Photochemistry undPhotobiology. 1966. Vol. 5. pp. 787-795. Pergamon Press Lid. Printed in Great Britain

THE DOSE-RESPONSE RELATION FOR THE PHOTO- REACTIVATION OF ULTRAVIOLET INACTIVATED STATIONARY PHASE TETRAH YMENA PYMFORMIS

JOHN CALKINS

Division of Radiobiology, Department of Radiology, The University of Kentucky College of Medicine, Lexington, Kentucky

(Received 6 September 1965; revised 3 May 1966)

Abstract-Some kinetics of photoreactivation of the lethal effect of U.V. light on Tetrahyrnenn pyriformis have been determined. The dose-response relation appears to be first order (single-hit). First-order photoreactivation kinetics have been found in some other systems and are difficult to reconcile with current knowledge about other aspects of photoreactivation.

INTRODUCTION THE average survival of an irradiated population as related to the radiation dose is often termed the inactivation or survival kinetics. By analogy with the inactivation kinetics one may also investigate the kinetics of photoreactivation.

A U.V. irradiated population can be regarded as having three components: organisms which (1) appear to be undamaged (the dark surviving fraction), (2) can be restored to the surviving population by photoreactivating light (the photoreactivating light dose may vary widely for different individuals) and (3) die with or without photoreactivating light (non-reactivable). The three components of an actual population may be easily resolved experimentally.

It is customary and convenient in investigating radiation kinetics to consider the radia- tion of the log of the fraction of the population surviving a given radiation treatment as a function of the radiation dose (Equation la). In general, if this semi-log plot is a straight line passing through the ordinate at 1.0 survival, the plot is termed single-hit, the simplest explanation for such behavior being that a single all-or-nothing event has produced the observed effect. Many plots are not single-hit but show a concave-downward dose- response curve which eventually approximates a straight line ; such a response is termed multi-hit or multi-target and suggests that an accumulation of events is required to produce the final inactivation.

Single-hit response can be mathematically expressed as follows.

n/no = e-kd (1) Where no is the number of organisms initially present, n is the number of organisms sur- viving after exposure to the dose d, k is a constant parameter of the inactivated population, and e is the base of natural logarithms. It is frequently convenient to consider the equation obtained by taking the natural log of Equation (1):

In (n/no) = -kd (14 787

788 JOHN CALKINS

The multi-target situation frequently seems to fit an equation of the form n/no = 1 - (1 - ekd)m (2)

where n, no, e, d have the same significance as in the single-hit case, k and m are parameters of a population, which suggests that there are a number of sites rn of radiation damage each with an individual sensitivity k to radiation. The parameter rn is sometimes called the target or extrapolation number. It is implicit in this model equation that if any one of the m sites of radiation damage remains uninjured, then the entire organism will survive.

The photoreactivation of a population of organisms inactivated by a fixed dose of U.V. light can be regarded in the same manner as inactivation. Where one would consider surviving fraction in a survival experiment, the fraction of the reactivable organisms not reactivated (i.e., those “surviving” the photoreactivation treatment) must be substituted when investigating photoreactivation kinetics.

If the number of organisms surviving without reactivation (dark survival) is No and the number of surviving with maximum reactivation is Nm the reactivable population is then Nm-No. If the number of surviving organisms after intermediate doses of photo- reactivation is represented by N , then the fraction of the reactivable population reactivated by a photoreactivating dose will be (N--N0)/Nm -No) and the fraction not reactivated will be

1 - [(N--No)/“m--No)l ( 2 4

In analogy with the inactivation case we would then express photoreactivation kinetics by Equations (3) and (4): For single-hit,

or taking the logarithm of both sides,

and for a multi-hit situation,

Where N , Nm and N o have the significance indicated above, D is the dose of photoreacti- vating light and in anology with the inactivation case, K is a parameter which relates to the sensitivity of individual lesions to reversal by photoactivating light, and L indicates the number of lesions in an individual, any one of which would be lethal.

