Photochemisrry and Photobiology Vol. 45, No. 3, pp. 389-398, 1987 Printed in Great Britain. All rights reserved

003 1 -8655187 $03 .oO +0 .oO Copyright 0 1987 Pergamon Journals Ltd

SPECTRAL DEPENDENCE OF SOME UV-B AND UV-C RESPONSES OF Tetrahymena pyriformis IRRADIATED

WITH DYE LASER GENERATED UV

JOHN CALKINS*, ED COLLEY and JOHN WHEELER Department of Radiation Medicine and School of Biological Sciences, The University of

Kentucky, Lexington, KY 405364084 and The Graduate Center for Toxicology, The University of Kentucky, Lexington, KY 40536, USA

(Received 21 July 1986; accepted 16 September 1986)

Abstract-We have generated UV-B and UV-C radiations using a flashlamp driven tunable dye laser combined with frequency doubling crystals. Using this novel UV source, we have investigated lethality and its modification by growth phase, photoreactivation and caffeine in Tetrahymena pyriformb at 254 nm and from 26G315 nm in 5 nm steps. From the observed responses we have constructed action spectra for lethality, with or without caffeine (a repair inhibitor) and under conditions of photoreactivation. We have also estimated quantum efficiencies for these responses. Our observations suggest that complex changes in response occur at several wavelengths over the UV-C and UV-B regions.

Action

INTRODUCTION

spectra in the UV-C range (200-280 nm) have provided very basic insight into the biological action of these wavelengths. However, negligible amounts of these short wavelengths are present a t the Earth's surface. Action spectra in the UV-B wavelength range (280-320 nm) are of critical importance for environmental hazard evaluation and for sunlight dosimetry (Rupert, 1982; Caldwell, 1982). Since solar UV, including significant levels of UV-B, is variable in spectral distribution (depending primarily on the amount of ozone enco- untered before reaching the Earth's surface) it is impossible to fully evaluate the potential biological actions of solar UV without reliable and detailed information as to how these actions depend upon the spectra of the incident radiation. Because the efficiency of biological action per incident quantum may vary by three or four orders of magnitude between wavelengths of 290 and 360 nm (MacKay et a/. , 1976; Setlow, 1974), the action spectrum will strongly influence any calculations of the potential effects of ozone depletion.

Quantitative data on UV-B action spectra are largely from bacterial studies. Gates (1930), Hol- laender and coworkers (see Zelle and Hollaender, 1955), and Luckiesh (1946) conducted UV action spectra studies on bacteria; the latter two extended from the UV-C into the visible range. Webb and coworkers have more recently conducted extensive action spectra studies (see Webb, 1977). Prior to the very recent studies by Peak et d. (1984), the bacterial action spectra in the UV-B range were largely confined to the arc-monochromator gen- erated Hg lines (particularly 280.4,296.7, 302.2 and

~ -

'To whom correspondence should be addressed.

312.9 nm). The monochrometers were often used with half bandwidth of 5 nm or more.

There are presently very few action spectra of eukaryotic organisms, especially those which include the critical UV-B range (28CL320 nm) (see Calkins and Barcelo, 1979). Aside from the early work of McAulay and Taylor (1939) and by Giese and Kimball and their coworkers (summaried in Giese, 1953), and the recent studies of yeast by Zolzer and Kiefer (1983), there are almost no action spectra studies of injury of eukaryotic organisms or small animals which include wavelengths greater than 300 nm. Action spectra studies of cultured mammalian cells, which include UV-B wavelengths, are receiving increasing attention (Kantor, 1984).

In principle, the tunable dye laser could simplify determinations of action spectra. We have obtained such a laser system and have begun using this device for the generation of UV-B biological action spectra, a use which is, to our knowledge, the first application of laser technology in this manner.

