THE ACTION OF SHORT RADIO WAVES ON TISSUES:I. EFFECTS PRODUCED IN VITRO

WITH SOME OBSERVATIONS ON THE ACTION OF HEAT ON TISSUE METABOLISM

FRANK DICKENS, M.A., PH.D., STANLEY F. EVANS, M.Sc., AND HANSWEIL-MALHERBE, M.D.

(From the Cancer Research Institute, North of England Council of the British Empire CancerCampaign, Royal Victoria Infirmary, Neuicastle-upon-Tyne)

The development of the thermionic radio valve during the past fifteenyears has enabled short radio waves below 15 metres in wavelength to begenerated with considerable intensity. It has been found that these ultra­short waves (U.S.W.) produce strong heating effects in tissues, and this heat­ing of electrolytes and of animal tissues has been systematically studied,especially by McLennan and Burton (1-3) and Patzold (4). Short wavediathermy thus rests on a secure experimental basis, and is now an acceptedpart of physical therapy.

The application of these waves to the treatment of cancer, however, is ina less satisfactory condition, and has not yet emerged from the stage ofpreliminary experimental work. In this sphere, the influence of the conceptof "specific action," i.e. action of the waves not to be accounted for by theirheating effect and usually associated with specific wavelengths, has been con­sidered by some workers to occupy a paramount position, while others havetaken the view that heat effects would alone suffice to account for the curativeaction observed in animal tumours.

It was with the intention of clarifying this unsatisfactory position that weundertook a comprehensive study of the action of these waves on normal andmalignant tissues. The present paper will set forth the results obtained whentissues in vitro are exposed to ultra-short waves, the results of in vivo workat present in progress being reserved for a later publication.

The first workers to apply these waves to pathological growths appear tohave been Gosset, Gutmann, Lakhovsky and Magrou (5), who in 1924 re­ported the effects of ultra-short waves on tumours produced in the geraniumby means of B. tumefaciens. In 1928 Schereschewsky (6) published the re­sults of an extensive series of experiments on the treatment of transplantedsarcoma in mice and fowls, with a wavelength of approximately 4.5 metres.It was shown that complete destruction of the tumour could be effected in 25per cent of the animals. At the time of this pioneer work, Schereschewskywas of the opinion that the temperature of the tumours did not rise sensiblyto the touch, and that heat alone could not account for the results; but in alater paper (7) he has described experiments showing that the treatmentcaused the temperature within the tumour to rise to 48-49° C., and producedreasons for considering that this increase is responsible for the cures effected.In 1930 Pflomm (8) reported results of the action of 3.2 metre waves on

603

604 FRANK DICKENS, STANLEY F. EVANS, AND HANS WEIL-MALHERBE

Jensen rat sarcoma. In a selected group of 11 animals, he found that a tem­porary regression could be produced, but that after the 30th day there wasagain slow growth of the tumours; the strain of rats used appears to havebeen unsuitable for therapeutic experiments with this tumour, for the inocula­tion yield was only 30-60 per cent. Finally Reiter (9) attracted consider­able attention by the publication of his experiments, which purported to showthat the Jensen rat sarcoma could be destroyed by high-frequency energy ofwavelength 3.4 metres under conditions such that all heat effects were ex­cluded. Wavelengths only a few decimeters above and below 3.4 metres werevery much less effective. Similar results were also shown to apply to Flexnercarcinoma and the 8-tumor of the Charite Krebsinstitut, for which also atrifling shift of frequency resulted in loss of destructive or even of growth­inhibiting action, whereas at 3.5 metres destruction was complete.

Shortly after the publication of these in vivo results, Reiter (10) produceda further paper on the in vitro action of these waves, which was the startingpoint of the present paper. He found that exposure of tumor tissue in vitroto the short wave field resulted in a fall of the anaerobic glycolysis of about50 per cent, and also an inhibition of the respiration by 25-30 per cent. Inhis experiments a large number of tissue slices, moistened with a little Ringersolution, were placed in a quartz dish between the electrodes. Although heremarks that the temperature of the control and treated pieces of tissue .waskept the same, i.e. 38-39° c., no details are available either of the method ofcooling, if any, or of the way in which this temperature was controlled. Onthis evidence, Reiter claimed to have demonstrated an immediate destructiveeffect, independent of heat. This was again only produced by the use of thecritical wavelength of 3.4-3.55 metres, according to the particular tissue.Since this was a primary, direct effect of the waves on metabolism, and oc­curred after only a few minutes' exposure to the radiation, it was claimed thatthis range of wavelengths is more rapid and intense in its action than eitherx-rays or radium, the effect of which appears only slowly after the period ofirradiation, as a secondary destructive action on metabolism .

. It will be seen that the whole basis of these statements is the assumptionthat the tissue does not become heated to any appreciable extent by the ex­posure to ultra-short waves. Great prominence has been given to these experi­ments, which have been extensively quoted as a striking instance of the specificaction of short waves.

