Phofochem’srry andPhotobiolo,qy. 1969. Vol. 10, pp. 61-66. Pergamon Press. Printedin Great Britain

RESEARCH NOTE

U.V. REACTIVATION OF PROTOZOA JOHN CALKINS and GASTON GRIGGS

Departments of Radiology and Zoology, University of Kentucky, Lexington, Ky. 40506, U.S.A

(Received 5 November 1968; in revised form 4 February 1969)

Two DIFFERENT biological responses have been termed ‘u.v. reactivation.’* Weigle [ 11 discovered that low doses of U.V. light delivered to the host bacterium promoted the survival of U.V. inactivated phage. This reactivation of viruses, termed U.V. reactivation (UVR), has been repeatedly observed[2] and differs from the more common type of virus reactivation, Host Cell Reactivation (HCR), especially with regard to the ability of UVR to function on single stranded DNA[3,4]; UVR of phage has also been ob- served following X-irradiation of the host bacterium [4]. Elkind and Sutton [5] found that irradiation of yeast cells with U.V. light either before or after X-irradiation tended to promote survival. These phenomena may be called, following Elkind and Sutton, U.V. protection and U.V. reactivation. The quantitative indifference as to whether the U.V. treatments were given before or after X-irradiation strongly suggested that the same mechanism was involved in these two phenomena. The type of reactivation observed by Elkind and Sutton has recently been repeated using yeast [6,7].

It has frequently been observed that HCR, a prominent form of radiation repair in bacteria and phage, is sensitive to normally-non-lethal concentrations of caffeine [8], while U.V. reactivation appears relatively resistant to caffeine but sensitive to protein synthesis inhibitors [4]. A caffeine sensitive recovery system has also been observed in protozoa[9,10] and in mammalian cells[ll]. These experiments have suggested that protozoans exposed to low doses of radiation are primarily dependent on the caffeine sensitive recovery system as distinguished from the relatively caffeine resistant repair system which functions after higher radiation doses [9,10]. The caffeine sensitive component of protozoan recovery has been termed the ‘N’ system (as it appears to be a normal and non-activated component of all cells in either log or stationary phase); the caffeine insensitive component of protozoan repair, which functions much better in log phase animals, has been termed the ‘T‘ system (denoting the necessity of this system to be triggered. or activated or induced by radiation damage or some equivalent stimulus). Some of the evidence suggesting that the repair systems of various micro- organisms are strikingly similar is described in [9] where it is specifically suggested that:

The ‘ T system of Tetrahymena = UVR of E. coli The ‘N’ system of Tetrahymena = HCR of E. coli

and photoreactivation (PR) is similar in the two organisms.

*We have considered ‘reactivation’ from or by radiation treatment to imply a measurable reversal of the pre-existing radiation damage. Radiation survival curves where (with increasing dose) survival first falls, then rises and finally falls again, (note Fig. 2(B)) are, by definition, instances of radiation reactivation. The type of reactivation considered in this paper is fundamentally different from photoreactivation or ‘liquid holding’ type reactivation where the reactivating agent is a relatively mild and usually completely harmless treatment. X-ray and U.V. (germicidal) reactivation are instances of reversals of radiation damage by agents that are almost invariably harmful to living organisms.

61

62 J. CALKINS and G . GRIGGS

The U.V. reactivation of phage is clearly mediated by the post-irradiation repair processes. A direct modification of the primary lesions (by the reactivation treatment) can hardly account for variations in response since the irradiated virus need not be present in the host bacterium during the U.V. reactivation process. The U.V. reactivation phenomena in yeast (and protozoa) is somewhat more equivocal. Since the inactivation and reactivation processes involve one and the same organism one cannot rule out the possibility that the ‘reactivating’ radiation may modify the initial radiation injury to reduce its effective magnitude. However, we suggest that all U.V. reactivation pheno- mena may derive from the same process, the activation of a repair system.

Ono and Shimazu[4] suggest that “. . . UVR can be initiated by induction of a new reactivating enzyme system after U.V. irradiation”. and Kneser, Metzger and Sauerbier [8] refer to the ‘triggering’ of UVR. Since it was proposed that protozoa have a triggered or induced repair mechanismr9, lo], ‘u.v. reactivation’ should also be manifest in protozoans. This report presents data indicating that such is indeed the case if the proper conditions are satisfied.