MATERIALS AND METHODS

Radiation sources. We have described the charac- teristics and operation of our primary radiation source, a tunable dye laser, in earlier publications (Calkins ef al . , 1983; Wheeler and Calkins, 1985). The average power output of the laser can be controlled by the charge on the high voltage condenser and the rate of firing; normal operation would use a high voltage of 1S18 kV and repetition rates of 2 4 Hz. The instantaneous power in each pulse is much higher since the pulses last only 0.3 microseconds. Our average power density (irradiance) incident on the sample for the UV-C irradiations was 0.7-10 w m-' and 5-200 w m-2 for UV-B irradiation. Even lower average irradiance was often used when the repair inhibitor, caffeine, was to be used. The laser radi- ation was incident from above on a small (5 or 10 mm in diameter) open sample holder. Experimental techniques, when germicidal lamps were used as an ultraviolet radi- ation source, are as described in Calkins (1968).

389

390 JOHN CALKINS et al.

During the course of the studies, laser dosimetry methods have changed. In all cases, the laser output has been monitored throughout the experiments. The main beam (visible) and frequency doubled beam (UV) emerge from the doubling crystal together. We separate the two beams using a 60” quartz prism and a Schott glass UV transmissive filter; initially we monitored the visible beam during UV irradiations and computed UV exposures from a calibration curve relating main beam power to UV power. A direct integrating UV monitor was developed and used for the more recent experiments (Hazle et a f . , 1984). A constant fraction of the UV beam reflects off the face of a 60” dispersing prism or a beam splitting plate. A transducer, a photocell which measures the fluorescence generated by the deflected UV, has been used to directly monitor the UV irradiance during irradiation; the energy delivered (irradiation = time integral of the irradiance) was determined by electronic integration of the monitor signal (Hazle et al., 1984).

All dosimetry was based on measurements using a Sci- entech model 361 power meter to calibrate the monitor system. The sensor for the power meter is an absorber- thermopile type device with a built-in heater. Calibration of the Scientech system was checked by applying a known power through the heater system. Operating wavelength determinations were made using a Beckman DU spec- trophotometer which was calibrated using mercury lines from a small mercury vapor discharge calibration source and a HeNe laser. The optical density of samples was measured using a Carey spectrophotometer. Our samples were optically “thick” and consequently were magnetically stirred throughout irradiations; we used the Morowitz (1950) correction to calculate average irradiance to the sample.

Biological methods. The strain of Tetrahymena pyri- formis (SlZ), and the general culture methods described by Calkins (1968) were used. The animals were grown in lettuce medium inoculated with Escherichia coli. The Tetrahymena to be irradiated were primarily in “log- arithmic” (log) growth phase; some experiments used stationary phase animals as noted. Data were analyzed allowing for control mortality (@15% in stationary phase experiments). Log phase control animals would very rarely die upon isolation either in normal medium or medium containing caffeine, so no control mortality correction was needed.

Cultures to be irradiated were diluted 1:1 with distilled water prior to exposure. Samples (a variety of sample sizes were used in the course of the experiments 0.4,0.2 or 0.1 me in volume) contained 5@200 animals depending on the experiment to be conducted. After irradiation, samples were often split; one portion being given photoreactivation o r caffeine treatment, while the other portions were assayed for survival without further treatment. Irradiated animals receiving photoreactivating light were treated for 20 min immediately following laser irradiation, using two 40 W Westinghouse fluorescent “AGRO-LITES” located 20 cm above the sample. Caffeine treatment (caffeine incorporated in the normal medium at a concentration of 0.02% wtivol) began immediately following laser irradiation, and was continued throughout the survival analysis period. Tetrahymena were isolated individually into depressions containing - 0.25 m4 of fresh medium; the excess treated animals were retained and observed thus permitting estimates of survival levels less than 1%. Disposable polyethylene depression plates, 32 depressions per plate, were used for isolation; the medium in each depression would support growth of the single isolate to 30C-1000 animals. The depressions were checked period- ically until growth to the limits of food occurred (our definition of survival) or the isolate died out.