The only work dealing with the transplantation into animals of tumourmaterial, after subjecting it to ultra-short wave irradiation in vitro, appearsto be that of Raffo (11-13) and of Rasche and Collier (14). Roffo placedvessels in which were growing cultures of cancer cells, contained in incubatorsat 380 C, at the antinodes of stationary ultra-short oscillations along a pairof Lecher wires. Various results were obtained, showing a difference in theeffect of ultra-short waves on growth in vitro of normal and tumour tissue.When the tumour cell cultures were transplanted into rats, the irradiatedtumours failed to grow in a large proportion of the animals, while the controlsgrew in nearly every case. Again in these experiments, no indication ofspecial precautions to control temperature of the treated cultures is given.The German workers Rasche and Collier (14) evidently realised the need

THE ACTION OF SHORT RADIO WAVES ON TISSUES 605

for drastic temperature control, for they describe an apparatus by means ofwhich cooling water flowed around the specimen. This was diluted asciticfluid obtained from mice bearing a strain of the Ehrlich carcinoma which givesrise to peritonitis carcinomatosa, and they observed that when the asciticfluid after exposure to 3.5 metre radiation was inoculated into mice, theseanimals died of carcinoma just as the controls did.

That so few workers have attacked this problem, and that the few resultsavailable are so contradictory, is probably to be explained by the difficulty ofthermal control, particularly where solid tissues, and not isolated cells, areused. In this paper we describe a technique which enables these difficultiesto be overcome, and its application to the problem of in vitro action.

250V'"

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FIG. 1. OSCILLATOR CIRCUIT

V, V, = Oscillator valves. V, V, = Rectifying valves. L, = Anode inductance. L, = Gridinductance. L, L, L, L, L, L. = H.F. chokes. M, M, = Filament voltmeters. M, = High­tension voltmeter. M, = Milliameter. C = 1 mfd. smoothing condenser. R = 100,000 ohmbleeder resistance. T, = High-tension transformer. T, = Rectifier filament transformer. T, =Oscillator filament transformer. T, = Tapped autotransformer.

In addition to the present investigation dealing with this aspect of tumourtherapy, we intend in future publications to consider the broader and moredifficult question of the mechanism of action of these waves in the livinganimal, where the effects due to heat cannot be separated from any othereffects which may be produced by the ultra-short waves.

EXPERIMENTAL TECHNIQUE

Design of Oscillator: The circuit of the oscillator is shown in Fig. 1. TwoMullard TX4-400 valves are used in a push-pull self-oscillatory circuit. Thehigh tension supply is rectified by two G. U. 2 mercury arc rectifiers (General

606 FRANK DICKENS, STANLEY F. EVANS, AND HANS WEIL-MALHERBE

Electric Company) and can be varied from 500 to 2500 volts by an auto­transformer in the primary circuit of the high-tension transformer. Thewavelength is altered by changing the anode and grid inductances, the mini­mum attainable being 3.0 metres. Below this the valves cannot be made tooscillate. It was found that on these very short waves, the efficiency wasgreatly reduced by the presence of a grid leak and in the final version of thecircuit this was accordingly omitted.

As tuned circuits for these wavelengths are necessarily of very smalldimensions and all leads must be kept as short as possible, the electrodes, if

2

3

sFIG. 2. FEEDER AND TERMINATING CIRCUIT

1. Anode coil of oscillator. 2. Tuned, coupled secondary circuit. 3. Terminating circuit. S.Specimen under exposure to ultra-short waves.

placed in the secondary circuit, must be close to the valves. This is alwaysinconvenient and may even vitiate the experiments owing to the heat radiatedfrom the valve anodes. A feeder system is therefore used to convey the high­frequency energy from the coupled secondary circuit to a third terminatingcircuit to which the electrodes are connected as shown in Fig. 2. The feederis a length of 5 ampere lighting flex and if the system is correctly adjusted theloss of energy is not large. An account of the theory and use of feeders isgiven by Ladner and Stoner (15).

The adjustments are simple; the two tuned circuits are adjusted to reson­ance and the power is regulated by alteration of the coupling between theoscillator and the secondary circuit. The wavelength is measured by aMarconi absorption wavemeter which has been calibrated against an absolutefrequency standard and enables the wavelength to be estimated to 0.01 metre.Lecher wire measurements were found to be very unsatisfactory, errors asgreat as 5 per cent being observed on some occasions despite all usual precau­tions to ensure accuracy. The output from this oscillator was fed to the ap­paratus used for all our in vitro experiments, which is described below.

Technique of Irradiation in Vitro at Constant Temperature: For workof this kind, where it is essential that portions of tissue should be maintainedfor a considerable time under conditions which are as nearly as possiblephysiological, the adaptation of the well known technique of Warburg (16),as used for studying the gas-exchange in surviving tissues, at once suggesteditself.

The apparatus finally evolved (Fig. 3) consisted of a box-shaped glass

THE ACTION OF SHORT RADIO WAVES ON TISSUES 607

vessel containing the tissue suspended in a suitable saline medium, and con­tinuously shaken so as to maintain equilibrium with the air, oxygen, oroxygen-CO" mixture with which the gas-space was filled. This vessel wasattached by means of its ground-joint to the usual Warburg open-ended ma­nometer which had on each limb a scale of 0-300 mm. and was filled withClerici solution of specific gravity 4 (Dickens and Simer, 17). The woodenstand of the manometer carried at its upper end the terminating circuit fromthe transmitter, consisting of an inductance and a variable capacity (Fig. 2,circuit 3); by this means the leads to the two electrodes, which were connected

FIG. 3. ApPARATllS FOR IRRADIATION IN VITRO AT CONSTANT TEMPERATURE

across the variable condenser, were kept very short. These electrodes con­sisted of flat copper sheets placed externally to and on opposite sides of therectangular glass vessel containing the specimen.

