IO4 IO5 IO6

NUMBER OF CELLS INJECTEDChart 11. Height of ESR signal from mouse whole blood as a func

tion of the number of AKR/J leukemia cells injected. Samples wereobtained at 1,2, and 4 days after injection.

definite increase in the mean peak height of the ESR signal. By Day 2, even the mice receiving as few as 100 cellsshowed a very significant increase, and this increase did notchange very much by Day 4.

The molecular species giving rise to the blood ESRspectrum is not known. The g factor and line shape suggestthat it is a transition metal ion, possibly copper. Fractiona-tion of the plasma has shown that it is in the globulin fraction and is not dialyzable. We performed experiments onhuman plasma and showed that, in patients, a prominentsignal of similar shape is definitely due to ceruloplasmin.In that experiment we correlated chemical ceruloplasmindeterminations with ESR signal heights. We also wereable to obtain hyperfine structure in the spectrum that confirmed our assignment. Bearing in mind the dangers ofinterspecies correlation, we suggest that here too we areobserving ceruloplasmin.

DISCUSSION

Brain, Thymus, and Muscle

The lack of a significant difference in signal heights between control and leukemic mice reflects the observationthat the disease does not actively involve these organs. Wecannot compare these results directly with the work on anAKR leukemia by Chumakov et al. (4), since they lookedonly at the ESR signals from liver and kidney. However,the study of Saprin et al. (21) with La leukemia in micedid include ESR results from brain. Their results showeda free radical signal which linearly decreased in amplitudewith time, reaching about 70% of its initial value by thefinal day of the experiment. This linear drop did not correlate well with the development of the La leukemia, as it.

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Swariz et al.

like the AK.R, takes several days to develop. In addition,their histológica! study of the brain tissue showed that noLa leukemia cells were present. They also noted fluctuationsin the levels of free radical signals in kidney and lungs whichdid not appear to correlate with the development of thedisease.

Spleen, Liver, and Blood

Spleen. The free radical content of the spleen, as measured by the ESR spectra at 0. l mW, did not increase duringthe development of the leukemia, in contrast to the findings of some of the Soviet workers (4, 19 21). We observedan increase in the magnitude of transition metal paramagnetic species, as well as a change in the type of paramagnetic species. This increase (as measured by peak-to-peakheights and subject to the limitations discussed in "Results") rather closely paralleled the development of the

leukemia, as measured by the spleen weight, progressinguntil the animals were preterminal. The change in theshape of the ESR spectrum also seemed to be related tothe extent of the leukemic infiltration of the spleen, although the presence of a shoulder in some of the controlanimals suggests that this change may not be specific forleukemia. The high-power spectrum appears to be a mixture of at least 2 types of paramagnetic transition elements, but the precise number and nature of the species involved cannot be determined from the data we have available.

Liver. In the liver there is simply a general decrease inall signals which parallels the systemic spread of the leukemia. We believe that this may be due to a loss of normally functioning tissue as leukemic cells infiltrate thisorgan.

Blood. The main peak (g factor of cross-over point =2.05) seen in frozen blood increased in amplitude significantly above that of the controls. The blood changes preceded other ESR changes and other indicators of leukemiaand rapidly reached a plateau; in fact, they appeared before there was time for many cell divisions of the leukemiacells (generation time, ~12 hr). All of this suggests thatthe observed blood ESR changes may not be directly relatedto the malignant process but may reflect a more generalcellular response.

There is some other unpublished evidence that is consistent with the hypothesis that blood changes are a nonspecific systemic response. Dr. Finch of the Naval Medical Center in Bethesda, Md., has kindly made available tous blood ESR data obtained after injections of ascitestumor cells into mice. He observed an early rise in ESRsignal height. Then his attention was drawn to the fact that1 control animal that had a middle-ear infection alsoshowed a very large increase in the main peak. On thisbasis, he took a small number of animals and gave theminjections of a typhoid vaccine. After inoculation of thetyphoid vaccine there was also an increase in the mainpeak, quite similar to that seen with the tumor cells.

All of this suggests that a systemic response, perhapsimmunological, may be involved in the observed ESR

changes in blood. If so, it is not clear what aspect of the immune response is represented by these paramagneticchanges. The rapidity of the ESR response exceeds anyclassical nonsensitized immunological serum responses. At24 to 48 hr, the antigen-recognition aspect of the immuneresponse should be occurring, but peripheral blood changesdue to this part of this immune response have not beenpreviously reported. Antibody production, which doeslead to detectable serum changes, occurs much later. Ifthe ESR changes are part of an immune response, theyprovide information on an aspect of the immune responsethat has not previously been amenable to study in serumsamples. More likely, the observed blood changes aredue to a nonspecific stress response. If the spectral similarity to ceruloplasmin is borne out, this may simply be arise in this nonspecific reactor protein.

The above discussion on the origin of the blood ESRchanges may be applicable to the observed findings in human patients (26). Ceruloplasmin is known to increase inhuman cancer patients (23).

The results from the tissues (spleen and liver) directlyinvolved in the leukemic process do not support the results of Saprin et al. (19-21). No early rise in free radicalswas observed; there was a decrease in free radicals as theleukemic cells infiltrated and replaced normal cells. In thespleen there was an increase in a paramagnetic transitionelement, but the kinetics of this increase was quite different from the reported by Saprin et al. for free radicals.They found the maximum increase with tumor involvement was very early, while we observed changes thatparalleled tumor development.

There are several possible explanations for the discrepancy between our data and that of Saprin et al. They usedlyophilized samples and may have studied some parameter related to interaction of oxygen with cell biochemi-cals and not directly related to physiologically occurringfree radicals (9, 24). The experimental systems they usedmay also not be directly comparable to ours. A direct comparison, with the use of the same tumor system and experimental conditions, would seem to be necessary toachieve a basis for a rigorous comparison of results.

One useful firm conclusion can certainly be drawn fromour work. On the basis of their results, Saprin et al. postulated that an early rise in free radicals might be characteristic of all developing tumors. Our results clearly show thatthis is not true for at least 1 leukemia. Previous studieshave indicated that it is not true for some hepatomas (7,11,29,30).

Additional studies should establish whether systemssuch as the AKR/J leukemia typify the rule or the exception, in regard to free radical changes in cancer.

ACKNOWLEDGMENTS

We should like to acknowledge the assistance and facilities providedby Dr. Mortimer Bortin of the May and Sigmund Winter ResearchLaboratory of the Mt. Sinai Medical Center, Milwaukee, Wis. The technical help of Evangeline R. Reynolds, Anita Lapp, and Donna Eastmanin the mouse leukemia experiment is also gratefully acknowledged.

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Paramagnetic Changes in AKR Leukemia by ESR

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1973;33:2588-2595. Cancer Res   Harold M. Swartz, Colin Mailer, Sunandini Ambegaonkar, et al.   ResonanceAKR/J Leukemia in Mice as Measured by Electron Spin Paramagnetic Changes during Development of a Transplanted

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