P/~ofoc/renris,ry and Photobiology. 1964. Vol. 3, pp. 143-151. Pergamon Press Ltd. Printed in Great Britain

THE LETHAL EFFECTS OF RADIATION ON SIX SPECIES OF PROTOZOANS

JOHN CALKINS* M. D. Anderson Hospital and Tumour Institute, The University of Texas,

Houston, Texas, 77025

(Received 18 November, 1963)

Abstract-The lethal response of six strains of protozoa to both ionizing radiation and U.V. light has been established. Values have been determined by observation of isolated animals and death was considered to be failure of irradiated individuals to produce unlimited progeny regardless of the actual time of death following irradiation. The strains were selected to give a large variety of size. Lethal doses of U.V. radiation have been determined in the same units used for ionizing radiation (the rad). The results reported here have been supplimented by values from the literature and plotted in the form of a graph of LD-50 value versus individual organism size.

PROTOZOA are exceptionally good subjects for radiaticn research and have been extensively studied. They grow rapidly in simple media; they are single-celled animals which can grow to large numbers by simgle binary fission. These single cells are large enough to be indivi- dually observed following irradiation; in some cases, organelles may be transferred among experimental animals. The properties of protozoa have provided, and doubtless will continue to provide, information on the radiation response of biological systems which can only be obtained with great difficulty in other organisms. At times, the abundance of information available from irradiated protozoa tends to obscure some of the more basic aspects of radiation response. Radiation research with the smaller microorganisms, such as bacteria and viruses, has proceeded by means of techniques which, in essence, define survival as the ability of an irradiated individual to produce an unlimited number of progeny. The size of many protozoa permits actual observation of the presence or absence of a living animal; thus, a number of radiation studies have used criteria of survival similar to those widely used for higher animals, i.e. survival of irradiated animals for some arbitrary period of time following irradiation. Although thih criterion of survival provides some information regarding the radiation response, it has two serious defects. First, it tends to imply that “survivors” thus defined are normal animals. It is well known that, following irradiation, protozoa often “survive” for long periods before dying; occasionally there are a few divisions before death. Secondly, the use of the arbitrary time criterion of survival frustrates attempts to compare radiation responses of protozoa. There is no obvious period of time which an irradiated animal should live in order to be considered a survivor. Various investigators have chosen a wide variety of times.

*Present address: Department of Radiology, The University of Kentucky, Lexington, Kentucky. 143

144 JOHN CALKINS

While there are many obstacles which prevent the use of the microbiological (unlimited numbers of progeny) criterion of survival with higher animals, it is not excessively difficult to require this criterion for irradiated protozoa. It is reasonable that a survivor should be sufficiently viable to multiply after irradiation to numbers limited only by the food supplied. The microbiological criterion of survival permits comparison of the radiation response of different varieties of protozoa, or of the same protozoa irradiated with different radia- tions which might produce lysis of the animals at quite different times following irradiation.

By using the microbiological criterion of survival, the radiation response of six proto- zoans following ultraviolet and X-irradiation will be reported. In this paper only the simple survival or nonsurvival of the irradiated animals will be considered; other observations on the behaviour of irradiated animals were made in the process of obtaining the survival data and will be reported elsewhere. Data from the literature have been incorporated into a graph (Fig. 3) in which it is attempted to compare the lethal radiation response of the widest possible variety of protozoa. Not all information found in the literature uses the microbiological criterion of survival; only those data are included in Fig. 3 which, from the description of the experiment, seem to be approximately equivalent to results obtained when the microbiological criterion is used.

MATERIALS and METHODS

Biological. The strain of Chilomonas paramecium was purchased from the Carolina Biological Supply Company. The remainder of the animals were isolated from Texas water in the spring of 1960 and have been grown as stock cultures since that time. Some specimens have been stained with nuclear stains and also silverline staining but identifica- tion of the animals has been made primarily on the basis of size and morphology. The strains have been identified as the four ciliates Paramecium aurelia, Tetrahymena pyriformis, Euplotes patella, and Cyclidium litomesium, and a small flagelate of the Monas sp. The animals were selected to give a range of sizes, particularly including the smaller protozoa which have not been extensively examined for radiation response.

All animals grow in a bacterized lettuce medium similar to that described by Sonne- born(21) for growing Paramecium aurelia. Paramecium, Tetrahymena, and Cyclidium were grown in medium inoculated with E. Coli Blr. Euplotes, Chilomonas, and Monas grew in media without specific inoculation of bacteria; however, since bacteria were not eliminated from the cultures, these three carried their own bacteria into the media. Euplotes grew best in a medium also containing Chilomonas parameciutn.