Two sizes of vessel were used according to the quantity of tissue and ofsolution employed; their volumes were 40 c.c. and 7 c.c. respectively. Theelectrodes were of such size that the sides of the vessel were just covered andwere held in position by means of rubber bands stretched around electrodesand vessel after the latter had been attached to its manometer by the groundjoint.

In the usual Warburg apparatus, the vessel is immersed in a water ther-

608 FRANK DICKENS, STANLEY F. EVANS, AND HANS WEIL-MALHERBE

mostat, in which it is continuously shaken. With this electrode system water,on account of its electrical properties, was found, as expected, to be quite un­suitable for a cooling bath. After several trials medicinal liquid paraffin wasselected as most suitable, benzene being excluded on account of its inflammablenature. The bath consisted of a glass tank 40 X 20 X 20 em. filled to withina few centimeters of the top with liquid paraffin; it could be cooled as desiredby immersion of tins containing ice and salt into the paraffin. This simplecooling arrangement was quite adequate. The paraffin was vigorously stirredthroughout by a motor-driven propeller, and the usual shaking apparatus wasprovided for the manometer and its attached vessel and electrode system.By this means the vessel, immersed in the paraffin bath, could be shaken at arate of 120 complete swings per minute, with a throw of 5-10 em. (Fig. 3).

Manometric Control of Temperature: The purpose of the manometer inthese experiments was not, as in the usual manometric experiments with theWarburg apparatus, to measure metabolism by means of the gas exchange, butmerely as an indicator of the prevailing temperature within the vessel. Thesystem was used, in fact, as a type of constant-volume air thermometer.

It was at once apparent that the heating effect of the short-wave field,produced by the transmitter described, was very great; in spite of the vigorousshaking and extremely efficient cooling secured by the above apparatus, thetemperature within the vessel rose high above that of the bath immediatelyafter switching on the current. This could clearly be followed from the rapidrise of pressure which occurred, and the rise of temperature was also measuredby means of a small clinical mercury thermometer which was introduced intothe vessel through a small tubulure so that its bulb was immersed in the liquidcontained in the vessel. This thermometer was held permanently in placeduring the experiment by a firmly inserted rubber bung.

The increase of pressure observed is due to several causes: (a) increasedtemperature of the liquid results in increase of vapour pressure, accompaniedalso by expulsion of dissolved gases according to the alteration of the gasabsorption constants with temperature; (b) thermal expansion of the gas(that of the liquid could be neglected by comparison) and (c) any change ofpressure caused by gaseous metabolism of the tissue. Relative to a and b,c can be kept small, and it was roughly compensated by comparison with thepressure changes occurring in another similar vessel containing a similar quan­tity of tissue and solution, but shaken in a separate (water) bath at 37.5 0 C.

Owing to the fact that the U.S.W. field heats the liquid more than the gasin the irradiated vessel, it was not possible to calibrate the arrangement bymerely immersing the vessel, attached to its manometer, in baths at differenttemperatures and noting the pressures developed. This method leads toincorrect temperature-readings, since by this means both the gas and liquidare heated to the same extent. The manometer readings were thereforecalibrated empirically, by the use of the mercury thermometer already men­tioned. Patzold (18) has shown that when immersed in a substance of highdielectric constant, a mercury-in-glass thermometer gives a quite reliablereading of temperature in the U.S.W. field.

The calibration showed that with the arrangement described and with aninitial bath temperature of about 6° C, a positive pressure of about 300 mm.

THE ACTION OF SHORT RADIO WAVES ON TISSUES 609

Clerici (or 1200 mm. water) was shown when the temperature of the specimenwas 37.50 C., a very convenient increase amounting to the full scale-readingof the manometer. This manometric control proved of the greatest value inensuring constancy of experimental temperature with slight variations of theoutput from the transmitter; these fluctuations could be immediately and con­tinuously compensated by a slight tightening or loosening of the couplingbetween the primary and secondary circuits so as to keep the manometerreading constant during the experiment. In this way readings of within adegree or two of the desired level were regularly maintained during experi­ments lasting one to two hours, and the difficulty of keeping a continuouscontrol of the temperature of the vessel contents, without interrupting theshaking mechanism, was thus completely overcome.

Maintenance of Physiological Conditions: In any experiment of this kind,it is important to eliminate as far as possible any other source of damagewhich might act concurrently with the ultra-short waves upon the cells. Themost essential point, the elimination of heat action, was achieved by the ar­rangement already described. In addition, the following points were adheredto:

(a) Preparation of the Tissue: Immediately after the death of the animal,the tissue, which in the case of tumours was taken only from macroscopicallynon-necrotic specimens, was dissected and was used either as thin slices of0.3 to 0.5 mm. thickness, cut with the razor, or as a mince prepared by a micro­mincer as used for transplantation of tumours. For metabolic experimentsonly slices are suitable; the mince was reserved for certain transplant experi­ments where no measurement of metabolism was made.