METHOD

The irradiation and culture of the protozoans and the strain of Tetrahymena pyriformis were as previously described [9,10]. The strain of Colpoda sp. (recently isolated from Mammoth Cave by Dr. S. Gittleson and Mr. R. Hoover) has proven to be interesting in that it appears to lack the capacity to repair ultraviolet radiation injury through the photoreactivation system. All animals were grown in a baked lettuce and trypticase broth medium with E. coli as the food bacterium. Log phase protozoans were irradiated using soft (23 KV effective photon energy) X-rays at a dose rate of 125 krad./min; 253-7nm U.V. was at 20ergslmm21sec, when caffeine was used it was added (to the normal medium) after U.V. irradiation at a concentration of 0.02 per cent; unirradiated animals show no reduction of viability in medium containing this concen- tration of caffeine. Control and irradiated animals were isolated in a small drop of med- ium and observed intermittently until they had either lysed or grown to the limit of the food in the drop. The surviving fraction was computed; all Colpoda and almost all Tetrahymena results were determined from animals isolated one per drop of medium. Where very low survival was anticipated part of the irradiated Tetrahymena were isolated one per drop and some were isolated in groups of 2 per drop. Many experi- ments have shown that single or multiple isolation has no effect on the probability of survival of the individual irradiated animal [ 101.

The Colpoda were first X-irradiated then U.V. irradiated. Since ‘u.v. reactivation’ of Trtrahymenu can only be demonstrated in caffeine treated organisms the order of irradiation was reversed. The inhibitory effect of caffeine on recovery is rapidly lost [9] so caffeine must be added immediately after the first irradiation. X-irradiation can be given with caffeine present but, because of its high absorption of 253-7 nm, U.V. irradia- tions without caffeine are more desirable. Tetrahymena, therefore, were given U.V. doses first followed by immediate exposure to 0.02 per cent caffeine. Irradiated animals were then held at ice water temperature until they could be X-irradiated ( 15-30 min).

RESULTS A N D DISCUSSION

Figure 1 shows the dose-response relation for X-irradiated Colpodu and the effect of U.V. light added to the X-irradiation at 2 different dose levels. In all cases, rather than

U.V. reactivation of protozoa 63

\ t I 0 125 260

X-RAY DOSE IN KRAOS

Fig. 1. The survival of log phase Cofpodu sp. following X-irradiation (heavy curve) (dose rate 125 krad./min) and X-irradiation plus various doses of U.V. light (dashed line) dose rate 20 ergs/mm*/sec). U.V. doses are indicated on the dashed axis. Bars indicate typical standard error of the surviving fractions. Each point was determined by observing the survival of 28-56 animals. Points with downward pointing arrows indicate no survivor among the irradiated animals and are plotted at the survival level that would have been observed had one animal in

the group survived.

causing additional injury, lower levels of U.V. dose have improved the survival of X-irradiated animals.

Figure 2(A) shows the effect of U.V. irradiation of Tetrahymena and the effect of the same doses of U.V. light combined with 50 krad. of X-irradiation. Figure 2(B) shows the same treatments illustrated in Fig. 2(A) except that after U.V. irradiation, animals were held in the normal growth medium containing 0.02 per cent caffeine. Without caffeine Tetrahymena shows an essentially additive effect of X-ray and U.V. (Fig. 2(A)) but when the X-irradiation is combined with caffeine a large (-9 fold) increase in survival is produced by addition (preirradiation) of increments of U.V. dose (Fig. 2 (B)).

We believe the demonstration of U.V. reactivation requires special insight into the inactivation and reactivation processes. There are several requirements for observing radiation induced reactivation: ( 1) the irradiated population to be reactivated (the ‘primed population) must possess an activated repair system, (2) there must be sub- stantial injury, (3) but, not sufficient to activate the repair system, and (4) the ‘reactivat- ing’ radiation must be a relatively efficient activator, i.e. the activating action must be more beneficial than the additional radiation injury it produces.

X-rays and U.V. appear to differ in the relation of their activating capacity to their capacity to inflict further damage, U.V. being the more efficient activator. U.V. reactiva- tion of yeast is manifest by an actual increase in numbers of cells surviving a given X-ray dose with the addition of a small U.V. dose [5-71. From the X-ray survival curves in these reports we can deduce that if the U.V. dose were replaced by increments of X-rays fewer cells would survive. However, one should note that in most instances the yeast cells showing U.V. reactivation had been ‘primed for the phenomena by X- irradiating to a dose level producing an abrupt break in the X-ray survival curve; after the priming dose further increments of X-ray, although still producing a net de-

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64 J . CALKiNS and G. GRIGGS

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X.50 KRADS

g 010

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0 400 a00 lz00 1ooo ULTR*VKKFT DOSE IN ERGS/&