Analysis of biological data. All action spectra are based on survival: we have used the Litchfield-Wilcoxon (1949) analysis or Statistical Analysis Systems (SAS) Probit Pro-

gram (SAS Inc., Carey NC) to determine the exposure dose lethal to 50% of the treated population (LD,,) and its 95% confidence limits. Irradiated Tetrahymena, especially when treated with caffeine, may show anomalous forms of dose-response kinetics (see Calkins, 1967a, 1968, 1973) yielding “V-shaped” survival curves which may produce large confidence limits on LDso values when analyzed by either Litchfield-Wilcoxon or SAS method. When the “V- shaped” response was obvious, we have used the initial drop in survival of treated animals (survival falling below 50%) to determine the LD,,.

Neither the Litchfield-Wilcoxon method nor the SAS program can determine the LDSo without a minimum of two data points between 0 and 100% mortality. We have graphically determined LD,,, in some instances where the general limits of response were well defined but there were not enough data points to analyze for the 95% confidence limits of LD,,,.

RESULTS

Survival curves

Figure 1 illustrates representative survival data. The horizontal error bars plotted at 50% survival indicate the 95% confidence limits of the LDso values for the set of data points. Vertical error bars are plotted for the 310 nm data points indicating the typical standard deviations of the plotted survival points, which is typical of most data points at the same survival level. Absence of any survivors, in the group tested, is indicated by a point with a downward pointing arrow plotted at the survival level which would have been observed if one isolate had survived. W e did not attempt to analyze the survival curves in terms of models such as the “mul- titarget” model beyond the fitting implicit in the LDso determinations. Photoreactivation tended to increase the LD50 by increasing the shoulder or threshold of response; and photoreactivation was effective at wavelengths of 305 nm and shorter. Our data are too limited t o decide if photoreactivation also changes the slope of the dose-response relation- ship. Photoreactivation provides no protection from lethality at 315 nm and shows only reduced pro- tection at 310 nm.

It is clear that post-irradiation growth in caffeine- containing medium dramatically sensitizes the Tetrahymena to UV at all wavelengths tested (Fig. 1). Caffeine treatment reduced or virtually abol- ished the threshold in the dose-response curve. Fol- lowing caffeine treatment many of the experiments provide data compatible with exponential (“single hit”) survival. The slope of the survival curve is also much steeper than the untreated response.

Variation of LDw with wavelength

The reciprocal of the LDS0 irradiations for log phase Tetrahymena and their means are plotted as a function of wavelength in Fig. 2 ; data for 254 nm germicidal lamp radiation treatment is also shown. The effects of photoreactivation and caffeine treat- ment are shown in Fig. 2.

UV action spectra of Tefruhymenu

315nm :-l 39:

305 nm

P r

305nrn

”\

310nrn

li, 20000 310nrn 40

t L 40000 BOO00

315 nrn

IR R ADI AT I o N i n J / m 2

Figure 1. Representative dose-response curves for UV-B killing of log phase Tefruhymena, (hexagon) indicates no further post irradiation treatment; (0) 20 min photoreactivation; ( V ) survival assayed in medium containing 0.02% caffeine. Horizontal bars at 50% survival level denote the 95% confidence limits of the LD,, determinations. Vertical bars on the 310 nm data indicate the standard deviation of the survival points. Points with downward pointing arrows indicate no survivors among the animals tested. Many no survivor and 100% survivors points have been omitted for clarity. The “no treatment” data at 300 nm illustrate a response which cannot be statistically analyzed for LD,,, limits but which

clearly define the LD,,,; graphical fitting of such data were used to determine the LD,, value.

CAFFEINE TREATED

.oooOl I ( 8 ‘ ‘ 2 ; o ‘ xm ’ 8 am ’ h 2 I 0 250 260 270 2 M

WAVELENGTH (rm)

Figure 2. The reciprocal of the LD,, irradiation level as a function of wavelength. Symbols signify the same post- irradiation treatments as in Fig. 1. Solid lines connect the mean values; bars indicate the SD of the mean. Filled symbols indicate the response from a typical germicidal lamp irradiation (254 nm) experiment; bars indicate 95%

confidence levels.