(b) Suspension of the Tissue in an Adequate Medium: Control experi­ments have shown that the metabolism of tumour tissue slices suffers by tem­porary absence of glucose as well as by insufficient buffering. A well buffered,glucose-containing, physiological salt solution (bicarbonate saline of Krebsand Henseleit, 19) was used in all metabolic experiments. In transplantationexperiments with minced tissue, the same medium buffered with phosphateinstead of bicarbonate was used.

(c) Adequate Gas Exchange and Diffusion of Metabolism: In metabolismexperiments, for the preliminary exposure to ultra-short waves before measure­ment of metabolism, the slices of tissue were suspended in 5 c.c. glucose­bicarbonate-saline, and were shaken in an atmosphere of 95 per cent oxygenand 5 per cent CO". The rectangular vessel of about 40 c.c. volume, alreadydescribed, with its external pair of electrodes and attached to its Warburgmanometer, was used as container.

EXPERIMENTS ON THE IN VITRO ACTION OF ULTRA-SHORT \VAVES

Two methods have been applied to detect any damage which might arisefrom the action of these waves: (1) measurement of the metabolism (respira­tion and aerobic and anaerobic glycolysis) of tumor and normal tissues afterexposure to the U.S.W. field; (2) inoculation of previously exposed tumourtissue into animals. By this latter any damage to the growth-properties ofthe cells could be detected.

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(1 ) Metabolic Experiments: The tissue slices, prepared as describedabove, were divided into two equal parts and transferred to two box-shapedvessels of similar shape and size containing 5 c.c. glucose-bicarbonate-saline.The vessels were attached to their manometers and gassed with oxygen with5 per cent CO~. One vessel, serving as control, was shaken in a water bathat 37.5° for the time of the experiment; while the other vessel was exposed tothe short-wave field in the manner already described. After completion ofexposure, both vessels were removed from the baths, the tissue transferred toother vessels, and the metabolism determined in the usual way (Warburg, 20).All measurements on metabolism are expressed in the generally adopted formof Q-values (Warburg, 20): Qo, = respiration; Qgb

2= aerobic glycolysis;

Q~l,. = anaerobic glycolysis. All three quotients are in cm. per mg. dryweight of tissue per hour.

The results of our metabolic experiments with tumour tissue are set outin Table 1. Before discussing them, we wish to point out that a long experi­ence of these measurements has taught us that they are likely to be somewhaterratic when, as in the present series, a period of one to two hours precedesthe actual measurement. This is the probable reason for the low respiration

TABLE II: Action of Ultra-short Waves on Metabolism of Brain Cortex

Temperature Metabolism of exposed Metabolism of control(0C) tissue tissue

Wave- DurationSpecies length of expo-

sure Initial Maximum(metres) (min.) temper- temper- Qo, Q81" Q~b, Qo, Q211, Q~1)2ature atureof bath reached

-- -------------Rat 7.2 60 19.0 ca. 37.5 -11.2 0.8 19.7 -10.7 1.7 17.8Guinea-pig 3.4 60 6.2 41.0* -10.1 0 21.8 - 8.1 0.6 21.7

* Temperature controlled by clinical thermometer.

recorded in Experiment 2, and of the low anaerobic glycolysis in Experiment 1,Table I, both occurring in the controls. We have therefore to consider theaverage values, rather than the result of a single experiment. It is obvious,however, that our results are far from showing the spectacular destructiveeffect which has been claimed by Reiter. If anything, there is a tendency fora slight increase of respiration and anaerobic glycolysis (e.g. Experiments 4and 6, Table I). But this is not a constant feature, and there are other ex­periments showing a slight tendency in the opposite direction (Experiments 3and 5, Table I). The highest power was applied in the last experiment(No.7), where the temperature difference between the bath and vessel con­tents amounted to 36.3 0

, and the results obtained in this experiment are almostidentical in the control and irradiated specimens.

The metabolism of brain cortex has been investigated as representativeof a normal tissue. Brain was selected, because its metabolism is most sus­ceptible to accelerating or inhibiting influences, particularly those of a physicalnature (Dickens and Greville, 21). The results (Table II) are the same asthose obtained with tumor tissue.

Summarising these experiments on metabolism, it is clear that no sig-

612 FRANK DICKENS, STANLEY F. EVANS, AND HANS WEIL-MALHERBE

nificant action of ultra-short waves can be observed even after exposure forone or two hours to the waves. Our results are quite contradictory to thoseobserved by Reiter (10) following exposures of only a few minutes' duration.This difference is to be attributed to the adequate control of temperature inour experiments.

(2) Transplantation Experiments: It appeared possible that without anyimmediate effect being produced on the metabolism of irradiated tumourtissue, some action could conceivably have been produced which might onlyslowly appear a considerable period after the exposure to the waves. In orderto control this point, we have used the growth on subsequent transplantationas a criterion of damage produced during the period of in vitro irradiation atconstant temperature.