The experimental results reported here were obtained from observations of isolated irradiated animals. The animals were isolated in double depression slides; the slides were placed in 15-cm petri plates, which were stored in stainless steel racks in a large animal jar. A small amount of water was placed in the bottom of the animal jar and the jar was covered by a tight-fitting lid made of Styrofoam, By this method, the evaporation of water from the medium containing isolated animals was maintained at such a low level that the animals could be left for weeks without further attention. One petri plate holds seven double depression slides and a stainless steel rack holds seven petri plates. Thus, one animal jar contains about 100 isolated animals. Experimental numbers could conveniently be made to coincide with full petri plates or jars.

Irradiated animals were usually observed daily following irradiation. Only when several days or weeks had elapsed following irradiation, were longer intervals between observations

The lethal effects of radiation on six species of protozoans 145

used. It seldom takes longer than three weeks to determine if an irradiated animal will survive in the microbiological sense.

The irradiated protozoa were found to be quite sensitive to traces of soap on the de- pression slides. This difficulty was eliminated by washing the slides in water only. The slides were then oven-dried and before use they were wiped with a cloth greased with silicone grease which tends to prevent the media from wetting the slide and eliminates the difficulties encountered with slides washed with soaps.

Physical. A General Electric Maxitron 250 X-ray unit was used for X-irradiation. The animals were irradiated in 3 ml of solution in the bottom of a flat-bottomed vial. The 3 ml made a layer 1 cm deep and the X-rays were incident through the bottom of the vial. An additional filter of 0.25 mm A1 was added. The machine was operated at 250 kVp and 30 mA, which produced a dose rate of 14.9 krad/min. The HVL of the beam was measured as 4.5 mm of A1 corresponding to an average energy of 39kV. The ionizing radiation dosimetry was done with ferrous sulfate dosimeter by procedures indicated by Shalek ef al. (I9). Since the X-ray machine would kill animals as a result of heating during long irradiations, an ice container was placed around the irradiation vial to maintain a temperature below 20°C. during irradiation. Following X-irradiation animals were iso- lated into unirradiated medium in the depression slides. Fresh medium was added as required during observations.

Ultraviolet irradiations were from GE 15 W germicidal lamps. A variety of intensities were used, which ranged from about 5 to about 20 erg/mm2/sec depending on the radiation response of the irradiated animal. Doses, in ergs/mm2, were determined with a vacuum photocell which had been calibrated from an NBS Standard Lamp by means of a varistor bolometer. Allowance was made for the spectral composition of the output of germicidal lamps; this output was measured with glass filters as 89.5 per cent in the U.V.

Suspension in a dilute salt solution, the method often employed for the U.V. irradiation of biological materials, was found to be unsatisfactory. No salt solution could be found which did not produce a detectable effect on the protozoa when they were suspended in a very low concentration. Some animals in salt solution tended to become sticky (conjugation of Paramecium aurelia is induced by the salt solution) ; so inany animals would have been killed in the process of isolation. It was found that if the normal growth medium was present in a sufficiently thin layer, it would not absorb a significant amount of the incident U.V. light. This condition could be obtained by isolating the animals in a very small drop of growth medium. The drop was then covered by a small square of polyethylene. The polyethylene absorbed and reflected 20 per cent of the incident 2537 A U.V. light. The complete layer of culture medium under the polyethylene absorbed only about 5 per cent of the incident 2537 A U.V. Since the protozoa were under a small fraction of a complete layer of medium, no correction for medium absorption was used. After the aninials were irradiated under the plastic tab in a depression, a couple of drops of un- irradiated medium were added. This method eliminates any transfers of u.v.-irradiated animals.

Observations following U.V. irradiation were made with nonphotoreactivating light. A red filter was used with a low intensity light. The animal jars containing u.v.-irradiated animals were covered with black paper to eliminate room light.

By the methods already discussed, the amount of U.V. light incident on the protozoa which is required to produce some biological effect can be determined. However, a different

146 JOHN CALKINS

type of u.v. dosage measurement is required in order to compare U.V. and ionizing radiation response. The amount of U.V. energy absorbed to produce a given level of biological effect is a quantity which can be compared to the ionizing radiation response. This quantity can be determined from the incident intensity if the average absorption properties of the animals are known. If (1) the average area an animal offers to the U.V. beam, (2) the fraction of photons incident on this area that are absorbed, and (3) the weight of an animal are known, then dosages can be expressed in ergs/gm or in rads ( 1 rad=100 ergs/g), the dosage unit commonly used for ionizing radiation dosimetry.