Our strain of Tetrahymena exposed to germicidal lamp radiation show the same relative sensitivities in log and stationary phase as laser (254 nm) exposed animals (Calkins, 1968); however, there is a dis- placement factor of about two between absolute sensitivity to the laser and typical germicidal lamp radiations (Fig. 2), the germicidal lamp radiation producing the greater killing.

Action spectra

The set of LDSOs for the three post-irradiation conditions, no post-irradiation treatment, pho- toreactivation and recovery in caffeine containing medium plotted in Fig. 2 permit the computation of three action spectra (Fig. 3). The response at 265 nm has been used as the basis of comparison and given the relative value of 1.0. Symbols and error bars in Fig. 3 indicate the mean of the various determinations of LDso divided by the cor- responding values at 265 nm. The data points have been corrected for the energy per quantum.

Wavelength dependence of effects of post- irradiation treatments

Our observations provide data regarding the potential for recovery from the lesions induced b j UV-B and UV-C radiations. Photoreactivation is well known to reverse pyrimidine dimers in DNA. and caffeine inhibits recovery from UV exposure in a manner which suggests inhibition of excision

392 JOHN CALKINS et al.

0.8

0.7

t

- -

I I I 1 I 1

21.0

0 4 $0. I

t ; - 2 - w.01 - ? !

w w - 2 .

w :

4 : w

,001 i

’0

t I I 1 I 1 I I 60 280 300 320

WAVELENGTH (nm)

Figure 3. Action spectra for the three different post- irradiation conditions; symbols signify the same treatments as in Fig. 1. “Average DNA” action spectrum of Setlow (1974) as plotted by Peak et al. (1984) is indicated by the

dotted line.

repair (Calkins, 1967b). Figure 4 illustrates the modification of LDso values by post-irradiation treatments. We divided the treated response (LDSo) by untreated in each experiment where both values were determined. The mean and standard deviation of the experiment by experiment computation is shown in Fig. 4. Photoreactivation is relatively inef- fective for improving survival a t 310 and 315 nm (Fig. 4A). This is due in part to the early lysis of animals. At higher dose levels many of the animals were lysed before they could be exposed to the photoreactivating light. There also may be other factors contributing to the low effectiveness of pho- toreactivation at 310 and 315 nm. Caffeine remains a highly effective sensitizer at the longer UV-B wavelengths (Fig. 4B).

The effect of growth phase on radiation sensitivity

There is a large difference in U V response between log and stationary phase Tetrahymena populations when irradiated using germicidal lamps (254 nm). The ratio of LDso values is approximately 3 with the stationary phase cultures being the more sensitive. A similar ratio was observed with laser generated 2.54 nm radiation (Fig. 5). However, as wavelength increases the difference narrows. At 265 nm the phases are equally sensitive; a t the longer wavelengths stationary phase animals are more resistant than log phase but the differences d o not exceed the uncertainties of our determinations.

(A) j: 3 -J

1.0 !1 0 B

I -.

Figure 4. (A) The wavelength dependence of the dose reduction factor (LDSD ratio) produced by photo- reactivation. This plot of dose reduction factor was deter- mined as the mean and SD of the values of all experiments where the LD,, with and without photoreactivation were simultaneously determined. The dose reduction factor is essentially constant from 254 to 305 nm but drops to 1.0 at 315 nm (i.e., no effects at 315 nm). (B) The dose enhancement (LD50 ratio) produced by post irradiation caffeine treatment as a function of wavelength. Data points were computed as in Fig. 4A. With the large varia- bility in the determinations most of the data points could be fit with a horizontal (dotted) line indicating equal sensitization at all wavelengths. The points between 260 and 310 nm fit a sloping (dotted) line indicating a variation of repair efficiency of about 3. Dotted lines were drawn “by eye”; the rationale for disregarding the 254 and

315 nm data points is given in the text.