Dulbecco(1,2) investigated the photoreactivation kinetics of the bacteriophages T-2 and T-3. When he plotted the log of the function labeled (a) above against the photo- reactivating dose he found the phage T-2 gave a straight line passing through the origin (in agreement with the single-hit reactivation, Equation (3a). The phage T-3 could be classified as having multi-hit kinetics,@) with the average number of hits required a function of the inactivating U.V. dose.

There are only a few other studies of photoreactivation kinetics analyzed in the manner used by Dulbecco. Jagger and Latarjet@) confirmed the single-hit type response Dulbecco had found for T-2. Ginsberg and Jagged4) found that the bacterium E. coli T-A-U- shows a single-hit curve for low U.V. doses but a multi-hit type curve after inactivation by higher U.V. doses. Jagger (personal communication) has investigated the photoreactivation kinetics of several other microorganisms and found some which appear to show the linear (single-hit) type kinetics while other show the curved (multi-hit) type of response. Some, like E. coli T-A-U- show both types of response, depending upon the dose.

1 - [ (N-No) / (Nm-N, ) ] = e-KD (3)

In { 1 - [ (N--No) / (Nm-No)] ) == -KD ( 3 4

I - [ (N-No) / (Nm-No) ] = 1 - (1 -e-KD) (4)

Dose-response for photoreactivation of U.V. inactivated stationary phase Tetrahymena pyriJormis 789

MATERIALS AND METHODS The strain of Tetrahymena pyriformis used in these experiments was collected in Texas

in 1960 and has been grown in lettuce medium inoculated with bacteria since that time; several E. coli strains have been used as the food organism. Test animals were isolated and grown in depression slides; the slides were kept in an inverted 15-cm petri plate. Large glass animal jars with Styrofoam lids provided storage containers for racks of petri plates. The animal jars were covered with black paper to exclude room light and had water in the bottom which prevented evaporation from the depressions containing the animals.

Stationary-phase animals, 1 per depression, were inoculated into fresh medium in the depressions. The animals, after the lag period, would grow rapidly. When the nutrients of the medium were exhausted (in about one day, N 60-120 animals per depression) the animals were observed to remain joined in the final stages of division for several hours. This resulted in a culture where almost every animal appeared to be in division; when “long division” was over the animals became thin as was typical of stationary phase, in contrast to the almost spherical shape of log-phase animals. Animals termed “early stationary phase” were used for all experiments and were considered to be those cultures used one day or less following the long division.

1.5”. Since even the best-balanced salt solution, of the number tested, produced obvious changes in animals suspended in it, a technique was developed for the U.V. irradiation of protozoa which eliminates the necessity of removing the animals from the normal growth medium. The culture medium, in thick layers, absorbs U.V. light strongly; however, if the layer of medium containing the animals is sufficiently thin, the absorption of the suspending medium can be neglected. Animals were collected from an early-stationary-phase culture and placed into a small drop of medium; a plastic “tab” (sheet polyethylene approximately one centimeter square and 0.001 in. thick) was placed over the drop spreading the medium out into a layer sufficiently thin to produce negligible absorption of the U.V. light.

The source of U.V. light was a bank of five 15 W germicidal lamps located in a cabinet with movable shelves. The U.V. intensity was continuously controlled by the use of a variable transformer (the light intensity is a function of the voltage applied to the lamps). An U.V. meter composed of a RCA u.v.-sensitive vacuum phototube, type 935, a 45 V battery, and a 15 PA full-scale microammeter permitted the adjustment of the light intensity to the desired level. The metering system was calibrated by intercomparison in the labora- tory of R. B. Setlow at Oak Ridge National Laboratory and the author would like to express his appreciation for this assistance.

While the absorption of the thin layer of medium containing the animal is insignificant when the plastic-tab technique is used, there is absorption and reflection by the tab which must be included in computation of dose of the animals. Measurements have shown the plastic tabs reduce the U.V. intensity by 20 per cent; doses corrected for the presence of the tab have been given in ergs/mm2 of net dose incident on the animal. The dose rate for the series of experiments reported here was 20 ergs/mm2 sec-l.

