Phofochernisfry ondPhorobiology. 1969. Vol. 10, pp. 445-449. Pergamon Press. Printed in Great Britain

RESEARCH NOTE

PHOTOREACTIVATION OF U.V. REACTIVATION IN PROTOZOA

JOHN CALKINS AND GASTON GRIGGS Departments of Radiology and Zoology, University of Kentucky, Lexington, Ky. 40506, U.S.A.

(Received 16 April 1969; in revised form 5 June 1969)

INTRODUCTION IN A PREVIOUS communication [ I] we noted that phenomena similar to U.V. reactivation of phage and yeast could be induced in protozoans under favorable conditions. Both Kneser, Metzger and Sauerbier (u.v. reactivating phage) [2] and Elkind and Sutton (u.v. reactivating yeast) [3] observed that U.V. reactivation could be photoreserved i.e. ‘photoreactivated’. We have also tested the photoreactivation capacity of U.V. reactivation of protozoa and find that protozoans which can photoreactivate the lethal effects of U.V. also photoreverse U.V. reactivation under favorable conditions. Our strain of Colpoda deficient in ability to photoreactivate the lethal effects of U.V. like- wise fails to demonstrate photoreversal of u.v. reactivation.

METHOD The methods of irradiation, culture, and analysis were essentially the same as

described in our previous report[l]. We have X-irradiated groups of log phase proto- zoans, subdivided the groups and administered various doses of U.V. light to induce U.V. reactivation, then further subdivided the groups and administered various doses of photoreactivating light. The U.V. reactivating radiation was, in all cases, given after the X-rays. Caffeine treatment immediately after U.V. or X-ray irradiation is required to demonstrate U.V. reactivation in Tetrahymena [ 11. Caffeine probably absorbs a significant amount of U.V. in our irradiation technique. Therefore, the absolute (but not the relative) dosimetry of U.V. to Tetrahymena was not precisely determined. All Colpoda experiments were conducted without caffeine and Colpoda experiments were not subject to the caffeine induced uncertainty in dose. The dose rates were 125 kradsl min for X-irradiation, 54 ergs/mm2/sec (incident on the medium containing animals: for U.V. irradiation; photoreactivation dose rates were not measured. The photo- reactivation light source was a bank of 8 closely spaced fluorescent lights 5 cm ovei the samples being reactivated; photoreactivation was at room temperature. 20-30 mic of photoreactivation under our conditions produces the maximal reversal of U.V. lethality.

After irradiation the animals were isolated one by one in drops of nutrient medium Escherichia coli or Aerobacter aerogenes were used as food bacteria; the protozoan5 will grow to 200-500 animals per drop. The experiments were observed until the isolated animal either could not be found in the drop (lysed) or had grown to large numbers. N o time limit was placed on recovery, but it is rare for an irradiated anima

445

446 J. CALKINS and G. GRIGGS

to live more than two weeks without resuming growth (surviving) or lysing (dying). Unirradiated animals show extremely high viability under our growth conditions. Over 1000 unirradiated animals have been singly isolated to test the control mortality; not one failed to grow to the limits of food in the drops. Since all isolated animals are expected to survive, no ‘normalization’ of surviving fraction has been applied. The caffeine concentration (0.02 per cent) was severely limiting on our irradiated animals but did not inhibit the survival of growth rate of unirradiated (log phase) animals. Sensitivity to caffeine is a complex response which will be considered in detail in future reports; however, the 0.02 per cent caffeine limits survival or growth rate of unirradiated Tetruhymena only after prolonged starvation and during the time of the first few post- starvation divisions (unpublished observations).

We have tested the response of Cofpoda cucullus (obtained from the Cambridge Botany School, Cambridge, England) for comparison with the Mammoth Cave strain of Colpoda.

RESULTS A N D DISCUSSION Figure 1 illustrates the effect of photoreactivation on C. cucullus given immediately,

5 and 20 min after U.V. reactivation. The photoreactivation of U.V. reactivation is not extremely large in C. cucullus and is dependent on the time between U.V. and photo- reactivation; however, the U.V. reactivation response is shifted to higher U.V. dose levels just as Kneser, Metzger and Sauerbier [2] observed with the photoreactivation of U.V. reactivation of phage. The peaked nature of the U.V. reactivation response

0 5 -

0 2 - c 0

0 - 0

u- 01-

c

2 0 0 5 -

c 7

- 7 -

X my dose, krods

k ig. I . Photoreactivation of U.V. reactivation in Colpodu errcullus; 7, X-ray survival without subsequent U.V. treatment; V, survival after 125 krads followed by the indicated doses of U.V. and no subsequent photo- reactivation. X; survival after X-ray, U.V. and a 30 min photoreactivation treatment beginning immediately atter U.V. 3. X-ray + U.V. + photoreactivating light 5 min after u.v.; 0 X-ray + U.V. + photoreactivating light 20 min after U.V. The vertical bars indicate typical standard errors; 28 singly isolated animals were used to determine each point in this figure. Points with downward pointing arrows indicate no animal in the

tested group survived but are plotted at the survival level corresponding to one survivor per group.

Photoreactivation of U.V. reactivation in protozoa 447

further complicates the relationship. At U.V. doses below the peak, photoreactivation lowers survival, while at U.V. doses above peak, photoreactivation increases survival.