DISCUSSION

We have examined the dependence of lethality on wavelength. In our studies we have varied a number of parameters t o better understand the injurious action of U V radiation. Irradiated Tetrahymena die as a consequence of radiation

UV action spectra of Tetrahymena 393

Figure 5. A comparison of the reciprocal of the LD,,, irradiation for log (open symbols) and stationary phase (filled symbols) Tetrahymena. Bars indicate SD of the determinations at wavelengths where both growth phases were tested. There was only a single stationary phase determination at 295 nm. Error bars were displaced for clarity. Data are for Terrahymena which were not given special postirradiation treatments; caffeine and pho- toreactivation treatments produced roughly similar effects on stationary phase animals as observed for log phase

responses (data not shown).

induced chemical (photochemical) changes, which will be termed “lesions”. Aside from the production of photochemical lesions, we show that lesions can be repaired. The role and wavelength dependence of repair also needs to be considered in exploring the details of the UV action spectra of this animal.

In general it is expected that factors which control the number of photochemical lesions induced in Tetrahymena operate before or during irradiation, while repair must occur after irradiation. Production of chemical changes by UV radiation requires an energy absorbing substance or “chromophore”; the identification of the chromophore(s) for lethality would help elucidate UV action. The close cor- respondence between many action spectra and DNA absorption suggests that DNA is the primary chromophore for lethality and mutation in the UV- C and UV-B wavelength range (Peak et al., 1984; Setlow, 1974). The similarity of the absorption spec- tra of nucleic acids (peaking at 265 nm) and the action spectra of killing of E. coli lead to the sugges- tion of the importance of nuclear material (Gates, 1930; Loofburrow, 1948). The action spectrum for killing of Tetrahymena (Fig. 3) does not fully resemble the DNA absorption spectrum; our obser- vations provide some limited comprehension of this unexpected result.

observed. It should be recalled that Gates (1930) produced two UV-C - UV-B action spectra which were quite different. The E. coli action spectrum closely following DNA absorption but the Staphy- loc~ccus aureus, action spectrum, obtained by the same technique, shows higher sensitivity at 280 and 290 nm than did the E. coli action spectrum. Proto- zoan action spectra have shown maximum sensitivity near 280 nm rather than at 265 nm (Kimball and Gaither, 1951; Giese, 1945). A recently published action spectrum for cultured rose cells showed an action spectrum of very unusual shape (Murphy et al . , 1985).

It is widely accepted that photoreactivation removes pyrimidine dimers and is not effective on any other lesion. Since photoreactivation produces a large enhancement of survival at all tested wave- lengths between 254 and 305 nm, we assume as a working hypothesis that over this wavelength range (without concurrent photoreactivation), pyrimidine dimers are the significant lesion. There are several possible factors which might modify action spectra so that the action spectrum produced does not fol- low the absorption spectrum of the primary chro- mophore, in our case presumably the DNA of this organism. (1) It is possible that the nucleus is shielded by UV absorbing materials, thus modu- lating the primary production of lesions and dis- torting wavelength relationships. (2) The effec- tiveness of repair processes could depend on chromophores other than the primary lesion. (3) The quantum efficiency for lesion production may vary within the wavelength range under study.

We have conducted many experiments using ger- micidal lamps (254 nm) as the radiation source, and have repeatedly found log phase animals to be about three times as resistant as stationary phase animals (Calkins, 1968). Since the stationary phase animals are much smaller but contain the same genome, we presumed the growth phase difference arose from cytoplasmic shielding of the nuclei in the larger log phase animals. The most UV absorbtive cytoplasmic components, such as nucleic acids and proteins, are better absorbers at 265 or 280 nm than at 254 nm. Thus it would be expected that responses from 265-280 nm would show at least the log-to-station- ary phase, ratio of sensitivity observed at 254 if shielding were a controlling factor. Figure 5 shows that stationary phase animals approach log phase sensitivity as wavelength increases from 254 nm; they are equally sensitive at 265 nm and become more resistant at 280 nm and longer wavelengths suggesting that the phase dependent sensitivity dif- ferences are not likely to arise from shielding.