Fig. 2. (A) Survival of log phase Tetruhyrnenapyriformis after U.V. irradiation and U.V. followed by X-irradiation (50krad.); (B) plot similar to (A) except for caffeine (CF) treatment. Bars indicate standard error of surviving fractions; 28-1 60 animals were used to determine each point. Points with arrows and dose rates were as in Fig. I . Labels on curves indicate treatments

and the order of their administration.

crease in survival, were notably less efficient for lethality than lower increments of X- ray dose. Patrick and Haynes [6] (the only yeast study not showing a break in the X-ray survival curve) still required a high priming dose (1 00 krad.) to demonstrate U.V. reac- tivation : U.V. after a lower priming dose (50 krad.) did not produce a net reversal of X-ray injury. The details of the process of ‘priming’ a population for U.V. reactivation are clearly relevant to the subsequent U.V. reactivation.

We have selected conditions which we think most favorable for the demonstration

U.V. reactivation of protozoa 65

of U.V. reactivation of protozoans. Figure 1 shows that Colpodu responds to X-ray in a manner which could be interpreted as a two component population (about 30-50 per cent sensitive and the remainder resistant). By selecting the proper X-ray ‘priming’ dose it appears that both components show U.V. reactivation. Increments of X-ray dose above the lower priming dose produce a slight amount of ‘X-ray reactivation’ but sub- stantially less than the U.V. reactivation.

The demonstration of U.V. reactivation in Tetruhymenu is even more subtile than in Colpodu. The non activated (N) repair system in Tetruhymena is so efficient there is no killing at radiation doses below the T system threshold. When caffeine is used, survival falls to a low level at low radiation doses and further irradiation restores a large fraction of the irradiated population to full viability[9,10]. We interpret this restoration of survival capacity to activation of the T system. Figure 2(B) shows that the surviving fraction of a Tetruhymenu population is reduced to 5 per cent by the com- bined action of 50 krad. of X-rays plus a postirradiation growth in 0.02 per cent caffeine containing medium. This treatment is near the low dose minimum in the X-ray survival curve[9]. Additional X-ray dose would improve survival and Fig. 2(B) shows U.V. irradiation added to the X-ray and caffeine treatment also improves the survival. Technically Colpoda shows ‘u.v. reactivation’ while Tetruhymenu shows ‘u.v. protec- tion’ or ‘X-ray reactivation’ (since the X-rays were administered after the u.v.). The interpretation we propose suggests that the order of irradiation is of little importance; the significant factor being the relation of the summation of the two radiation stimule relative to the threshold for induction of the triggered repair system, ‘u.v. reactivation’ seems the most appropriate terminology.

We suggest the observations we report can be explained assuming that the mechan- ism of U.V. reactivation of protozoa is the induction or activation of a repair system by radiation damage or some equivalent stimulus as has been suggested for U.V. reactiva- tion of phage[4,8]. We also note the induction of X-ray resistance by pre-irradiation with U.V. observed in yeast [13] and E. coli[ 141 clearly fits our interpretation of radiation response, i.e. the U.V. pre-irradiation inducing the T system which confers added resistance to the subsequent X-irradiation.

Observations from the literature[2,4,8], our results reported here and previously [7,9,10, 121 all suggest a multisystems model of response, i.e. triggered repair+ non- triggered repair + photoreactivation (The T-N-PR model). With modifications for rela- tive effectiveness of the different components in different organisms this model can explain and predict the behavior of a wide variety of organisms.

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

REFERENCES I . J . J . Weigle, Proc. NatlAcad. Sci., U S . 39,628 (1953). 2. C. S. Rupert and W. Harm, Advan. Radiation Biol. 2, I (1966). 3. E. S. Tessman and T. Ozaki, Virology 12.43 1 ( 1 960). 4. J . Ono and Y. Shimazu, Virology 29,295 ( 1 966). 5 . M. M. Elkind and H. Sutton, Radiation Res. 10,296 (1959). 6. M. H. Patrick and R. H. Haynes, J . Bacteriol. 95, 1350 ( 1 968). 7. J. Calkins and W. Todd, Intern. J . Radiarion Biol. 14,487 (1968).

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8. H . Kneser, K. Metzger and W. Sauerbier, Virology 27.2 13 ( 1 965). 9. J. Calkins, Intern. J . Radiation Biol. 13,283 (1967). 10. J. Calkins, Photochem. Photobiol. 8, 1 15 ( 1 968). I 1. A. M. Rauth, Radiation Res. 31, 12 1 ( 1 967). 12. J. Calkins, Intern. J . Radiation Biol. 12,297 (1967). 13. A. Sarachekand W. H. Lucke,Experentia9,374(1953). 14. K. C. Smith and A. K. Ganesan, Radiation Res. Suppl. 6 , 2 18 ( 1 966).