Figure 2(a) illustrates the response of the Mammoth Cave Colpoda to photo- reactivating light after U.V. irradiation alone. Figure 2(b) shows the results of corre- sponding photoreactivating treatment of C. cucullus. I t is evident that C . cucullus is able to reduce U.V. lethality by the photoreactivating treatment while the cave species cannot.

u v hse,ergs/rnrn'x id3

Fig. 2. Survival of U.V. irradiated Colpoda with and without photoreactivating light treatment. A, survival without photoreactivating light; 0, survival after 30 min of photoreactivating light given approx. 18 min after u.v.; bars and arrows have the same significance as in Fig. I . (a) cave Colpoda (b) Colpoda cucullus.

Figure 3 shows U.V. reactivation of the Colpoda isolated from Mammoth Cave. The data illustrates a lack of effect of photoreactivating light administered after 'u.v. reactivating' light.

Figurz 4 illustrates the response of Tetruhymena to three successive treatments ( 1 ) X-rays combined with caffeine (2) with U.V. added and (3) followed by photoreac- tivation at various times. As in our previous publication we find U.V. reactivation to increase survival. Photoreactivation immediately after U.V. reactivation has little effect; however, a delay of 20 min permits an effective reversal of the U.V. reactivation. If still longer times are allowed between U.V. and photoreactivation then the U.V.

reactivation cannot be reversed. We believe that the (20 min) time requirement between U.V. and photoreactivation

could be explained as a time for attachment of PR enzyme to the U.V. induced lesions responsible for U.V. reactivation, analogous to (but somewhat longer than) attachment times observed by Harm, Harm and Rupert[4]. The loss of photoreactivibility at longer times would probably indicate that the induction of the repair process had proceeded beyond the point where it could be reversed by repair of DNA. The time during which U.V. reactivation can be reversed, although rather short, is long enough to permit substantial repair through the non-activated ( N ) repair system in Tetruhymenu (see Fig. 2 of Calkins [ 5 ] ) and suggests that the total response of this organism is a dynamic combination of repair events.

448 J. CALKINS and G. GRIGGS

0.5 -

c 0 2 - I?

e IT 01-

c 0

r

f ? > L

0 0 5 -

O o 2 t G - 0 100 200 300

X-ray dose,krads

Fig. 3. U.V. reactivation of X-ray lethality and lack of photoreversal in Cofpoda sp. isolatedfrom Mammoth Cave, Ky. Symbols have the same significance as in Fig. 1, i.e. V, X-ray alone; V, 75 krads X-ray+u.v. (no PR); X, X-ray + U.V. + PR immediately after u.v.; 0, X-rays + U.V. + PR 5 min after u.v.; 0, X-ray +

I01

0 5

0 2

= 01 P

e ~ 0 0 5 G

c 0

L

> > 3

- L

0 0 2 cn

0 01

0 03:

U.V. + PR 20 min after U.V.

X+CF+U V X+CF+U V +PR

(+cF/

0 5 10 15 20

L 0 5 10 15 20

U.V. dose,sec - 25 50

-ray dose, krads

Fig. 4. Photoreactivation of U.V. reactivation in Tetrahymena pyriformis. Large groups of log phase animals were X-irradiated, treated with caffeine (CF), subdivided and given the indicated doses of u.v., further subdivided and given the indicated doses of photoreactivating light (PR) immediately after U.V. and after

the indicated time intervals. The vertical bars indicate typical standard errors of the survival values.

These experiments fit our proposal (1) that there are two ‘dark’ repair systems and U.V. reactivation ‘turns on’ a repair system (the ‘T’ system, a system which prob- ably involves induced protein synthesis [ 5 ] ) which then repairs X-ray damage; they

Photoreactivation of U.V. reactivation in protozoa 449

show that within certain limitations, the triggered repair system can be ‘turned off by the same mechanism (photoreactivation) widely observed to eliminate lethal U.V. injury in these and other organisms. U.V. reactivation in protozoa resembles U.V. reactivation in yeast and phage in a significant characteristic, i.e. its photore- activibility .

We would draw especial attention to certain aspects of these experiments. Photo- reactivation of U.V. reactivation is a transitory response in a complex set of interactions among multiple interacting repair systems. The recent observation that U.V. irradiation induces a DNA polymerase inTetrahymena[6] and the recurring suggestion that E. coli possess at least two ‘dark’ repair systems, one of which is dependent on induced protein (possibly repair enzymes) synthesis [2,7,8] renders our interpretation even more plausible. We suggest that more complex models of radiation response are needed to explain the conflicting and paradoxical responses so frequently observed by different investigators.

Acknowledgements -This investigation was supported by the James Picker Foundation on recommendation of the Committee on Radiology, National Academy of Sciences-National Research Council. We thank Dr. S. Gittleson for supplying us with the cave Colpoda strain and Mark Lantz and AM Williams for very able assistance in these studies.

REFERENCES 1. J. Calkins and G. Griggs, Photochem. Photobiol. 10,61(1969). 2. H. Kneser, K. Metzger and W. Sauerbier, Virology 27,2 13 ( 1965). 3. M. M. EIkind and H. Sutton, Radiation Res. 10,296 (1959). 4. W. Harm, H. Harm and C. S. Rupert, Mutation Res. 6,37 1 (1968). 5. J. Calkins, Intern. J . Radiation Biol. W, 283 (1967). 6. 0. Westergaard and R. E. Pearlman, Exp. Cell. Res., 54,309 (1969). 7. J. On0 and Y . Shimam, Virology 29,295 ( 1966). 8. K. Suzuki, E. Saito and M. Morimyo, Photochem. Photobiol. 9,259 (1969).