It can be reasoned that, if a single type of lesion were the controlling factor in UV-C - UV-B response then repair efficiency would be inde- pendent of wavelength. Various wavelengths might

While non-DNA-like action spectra are unusual there are other instances in which they have been

produce a particular lesion with different efficiency but at the end of irradiation the fate of these lesions

394 JOHN CALKINS et al.

would be the same regardless of how the lesion had been produced. Repair systems cannot be pos- tulated to repair a chemically identical lesion with a different efficiency. If there are wavelength depen- dent differences in repair efficiency then they must arise from different lesions or wavelength depen- dent actions on the repair system.

Figure 4A illustrates the wavelength dependence of photoreactivation efficiency. Lesions from 265-305 nm (presumably pyrimidine dimers in the DNA) are, within the resolution of our experiments, equally subject to photoreactivation. In this wave- length range lethally injured animals live from 1-3 weeks before lysis. It is clear that at 310 and 315 nm, photoreactivation is much less effective. At the LD5o exposure level and above, 310 and 315 nm radiation causes death by rapid disruption of the exposed animals in a manner analogous to the symp- toms of death most often observed in sunlight exposed animals (lysis in less than 1 day and often in a few minutes). From the nature of death and the repair ineffectiveness we conclude that for monochromatic radiation there is a change in the nature of the predominant lesion between 305 and 310 nm. However, we find that by changes in repair or other conditions we can shift the transition of major lesions to longer or shorter wavelengths. If there is maximal or concurrent photoreactivation then the threshold for DNA type (late) death is increased and, even in the 270-305 nm range, early death contributes to lethality. Thus, it is reasonable to assume that the “early death” pattern would predominate for real sunlight, which is more intense in the longer UV-B range and also would provide effective concurrent photoreactivation. We have no clear evidence as to the nature of lesions which cause early death in this animal. Caffeine treatment shifts 315 nm radiation or even real sunlight to the late death (DNA) type response (unpublished observations).

Figure 4B illustrates the effectiveness of caffeine as a repair inhibitor. There are large uncertainties in many of the determinations so most of the obser- vations could be fit by a horizontal line (dotted line fitted “by eye”) indicating no wavelength depen- dence of repair effectiveness. However, another interpretation can be made. Disregarding the response at 254 nm (an anomalous wavelength as noted above) the effect of caffeine inhibition of repair progressively increases between 260 and 310 nm (sloping dotted line). Since the production of dimers over this wavelength range is thought to be a direct photochemical action on DNA (Peak et al., 1984) the variability of the caffeine action could most easily be explained by wavelength dependent effect on the repair process. It has often been sug- gested that radiation might inactivate critical repair enzymes and thus lead to a “saturation” of repair capacity at some particular dose level. It is, however, difficult to attribute an important bio-

logical role to repair enzyme inactivation because enzymes in general survive exposure to both ion- izing radiations and ultraviolet light many times larger than the levels required to produce lethality. Our observations suggest that repair is wavelength dependent from causes other than destruction of repair enzymes.

It has been proposed and verified in many exper- iments that this strain of Tetrahymena has a damage- inducible-repair system (Calkins, 1967a) analogous to the induced repair system of E. coli (Calkins. 1967b). We propose that many of the paradoxical responses observed in this animal arise through the inducible repair system. Calkins (1968) showed that repair through the inducible repair system was rela- tively insensitive to inhibition by caffeine. If a par- ticular wavelength were more effective at inducing the inducible repair system then the organism would be relatively insensitive to caffeine as compared to wavelengths with a low inducing capacity. We interpret 260 and 265 nm to be very effective inducing wavelengths. The effectiveness of caffeine as a repair inhibitor at 280-295 nm could be attri- buted to a reduced capacity of these wavelengths tc induce the repair system.