After the animals were irradiated they were isolated into small drops of fresh medium in depression slides or placed in test tubes in a temperature controlled bath for photo- reactivation. The isolation and observation of U.V. irradiated animals utilized a low- power microscope and illumination with red light. All manipulations of irradiated animals were done under non-photoreactivating conditions. Photoreactivation was effected by a

Growth and U.V. irradiation were always at room temperature, 24.5

790 JOHN CALKINS

bank of eight 15 W fluorescent lights closely spaced and located 5 cm above the test tubes containing the animals. The animals were photoreactivated in a water bath at 19" since photoreactivation at room temperature required inconveniently short times. Following photoreactivating treatment, animals were isolated and observed in the same manner as non-photoreactivated animals.

It was necessary to observe the animals for about 2-3 weeks in order to determine the fate of the different lines. Lethally irradiated animals will make a few divisions (usually two) and then persist without division, eventually lysing in a period of 2-3 weeks with an occasional late recovery. The surviving lines will grow in a matter of a few days to the limit of the food provided, usually 150-300 animals. These animals then settle in a dense clump in the bottom of the medium-filled depression and remain motionless for many months; however, if fresh medium is added they will revive and swim about in a matter of a few minutes and are fully capable of continued growth when food is added.

RESULTS Figure 1 shows typical survival curves of U.V. irradiated Tetrahymena pyrijbrmis. The

lines were fitted to the data points using a computer and assuming Equation (2) as the mathematical model. The rather poor fit observed in many experiments suggests that

t 0.005 cdt 0

@00210 Ilk 2bo 4 0 4bo d o 660 ,oo Dose, ergs/mm2

FIG. 1 . Typical survival of u.v.-irradiated stationary-phase Terrahymenapyriformis. Different experiments are indicated by different symbols; curves were fitted to data points using the

multi-target equation as a model

Dose-response for photoreactivation of U.V. inactivated stationary phase Tetrahymena pyrgormis 791

Equation (2) is only an approximation to the inactivation dose-response relation for this animal. Although the value of the LD-,, (50 per cent inactivation dose) is reasonably uniform throughout the series of experiments, the obvious deviation from Equation (2) makes the computed value of m ranging from N 5 to N 20, of doubtful value. As can be seen in Fig. 1, survival shows a steep decline to 5-20 per cent survival followed by a leveling and then a subsequent rapid decline. The initial and final rate of inactivation suggest a value of m of 20 or more but the fitting of all data points to a single multi-target equation leads to a lower value of m. Although the inactivation resembles that of a heterogeneous population, there is reason to believe this is not the case; the survival kinetics of this animal are quite puzzling and are under active investigation.

Figure 2 shows typical survival curves for animals non-photoreactivated and following various doses (expressed in minutes) of photoreactivating light. In the experiments described here, 20 min of photoreactivation produced approximately maximum photo- reactivation.

0.5 -

0 2 -

01

005 -

-

002 -

c ._ O 0.01 -

e - V

005 - c

Ol

> ._ .- 2 0 0 2 - a v)

0.01 -

0.005 -

0.002--

0.001 -

PR 5

Dose, ergs/mm2

FIG. 2. The survival of stationary-phase Tetrahymenapyriformis after U.V. irradiation followed by various doses (expressed in minutes) of photoreactivating light.

Table 1 shows the observed survival data plotted in Fig. 2 and from these data it is possible to determine what has been called the Fixed PR Dose Efficiency of photo- reactivation. Following a given U.V. dose, a certain part of the inactivated population can be reactivated by a dose of photoreactivating light. The Fixed PR Dose Efficiency is determined by calculating the fraction of the reactivable population which is reactivated by a given dose of photoreactivating light. It is assumed that the animals not reactivated by the maximum dose of photoreactivating light are not reactivable and may then be

792 JOHN CALKINS

TABLE 1. SURVIVAL AND EFFICIENCY OF PHOTOREACTIVATION FOLLOWING VARIOUS TREATMENTS*

PR dose U.V. dose (min) ( e w / m z ) NoINI NI NI @(Equation (5))