The factors necessary for dosimetry are tabulated in the results section. The details of the methods used to obtain this information are being published elsewhere. In brief, the method is as follows: The weight and the effective area of animals have been determined from measured dimensions. The fraction of incident light which is either absorbed or scattered was determined by using a Beckman DU Spectrophotometer. The true absorp- tion in relation to the scattering was determined by measuring the scattered intensity at eight angles and by interpelation, the total scattered intensity for all angles is deduced. The true absorption is the difference between apparent absorption and the amount of light scattered by a specimen.

The’ biological end point which seems most appropriate for the lethal radiation effect on protozoa is the LD-50, the radiation dose which kills one half of the irradiated popula- tion. The statistical limits pf the LD-50 (95 per cent confidence limits) have been deter- mined for each experiment and are tabulated with the LD-50 in the results section. These limits are computed by using a “logit” model, a common analysis for biological phenomena which show a threshold-type response. The actual computation of the LD-50 and its

1.0

03

0.2

0. I

0.0s c 0

0

U.

m 5

._ - 0

0.02 - ._ t 1 0.01

0.005

I Monas sp

I I

Telrohymena pyriformis

I I I 1 -

100 200 300 400 500 600 Uose in’ Kllorads

FIG. 1. The X-ray survival curves.

The lethal effects of radiation on six species of protozoans 147

limits was performed on an IBM 1620 electronic computer by using as raw data the dosage and fraction of irradiated animals surviving a given dosage. The program required for this computation was prepared by Doctor Reimut Wette of this institution. The dose- survival relations can be plotted as graphs (Figs. 1 and 2). It is convenient and customary

D o s e in E r q s / m r n Z

FIG. 2. The U.V. survival curves.

to plot dose on a linear scale and the fraction of animals surviving, on a logarithmic scale. While the experiments reported here establish the LD-50 to a precision which seems adequate considering the nature of the subject material, the ultimate slope of the survival curve and the extrapolation number (two parameters of radiation response considered to be of theoretical importance) are not sufficiently defined by these experiments to be specified.

RESULTS and DISCUSSIONS Table 1 indicates the general properties of typical animals. The area tabulated is the

area subjected to a vertically incident beam used in the survival experiments. The elongated animals tend to maintain their long axis in a horizontal plane. Weight estimates are only approximations. In the rapidly growing population used for these investigations, there is a two-fold change in weight following division; in addition, growth factors which were not completely controlled can affect the average size of animals.

Table 2 is a tabulation of the LD-50 values for ultraviolet and X-irradiation. The U.V. results are expressed in both incident ergs/mm2 and in rads (absorbed dose). The values tabulated for LD-50 are the results of the best experiments performed; LD-50 values from other less precise experiments and values found in the literature have been generally consistent with the values reported here.

148 JOHN CALKINS

TABLE 1

Animal Fraction of incident photons

Weight * (g) Area (mp? actually absorbed (260 mp) - ~ ~~

Pavaniecium aureliu

Tetrahymena pyriformis

Cyclidium liromesurn

Euploies palelli

Monas sp.

Chilomonas paramecium

* density assumed 1.05

2.2 x 1 0 - 7 8000

9 -5 x 10-0 2200

2.15 X 10-B 250

1.1 x 1 0 - 7 3 500

1.2 x 10-10 30

3.1 x 10-O 280

TABLE 2

0.70

0.40

0.73

0.50

0.68

LD-50 X-ray LD-50 U.V. LD-50 u.V.* LD-50 u.v.* Animal krad ergs/mm* ergs/animals h a d

Paranrecium aurelia ~ ~~ ~~

281 &34 3660&330 20.4 930

Tetrahymena pyriformis 482&14 778h104 1 *2 J 250

Cyclidium litomesum 143f25 209533 0.021 98

Euploies patella 425f59 58402C1040 14.9 1350

Monas sp. 55j~13 485 2C 265 0.00584 490

Chilomonas paramecium 115A14 690& 110 0.132 430

* The limits on these columns are the same percentagewise as for the ergs/mm* column.

Figs. 1 and 2 show the dose response relation for each strain. Arrows pointing down- ward are plotted at doses at which no irradiated animal lived and are plotted at the survival level that would have been observed if one animal had survived. Curves are fitted to the points by “eye”.

Figure 3 shows the general trend of the lethal effect of ultraviolet and ionizing radiation. Organisms both larger and smaller than protozoa are more sensitive to ionizing radiation and even the response of protozoa seems to show a peak in resistance. The 1i.v. response generally parallels the ionizing radiation resistance. The U.V. LD-SO is usually several times the values for ionizing radiation.