TABLE I II: Transplantation of Tissue Irradiated in Vitro(Wavelength = 3.4 metres in all experiments)

Clinical Result of transplantationInitial bath thermometer no. tumours/no. animals

Date Tumour temperature reading at Time('C) end of exteri. (minutes)

ment('C) Irradiated Control

Sept. 27, 1935 Walker 256" 23.0 39.8 60 3/6 6/6Oct. 8, 1935 Walker 256 16.0 37.7 100 6/6 5/6Oct. 9, 1935 Walker 256 14.5 38.0 120 5/6 5/6Oct. 15, 1935 Walker 256" 18.6 45.2+ 90 8/9 5/6Oct. 16, 1935 Jensen sarcoma 19.5 41.0 85 6/6 5/6

28/33 = 26/30 =TOTAL TUMOURS 85% 87%

Growing Growing

"Mince. Others slices. t Temperature at end of experiment minus initial temperature givesa measure of the dosage. +During this experiment the temperature was 41° (manometric)except for a short jump to 45°.

For this purpose, the experimental procedure was the same as that alreadydescribed for our metabolism experiments, except that in two experiments weused a tissue mince, moistened with a small volume of Krebs-Ringer solutionin order to allow the tissue to be agitated sufficiently when the vessel contain­ing it was shaken. Before transplantation to the animals, the excess of salinewas drained off from the tissue-mince. Complete asepsis could not be assured,but the usual precautions in preparation of tissue, solutions, and glasswarewere taken, and non-infected tumours resulted from the transplant.

It was necessary first to calibrate the manometer readings against thereadings of the clinical thermometer immersed in the vessel contents, by meansof preliminary trial experiments. This calibration held only for a particularvessel and a particular quantity of tissue and solution. The two vessels usedheld 40 c.c. and 7 c.c. respectively. The larger vessel was used for sliceexperiments. In some instances both metabolism and transplant experimentswere made with the same sample of tissue. The smaller vessel (7 c.c. totalcapacity) was more suitable for those mince experiments where the quantityof tumor tissue available was small; quantities of 1.5 gm. moistened with 0.5

THE ACTION OF SHORT RADIO WAVES ON TISSUES 613

to 1.5 c.c. Krebs-Ringer solution proved suitable for use with this vessel. Inall experiments, the selected non-necrosed tumour material, whether slices ormince, was divided into two approximately equal portions, of which one wasused for the U.S.W. irradiation in the paraffin bath; the other was a controlportion and was shaken in a vessel similar in size and shape to that used forthe experimental portion, and under precisely similar conditions except thatthe temperature was maintained in the usual way, by shaking the vessel inthe ordinary Warburg thermostat at 37.5'° C, instead of exposing it to theU.S.W. field.

After exposure for a period of from one to two hours, during which thetemperature within the experimental vessel was controlled from the pressurereadings of its manometer, corrected as necessary by reference to the manom­eter attached to the non-irradiated sample, the tissue from both vessels wasinoculated into the rats. For this, the slices were cut into small pieces andinoculated by trochar; the mince was taken up in a Bashford syringe witha wide needle. The results are shown in Table III. Despite the wider tem­perature fluctuations to which the irradiated portion was necessarily exposed,there was no essential difference in the growth of tumours following trans­plantation from that of the control tissue which had been maintained through­out at 37.5 0 C + 0.02 0

, in the thermostat.Dosage in these experiments was very much higher than could have been

applied in vivo. The actual absorption of energy, measured by the heatingeffect on the vessel contents, and by a subsequent manometric determinationof cooling curve, amounted to about 8 to 12 watts, and this continued for oneto two hours.

Action of Heat on Metabolism

Although our negative in vitro experiments seem to reduce the problemof short wave action to the problem of heat action, the fact remains thatanimal tumours can be cured by exposure to a short wave field, as was firstshown by Schereschewsky and confirmed by other workers and by ourselves.

Since we have shown that, when the heating effect is eliminated, there isno in vitro action of ultra-short waves on metabolism or growth, it seemeddesirable to investigate the action of heat on the metabolism of tumour cellsin vitro. This problem has already been made the subject of an extensivestudy by Westermark (22). That author investigated the metabolism oftumour slices after heating for various times. He found that the first meta­bolic activity of the tumour cell to suffer was its glycolytic power. The res­piration for a long time remained unaffected and then showed a sudden fall.Westermark's experiments, however, are open to certain criticisms; in his ex­periments, too, the tissue, during incubation was not kept under physiologicalconditions. It was suspended in plain Ringer, without either buffer or glucose,and no adequate gas exchange was possible, since the tissue was kept in air,without shaking. Experiments which we have carried out under these con­ditions yielded results quite different from those in which the tissue was ex­posed to the same temperature but was kept under more nearly physiologicalconditions throughout. Furthermore, we believed that a truer picture of the

614 FRANK DICKENS, STANLEY F. EVANS, AND HANS WEIL-MALHERBE

TABLE IV: Effect of Incubation at 45° on Metabolism of Tumour Tissue

Tissue kept at 45· Control tissue keptfor I or 2 hours, I hen at 37.$0 throughoutTinw* Temper- transferred 10 37.S·

No. Tissue (rnin.) Mcdiunrt at ure(0(')