We draw especial attention to the experiments from 275-295 nm (Fig. 2). At most other wave- lengths three or four replicate experiments define the LD50 to about -t 10%. Even with many replicate experiments at these wavelengths there is still large variability. When we examine the detailed survival curves we find evidence of inducible repair. Figure 8A and B shows the seven most recent experiments irradiating log phase Tetrahymena with 280 nm radi- ation. It is clear that the LDSo values in Fig. 8A are not randomly distributed but are bimodal with a resistant group showing a LD50 value about twice the value of the sensitive responses. While most experiments show either the sensitive or resistant response, Fig. 8B shows a transition response. With exposures less than 100 J m-2 the sensitive response is followed, but above 100 J m-z the more effective repair is induced or triggered and the high dose response follows the resistant pattern.

Figures 8C and D demonstrate a similar transition in the response of stationary phase animals after exposure to 260 nm radiation. Panel C shows the transition from sensitive to resistant response at 50 J m-2. Panel D shows the average response of the two experiments illustrated in panel C (heavy line) compared to three other experiments, one sensitive and two resistant. We, as many other investigators of inducible repair phenomena (note observations of Bockstahler and Lytle, 1970; Peters and Jagger, 1981; Parry and Parry, 1973) find it very difficult to obtain exact replication of induced responses. Inducible DNA repair systems are doubtless among the most complex of biological phenomena even though they appear to be common and critical to organisms living in a variable environment.

UV action spectra of Telruhymenu 395

1000

NO TREATMENT z 4

W 2 1 PHOTOREACTIVATED t v - W

W L > - I = - 4 - W

WAVELENGTH (nm)

Figure 6. A least squares fit of the action spectra data between 290 and 315 nm (symbols as in Fig. 1). The Setlow “DNA” data line was reproduced from Peak et ul., 1984.

For slopes and correlation coefficients see Table 1.

One further approach to the analysis of our obser- vations is the determination of the efficiency with which each absorbed quantum produces lethality. The true absorption of incident radiation was esti- mated for this strain of Tetrahymena (see appendix) thus permitting the calculation of quantum efficiency, which is plotted in Fig. 7 along with similar data on bacteriophage and the inactivation of Paramecium. Between 254 and 300 nm the quan- tum efficiency is essentially constant except for the case of caffeine treated animals. If caffeine is wave- length dependent in its repair inhibition then a simi- lar effect would be expected on quantum efficiency. Dimers are produced in E . cnli by wavelengths as long as 365 nm (Tyrrell, 1973). Over a wavelength range extending to 334 nm, Peak et al. (1984) observe dimer production closely approximating DNA absorption; an observation which implies the quantum efficiency for dimer production remains constant across this wavelength range. Assuming dimers to be the critical lesion, we are unable to explain the precipitous drop in quantum efficiency at wavelengths of 300 nm and longer. One possible explanation of our observations is that at 300 nm and longer wavelengths, other substances absorb the incident radiation but produce no effect, thus reducing the quantum efficiency. Since the animals have little true absorption below 300 nm (cross section 10% of area) (see appendix) this explanation is not entirely satisfactory. It is possible that there is a more effective coordination of lesions and repair at longer wavelengths. Perhaps dimers, although

I I I I 260 280 300 d0

WAVELENGTH (nm)

Figure 7. The quantum efficiency for lethality in Tetrahy- menu as a function of wavelength. Quantum efficiency for lethality for phage T1 (0) and T2 (0) refer to the right hand scale, and are from Zelle and Hollaender (1954); quantum efficiencies for lethality in Paramecium (0) are from Giese and Leighton (1935), other symbols are the

same as in Fig. 1.

formed and lethal when repair is suppressed by caffeine (Fig. 4B), are more effectively repaired through the non-induced (presumably excision type) repair systems because of the non-dimer effects of 310 or 315 nm radiation which do not occur with 265 nm irradiation. “Photoprotection” (Jagger et al., 1964) could be such a mechanism, however, photoprotection was not observed to be induced by 365 nm radiation (unpublished observations).