There is no obvious significance to the information plotted in Fig. 3 but the data in this figure represent important aspects of the response of protozoa to radiation. If we hope to understand the factors involved in radiation response of these or other living organisms we need to establish the quantitative response to radiation; Fig. 3 is an attempt to collect and represent such information in a graphical form.

The lethal effects of radiation on six species of protozoans

r I I

4

I

U D -2 e Y Y 0 0 0 0 0

'0 TJ

Y Y E e

0

'0

149

1 50 JOHN CALKINS

TABLE 3. SUMMARY OF RADIATION KILLING OF PROTOZOA

U.V. LD-50 X-ray Animal Weight (9) U.V. LD-50 ergs/mm2 G a d s * LD-50 Krads

Amoeba proteus Aslasia longa Chilomonas paramecium

Colpidium colpoda Cyclidium litomeunt Didiniurn nasuturn Dunaliella salina Entamoeba histolytica Euplotes patella E. raylori Monas sp. Paramecium aurelia

P. bursaria P. calkinsi P. caudalurn

P. multimicronucleaturn P. trichium Pelomyxa carolinensis P. illinoisensis Spirostomunz ambiguum Stentor coeruleus Tetrahymena pyriformis

Tillina ntagna

1 *1 x 10-6 2.7 x 3.1 x 10V

1.5 x 10-7

1.0~10-7 2.25 x 10-9

1 .i x 10-7 4 x 10-7

2.2 x 10-7

2.15 x 10-0

2.7 x

1.2 x 10-10

3 x 10-8 1.5 x 10-8

7 x 10-7

6 x lo-' 4 x 10-8 2 x 10-6

1.25 x 10-6

2 . 4 ~ 1 0 - ~ 3.65 x 10-7

9.5 x 10-9

3 -1 x 10-6 ~~

* Computed using optical properties of similar material where exact data was not available.

Acknowledgement-This work was supported in part by training grant CRT-5047 from the National Cancer Institute of the National Institutes of Health.

R E F E R E N C E S 1 . A. BACK and L. HALBERSTAEDER, Am. J. Roentgenol. Radium Therapy Nucl. 54,290 (1945). 2. J. BKIDGEMAN and R. F. KIMBALL, J . Cellular Conip. Physiol. 44,431 (1954). 3. M. G. BROWN, J. M. LUCK, G. SHEETS and C. V. TAYLOR, J. Gen. Physiol. 16, 397 (1933). 4. E. W. DANIELS, J. Exp. Zool. 130, 183 (1955). 5. E. W. DANIEIS, Ann. N. Y. Acad. Sci. 78, 662 (1959). 6. E. W. DANIELS and H. H. VOGEL, JR., Radiation Research 10, 584 (1959). 7. H. S. DIJCOFP, Physiol. Zool. 30, 268 (1957). 8. A. C. GIESE, J. Cellular Conip. Physiol. 13, 139 (1939). 9. A. C. GIESE, C. L. RRANDT, R. IVERSON and P. H. WELLS, Biol. Buil. 103, 336 (1952).

10. A. C. GIESE and P. A. LEIGHTON, J. Gen. Physiol. 18, 557 (1935). 11. A. C. GIESE, J. Cellular Conip. Physiol. 12, 129 (1938). 12. R. F. KIMBALL, J. Protozool. 5, 151 (1958). 13. R. F. KIMBALL and N. GAITHER, 1. Cetfular Con7p. Physiol. 37, 211 (1951). 14. D. MAZIA and H. I. HIRSHFIELD, Exp. Cell. Research, 2, 58 (1951). 15. M. S. ORD and J. F. DANIELLI, Quart. J. Microscop. Sci., 97, 29 (1956). 16. E. L. POWERS, Ann. N. Y. Acad. Sci. 59, 619 (1955). 17. H. J. RALSTON, Am. J. Cancer 37,288 (1939).

The IethaI effects of radiation on six species of protozoans

18. H. W. SCHOENBORN, Physiol. Zool. 26, 313 (1953). 19. R. J. SHALEK, W. K. SINCLAIR and J. C. C a m s , Radiation Research 16, 344 (1962). 20. D. C. SHEPARD, A. C. GIESE and C. L. BRANDT, Radiation Research 4, 154 (1956). 21. T. M. SONNEBORN, J. Exp. Zool. 113, 87 (1950). 22. C. WELLS, J . Cellular Conip. Physiol. 55, 207 (1960). 23. R. W I C ~ E R M A N , J . Protozool. 8, 158 (1961). 24. R. WICHTERMAN and F. H. J. RGGE, Biol. Bull. 106, 253 (1954).

151