Uo, U()' Q~i), Qo, UO

' U~l)2CO, CO2----------

I j.R.S. I. 20- 40 B.S. 45 -19.0 34.2 39.3 -14.2 19.5 42.140- 60 -17.3 40.2 39.3 -14.5 18.3 38.8

II. 20- 40 37.5 -10.9 26.5 22.1 -16.0 21.0 39.140- 80 -12.0 29.8 23.8 -12.7 17.5 37.0

- ----------2 j .R.S. I. 20- 40 B.S. 45 -19.4 55.2 56.5 -13.4 23.9 31.6

40- 60 -20.7 65.0 58.4 -12.7 24.6 32.4

II. 20- 40 37.5 - 6.5 37.5 31.9 -11.9 25.5 31 ..140- 80 - 4.9 32.4 30.1 - 9.3 23.7 30.0

------------ ----I. 20- 40 P.S. 45 -11.3 -10.8

40- 60 - 6.0 - 8.4

II. 20- 40 37.5 - 2.4 - 8.740- 80 - 1.8 - 7.9

- --- ----- ----.-------"-----

3 j.R.S. I. 20- 40 P.S. 45 -l.1.5 -13.1 -10.540- 60 -11.0 -10.3 - 9.9

--------"-- "'II. 20- 40 37.5° 45° - 7.5 - 9.2 - 8.0

40- 80 - 5.4 - 6.4 - 7.3- --- ------ --- -----

4 Mouse sarcoma I. 20- 60 B.S. 45 -16.7 34.9 31.0 - 6.6 6.0 25.5S 37 60-100 - 7.5 27.7 24.8 - 4.9 6.6 24.1

100-120 - 4.8 17.7 15.7 - 3.9 6.8 24.4

II. 20-100 37.5 - 1.3 3.5 5.0 - 2.2 10.8 24.0- --- -----------

5 Walker carci- I. 20- 60 B.S. 45 -10.2 34.8 37.9 -15.2 19.7 31.2noma 256 60-100 - 4.2 34.9 31.6 -12.9 16.8 28.7

100-120 - 3.5 27.6 25.8 - 9.9 14.2 26.0.-

* The times in this column refer to two separate periods of measurement. I is reckoned from0' = the introduction of the tissue into the 45° bath. II is reckoned from 0' = time of transferto 37.5° after regassing. A period of ten to fifteen minutes elapsed between end of I and beginningof II. In experiment 3 in period II the left hand figures for respiration were taken after transferto 37.5° and the right hand figures in a separate specimen kept at 45° throughout.

t B.S. = Bicarbonate saline with 0.2% glucose. P.S. = Phosphate saline with 0.2% glucose.

effect of heat could be obtained when the metabolism was not only measuredsubsequently to the heating, but during the actual exposure to increased tem­perature. Our experiments were therefore done in two thermostats, one beingkept at 37.5 0 and the other at 45 0

• After a suitable period the manometerswere removed from the baths, the taps opened and the manometers again filledwith the gas mixture before they were all put back into the thermostat at37.5 0

• The measurement of metabolism was then continued at 37.5 0 forone hour.

Metabolism of Tumour Slices at 45': As was to be expected, the metab­olism of the tissue kept at 450) was considerably accelerated compared with

THE ACTION OF SHORT RADIO WAVES ON TISSUES 615

TABLE V: Effect of Incubation of Tumour Tissue (Jensen Rat Sarcoma) at 50° for Five Minutes(Metabolism measured at 37.5° in bicarbonate-glucose-saline)

rncubated sample Control sample

Qo, Qgb, Q~l), Qo, Qg[), Q~1)2

-3.8 10.7 9.0 -8.0 18.7 32.8

that at 37.50. In most experiments, however, the rate of metabolism declinedfaster than at 37.5° as a consequence of the damage done by the unphysio­logical temperature.

The typical effect on respiration was an increase of 50-100 per cent, whichremained fairly constant during the first sixty minutes (Table IV, 1,2; TableVI, 2). This typical increase was not found in all experiments with tumourtissue. There were tumours with an apparently abnormally susceptible res­piration, which was damaged by heat from the very beginning. This wasobserved especially in those cases where the respiration of the control wasunusually high (Table IV,S; Table VI, 1). Respiration in phosphate bufferwas found less resistant to heat than in the more physiological bicarbonatebuffer (Table IV, 2).

The initial rate of anaerobic glycolysis showed an increase of about SOper cent on an average, followed by a slow fall, due to the combined effect ofheat and anaerobiosis.

The most constant feature of these experiments was a rise of aerobicglycolysis to the level of anaerobic glycolysis. This phenomenon, usually de­scribed as inhibition of the Pasteur reaction, is known to occur as a conse­quence of certain damaging influences upon the cell and is often the first signof disturbance. For example, it was shown by Kubowitz (23) to result infrog retina from an increase of temperature to an unphysiologicalleveI.

Metabolism of Tumour at 37.5' After Exposure to 45'": This effecton the Pasteur reaction persisted even when the tissue, after one hour's heatingto 45", was transferred to a temperature of 37.5). The aerobic glycolysis ofthe previously heated tissue was usually higher than in the control (Table IV,1,2. Table VI, 1). On the other hand, the respiration, which in some ex­periments is little affected by the previous heating, is in other cases seriouslyinjured. We conclude, therefore, in contrast to Westermark, that the res­piration of tumour cells is more susceptible to heat than the glycolysis.