1 1 1 5 5 5 5

10 10 10 10 10 20 20 20 20 20

200 400 600 200 400 600 800 200 400 600 800

1000 200 400 600 800

1000

0.357 0.038 0.0076 0.357 0.038 0.0076 0.00079 0.357 0.038 0.0076 0~00079 0.00079 0.357 0.038 0.0076 0.00079 0.00079

0.858 0.214 0.0183 1 .O 0.823 0.427 0.113 0.965 0.893 0.535 0,143 0.052 1 .O I .o 0.75 0.276 0.095

0.78 0.183 0.0 I4 1 .o 0 8 2 0.565 0.43 0.95 0.89 0.71 0.52 0.55 1.0 1 .o 1 .o 1 .o 1 .O

2 c

1.c

0.5

U c

5

?!

.- - ” 0.2

i 0.1 - 2 C ._

005 e lL

002

0.01 5 I 1 l I l l l 10 15 20 25 30 35

Photoreoct ivotion dose, min

FIG. 3. Photoreactivation kinetics plotted in the manner of Dulbecco. After a given dose of U.V. irradiation (600 ergs/mm2 in these experiments) the dose-response relation for photo- reactivation is plotted. The log of the fraction of the population reactivable but not reactivated In { l-[(N-NO)/(Nm-NO)] 1, is plotted against dose. Straight lines (implying single-hit or first-order kinetics) fitted to data “by eye”; multi-target model fits (Equation (4)) are indicated by dotted lines with the extrapolation number (L) given each set of data. Since the obvious differences in slope of this plot in various experiments prevented pooling of data, results

from four separate experiments (indicated by the various symbols) are shown.

*Nr is the number of organisms initially administered.

Dose-response for photoreactivation of U.V. inactivated stationary phase Tetrahymena pyr$ormis 793

excluded in determining the efficiency of photoreactivation. The efficiency of photo- reactivation is given by Equation (5).

N, No and N,,, having their previous meaning. Figure 3 shows the photoreactivation kinetics plotted in the manner of Dulbecco.

Four separate experiments are shown; many other experiments gave the same general results. The dotted curves fitted to the data using Equation (4) utilize two parameters L and K and therefore would be expected to fit the data better than a single parameter implied by the solid lines. The data points at both low and high photoreactivating doses are critically dependent on the behavior of one or two isolated animals. If L is significantly different from 1 it appears to be less rather than greater.

DISCUSSION The rate of photoreactivation, which is defined by DuIbecco(l) as being proportional

to the slope of the photoreactivation curve as plotted in Fig. 3, varies from experiment to experiment. While there is a rather high scatter of data points in any individual experiment it is evident that the photoreactivation kinetics are very nearly first order.

Current concepts of the nature of the U.V. lesion would indicate that a living organism contains many “targets” or ‘‘sites’’ each of which when inactivated by U.V. light may result in death. Therefore, many lethally irradiated micro-organisms would contain “excess” lethal lesions. Assuming there are a large number of “sites” for the lethal lesion, then after a particular exposure to U.V. light a population will have a distribution of the lethal lesions, some having 0, some 1, some 2, etc.

In the cases of organisms showing “single-hit’’ survival curves the distribution can be calculated by the Poisson equation. Table 2 indicates the distribution of lethal lesions of a population at four different levels of survival; the fraction of the population surviving would, of course, be that fraction of the population with no lethal lesions.

Organisms which show a multi-hit survival kinetics (including T. pyriformis) cannot be treated in the simple manner used for the single-hit case; however qualitative reasoning indicates that for the same survival level the part of the population with only 1 lethal lesion (the part of the population which would be expected to show a single-hit photo- reactivation kinetics) would be even smaller than in organisms inactivated in a single-hit manner.

At 1 per cent survival (the approximate levels used in this series of experiments) the maximum part of the inactivated population expected to show single-hit recovery would be even less than the 5 per cent (Table 2) expected in the single-hit inactivation case.