Metabolism of Tumour Tissue at 37.5° After Exposure to 50° for FiveMinutes: We have seen in unpublished in vivo experiments that tumours(J. R. S.) can be destroyed by very brief treatment by ultra-short wavesduring which the temperature of the tumour rises to about 50. Since datawere not available for the effect of very short exposures at this temperature onthe metabolism, we determined this for the Jensen rat sarcoma. The tumourslices were kept in a glucose-bicarbonate saline at SO) in a small cylinder im­mersed in a water bath while oxygen/S per cent CO" was bubbled through thesolution. After five minutes the tissue was removed for measurement of themetabolism at 37.5" (Table V). It will be seen that a severe destruction ofrespiration and glycolysis occurred and that the Pasteur reaction was again

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THE ACTION OF SHORT RADIO WAVES ON TISSUES 617

completely inhibited. Corresponding with the damaged metabolism, we havefound that in transplant experiments following exposure to 50° for fiveminutes carried out under similar conditions, there was no growth in mice.inoculated with Crocker sarcoma 180, while the controls grew in 100 percentof the animals.

Effect of Unphysiological Conditions: In order to analyse the divergentresults obtained by Westermark and by ourselves the following experiment wascarried out: slices of the same tumour were divided into four parts. Two ofthem were shaken in bicarbonate saline containing 0.2 per cent glucose in anatmosphere of O2/5 per cent CO2, at 37.5° and 45° respectively, as in the

.experiments already described. The remaining two portions were incubated,without any shaking, in plain saline and in an atmosphere of air, at 37.5° and45° respectively. On comparison of the two samples which were heated to

TABLE VII: Effect of One Hour's Incubation at 45" on the Metabolism of Brain Tissue

Tissue kept at 45" for one hour, Tissue kept at 37.5"Time" then transferred to 37.5" . throughout

No. Species(min.)

Temp. Qo. Qgb, ~ebs Qo. Q8b, ~ebs----------

1 Rat I. 20-40 45 -42.6 32.2 18.0 - 7.0 3.0 12.94<Hi0 -34.6 33.8 13.0 - 9.6 2.5 12.0

II. 20-40 37.5 -1.2 2.7 3.2 - 7.8 0.3 9.840-80 -1.1 2.4 5.1 - 8.4 1.0 10.6

-- ------------2 Rat I. 20-40 45 -30.5 21.0 24.7 -11.6 4.8 16.8

40-60 -27.6 26.8 17.9 -10.4 1.6 17.9

II. 20-40 37.5 - 5.8 7.8 8.2 -12.2 3.1 18.340-80 - 3.9 5.2 7.8 -11.5 28 16.9

------3 Guinea-pig I. 20-40 45 -18.8 10.3 32.2 - 8.9 0.9 19.2

40-60 -21.0 11.8 30.0 - 9.0 1.3 22.1

II. 20-40 37.5 - 8.0 3.5 11.7 - 9.3 1.3 18.040-80 - 8.2 6.7 12.3 - 7.4 1.8 19.7

• See note to table IV.

45° it is obvious that the glycolysis is far more damaged in the sample whichhad been incubated in plain saline (Table VI). That this is no pure heateffect is shown by the fact that the same changes, although less pronounced,are observed even in the control tissue which had been incubated at 37.5°under these conditions.

Metabolism oj Brain Tissue During and Ajter Incubation at 45°: Experi­ments with brain slices revealed in principle the same result of heat as withtumour tissue (Table VII). The increase of respiration and aerobic glycol­ysis was especially remarkable with rat brain. The effect on the Pasteurreaction is seen again, persisting even after the transfer to 37.5°.

Susceptibility oj Different Tissues to Heat: For a possible therapeuticapplication of heat, the question is of importance whether or not tumour cellsare more susceptible to heat than normal cells. The respiration of various

618 FRANK DICKENS, STANLEY F. EVANS, AND HANS WEIL-MALHERBE

tissues was therefore measured at 450 until it had fallen to a small constantfraction. With most tissues a short initial rise of respiration was observed,followed by a more or less sudden fall. Fig. 4 shows the respiration of varioustissues expressed in Q-values, calculated from the readings for twenty-minuteintervals, except for the first two points, which are based on ten-minutereadings. The readings began ten minutes after placing the manometers inthe bath. Fig. 5 shows the respiration at 4S0 expressed in percentages of thenormal respiration at 37.50 of the same tissue. The highest relative increaseis observed with brain. Although the curves of all tissues are very similar,tumor tissue shows the slowest decline of respiration. After seventy minutes

60 100 120

FIG. 4. RESPIRATION OF DIFFERENT RAT TIsSUES AT 45°, EXPRESSED IN Qo: VALUES(PHOSPHATE SALINE)

A = Brain. B = Kidney cortex. C = Jensen sarcoma. D = Liver. E = Testis.

the respiration of tumour has fallen to 50 per cent of its normal value whilethat of most other tissues has fallen to about 20 per cent. Although this re­sult was obtained twice, it may not be justifiable to generalise from it, since wehave seen that the susceptibility of tumour respiration to heat is very variablein different tumours. But on the other hand, there seems to be little groundto assume any higher susceptibility to heat in tumour than in normal tissuein vit,o.