Dulbecco(2) considered a number of alternative explanations of the unexpected first order (single-hit) nature of the photoreactivation kinetics of T-2. The same consideration could apply to Tetrahymena pyriformis. He considered four alternatives : (1) The organism has only one site of damage, and reversal of the injury to this single

site leads to survival. (2) Photoreactivation has a trigger mechanism which, by the absorption of one quantum,

reverses all lesions.

794 JOHN CALKINS

TABLE 2. CALCULATED DISTRIBUTION OF LETHAL LESIONS IN AN U.V. IRRADIATED POPULATION SHOWING SINGLE-HIT (EQUATION 1 ) TYPE INACTIVATION KINETICS

Number of lethal lesions

0 1 2

0.100 0.230 0.265 0.203 0117 0.054 0.02 1 0.007 0.002

Fraction of the total population

0050 0.010 0.150 0.046 0.224 0.106 0.224 0.163 0.168 0.187 0.101 0.172 0050 0.132 0.021 0087 0.008 0.050

0.001 0.007 0.024 0.055 0.095 0.131 0.151 0.149 0.129

(3) Photoreactivation, rather than reversing the lesions, provides an abnormal alternative path which permits survival without removing the actual lesions.

(4) There is a distribution of requirements (sensitivity) for photoreactivation among the reactivable population which is of such a nature that single-hit kinetics are simulated.

None of the above postulated mechanisms have independent confirmation. Postulate (4), that the apparent single-hit nature of photoreactivation kinetics arises from a certain distribution of sensitivities of the inactivated organisms for photoreactivation, could, if true, fit best with current theories of the biological action of U.V. light and its photoreversal. If there were some underlying basis for such a distribution of sensitivities, it would depend on the physical or chemical nature of the U.V. lesions, and it should be possible to distort the distribution of lesions in a population to render the reactivable population more homogeneous. One could then investigate the kinetics and observe if they were converted to the multi-hit type. However, it seems rather artificial to suppose that the distribution of reactivability of several quite different organisms would be exactly such as to produce single-hit kinetics.

If nothing else were known about photoreactivation, the trigger mechanism would seem to be the simplest explanation of single-hit kinetics. Experiments in vitro strongly support a lesion-by-lesion reactivation quite different from a trigger mechanism(5) One could imagine that the photoreactivating-enzyme : u.v.-damaged DNA complex, when triggered by the absorption of one photon of reactivating light, would lead to the correc- tion of enough damaged DNA to render the organism viable . Because of more “triggers” following higher U.V. doses an increasing Fixed PR Dose Efficiency would be expected in contrast to the observed reduction of PR efficiency with increasing U.V. dose (Table 1). Without any independent experimental evidence to substantiate the trigger model, it seems pointless to propose modifications which could explain the declining efficiency.

Studies of photoreactivation kinetics reveal that there are substantial unresolved problems involved in photoreactivation.

Acknowledgements-I would like to acknowledge the assistance of Yvonne Gover, Teddie Hamman, and Doris Gaines in the experiments reported here. I thank Drs. J. Jagger, C. S. Rupert, W. Harm and K . Haefner for interesting discussions of this material prior to its publication. This research was supported by Grant GM 12030-02 from the Institute of General Medical Sciences of the NIH. Facilities of The University of Kentucky Computing Center were used in the analysis of the experiments.

Dose-response for photoreactivation of U.V. inactivated stationary phase Tefrahymenapyriformis I95

REFERENCES 1. R. DULBECCO, J. Bucteriol. 59, 329 (1950). 2. R. DULBECCO, Radiation Biology, Vol. 2, chap. 12, p. 455 (A. Hollaender, Ed.) McGraw-Hill, New

York (1956). 3. J. JAGGER and R. LATARJET, Ann. inst. Pasteur 91,858 (1956). 4. D. M. GLNSBERG and J. JAGGER, J. Gen. Microbiol. 40,171 (1956). 5. C. S. RUPERT, Photophyxiofogy Vol. 2, chap. 19, p. 283 (A. C. GIESE, Ed.) Academic Press, New York

(1 964).

D