In vivo other factors, such as vascular supply and the defensive mechanismof the host, have also to be considered. It is therefore still possible that in vivothe tumour may be more vulnerable than normal tissues j this problem is atpresent being investigated.

THE ACTION OF SHORT RADIO WAVES ON TISSUES 619

SUMMARY AND CONCLUSIONS

The action of ultra-short waves of 3.4 and 7.2 metres wavelength on themetabolism and growth of tumour tissue has been studied in vitro.

An adaptation of the Warburg apparatus made it possible to eliminate anyheat effects during the exposure and so keep the tissue under physiologicalconditions of temperature, medium and gas exchange.

(1) No effect on metabolism was observed after the tissue had been ex­posed to intense fields during one to two hours. It is concluded that the

Percentage ofnormal resp,

at 37.5°240

200

160

120

80

40

Min. 10 20 40 60 80 100 120

FIG. 5. RESPIRATION OF DIFFERENT RAT TISSl:ES AT 45°, EXPRESSED IN PERCENTAGE OF

RESPIRATIOS OF CONTROL SAMPLES KEPT AT 37.5° (PHOSPHATE SALINE)

A = Brain. B = Jensen sarcoma. C = Kidney cortex. D = Testis. E = Liver.

effects on metabolism described by Reiter (10) were really due to heat andnot to a " specific action."

(2) Tumour tissue, after exposure to the waves in vitro, showed no in­hibition of growth when transplanted into animals. The total energy applied(intensity of the field X duration of exposure) was much greater in theseexperiments than it would have been possible to apply in the living animal.

(3) The action of heat on the metabolism of tumour and brain tissue hasbeen studied. At 45°, respiration, at first increased in most experiments,shows a variable degree of resistance to heat: whereas it is quickly andseverely damaged in some cases, it is but little affected in other cases by one

620 FRANK DICKENS, STANLEY F. EVANS, AND HANS WEIL-MALHERBE

hour's exposure. Anaerobic glycolysis, after an initial increase, tends to fallto a variable extent. The most constant and characteristic feature of theseexperiments is the inhibition of the Pasteur reaction, the aerobic glycolysisbeing increased to the anaerobic value or even above. This inhibition of thePasteur reaction persists as a sign of irreversible damage even when the tissueis transferred to the physiological temperature of 37.50

• At 500 both metab­olism and growth are severely injured even after exposure for five minutes.

The results of Westermark (22), who found that the glycolysis of tumourswas more susceptible to heat than the respiration, are shown to be due to anunphysiological medium and inadequate gas exchange during incubation.

Tumour tissue is in vitro not more susceptible to heat than normal tissue.The results contained in the present paper show that there is no detectable

in vitro effect of ultra-short waves on metabolism or growth, when heat effectsare rigorously eliminated. On the other hand, the action of heat explains theresults obtained by Reiter. Work at present in progress on the in vivo effectsof ultra-short waves will be presented in a later paper, and the nature of thecurative action on tumours discussed.

NOTE: We wish to express our indebtedness to Prof. W. E. Curtis, F.R.S., of ArmstrongCollege, Newcastle-upon-Tyne, for his deep interest in these experiments.

This research was undertaken at the request of the Scientific Advisory Committee ofthe British Empire Cancer Campaign. We are grateful to the Campaign for a special Short­Wave Research Grant which made this work possible.

REFERENCES

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bioI. 91: 626, 1924.6. SCHERESCHEWSKY, J. W.: U. S. Pub. Health Rep. 43: 927,1928.7. SCHERESCHEWSKY,]. W.: Radiology 20: 246, 1933.8. PFLOMM, E.: Miinchen, med. Wchnschr. 77: 1854,1930.9. REITER, T.: Deutsche med. Wchnschr. 59: 160, 1933.

10. REITER, T.: Deutsche med. Wchnschr. 59: 1497, 1933.11. ROFFO, A. E.: Bol. Inst. de med. exper. para el estud. y trat. del cancer 9: 210,1932.12. ROFFO, A. E.: Bol. Inst. de med. exper. para el estud. ytrat. del cancer 9: 542,1932.13. ROFFO, A. E.: Bol. Inst. de med. exper. para el estud. y trat. del cancer 10: 538, 1933.14. HASCHE, E., AND COLLIER, W. A.: Strahlentherapie 51: 309, 1934.15. LADNER, A. W., AND STONER, C. R.: Short Wave Wireless Communication, London,

Chapman and Hall, 1932, Chapter XI.16. WARBURG, 0.: Metabolism of Tumours, London, Constable & Co., 1930.17. DICKENS, F., AND SIMER, F.: Biochem. J. 25: 973, 1931.18. PATZOLD, J.: Strahlentherapie 54: 362, 1935.19. KREBS, H. A., AND HENSELEIT, K.: Ztschr. f. physiol. Chern. 210: 33, 1932.20. WARBURG, 0.: Biochem. Ztschr. 152: 51,1924.21. DICKENS, F., AND GREVILLE, G. D.: Biochem. J. 29: 1468, 1935.22. WESTERMARK, N.: Skandin. Arch. f. Physiol. 52: 257, 1927.23. KUBOWITZ, F.: Biochem. Ztschr. 204: 475, 1929.