Setlow (1974) demonstrated (regarding car- cinogenesis) that (assuming the “DNA” action spec- trum and September noontime sunlight incident on Gainesville, Florida) the biological effect of solar UV would, for practical purposes, be produced by wavelengths between 290 and 320 nm. Longer wave- lengths fail to contribute because of the low bio- logical effectiveness while radiation less than 290 nm is insignificant because of the vanishingly small solar irradiance. From 290-320 nm the Setlow “DNA” action spectrum and our data can be fit by a semilogarithmic relationship (Fig. 5 and Table 1).

396 JOHN CALKINS et al.

A LOG PHASE loo

fn fisE’ooTh STATIONARY 2 6 0 nrn STATIONARY 2 6 0 nrn

I I 100 200 300 200

IRRADIATION i n

D PHASE

Figure 8. Dose-response observations indicating a transition of response suggesting the induction or triggering of repair at defined threshold levels. Dose-response data are plotted as in Fig. 1; experimental points from the same experiment are connected; dotted lines indicate no survivor at the next higher dose point. Panels A and B show the response from the last 7 log phase experiments conducted at 280 nm.

Panel A shows a biomodal response with the resistant response (4 experiments) indicating a LDSo almost double the value of the 2 sensitive experiments. Panel B, illustrates a transition experiment. Response at doses below 100 JimZ follow the sensitive response pattern (dotted average from panel A); the threshold for induced repair is exceeded above 100 J/mZ and response follows the resistant pattern from panel A (dotted line).

Panels C and D illustrate a similar transition for stationary phase Tetrahymena exposed to 260 nm irradiation. Panel C shows 2 experiments. The S.D. of each data point is indicated by the vertical error bars. Panel D (heavy line) shows the average transition response from panel C superimposed

on one sensitive and 2 resistant experiments.

We find a slope slightly steeper than the “DNA” action spectrum but with maximal photo- reactivation, which doubtless occurs in natural sun- light, the slope becomes less. We think the slope function defines weighting functions which may be useful for modeling the role of solar UV in nature and predicting the effects of ozone depletion.

In summary we find: (1) the wavelength range 254-260 nm to produce qualitatively different effects from 265-300 nm exposures. (2) Photoreactivation

to provide approximately constant dose reduction from 254-305 nm suggesting pyrimidine dimers to be the most significant lesion produced by mono- chromatic radiation over this range. (3) Inhibition of repair by caffeine is minimal at 260 and 265 nm but shows increasing effectiveness as wavelength increases, an observation which we attribute to vari- ations in capacity to activate or induce repair. (4) Photoreactivation is ineffective at 315 nm and of reduced effectiveness at 310 nm indicating that a

Table 1 . Linear regression of the logarithm of effectiveness vs wave- length between 290-315 nm

No postirradiation Caffeine Photo-

treatment treated reactivated “Setlow DNA

Slope -0.2953 -0.2978 -0.2332 -0.2606 Correlation 0.9915 0.9909 0.9656

coeff.

*The Setlow “DNA” response used was deduced from Peak et al., 1985. The points were evidently fitted to a straight line over this wavelength range so the correlation would be 1.0. The slope of the Setlow DNA is approximately 12% less than our value, the slope of the Dhotoreactivated response is about 26% less.

UV action spectra of Tetrahymena 397

change of the predominant lesion occurs in this wavelength range. (5) Our action spectra over the critical 290-300 nm wavelength range are slightly steeper than the “DNA” action spectrum; however when the probable effects of concurrent pho- toreactivation are included the slope would be reduced; thus the predicted effects of ozone depletion would be less than would be predicted using the DNA type action spectrum.

Acknowledgements-We thank Ms. Cindy Keller for her assistance in preparation of the manuscript. This work was supported in part by grants from the National Institute of Health (GM285351) and RR01620) and cooperative agreement No. CR810294 from the United States Environ- mental Protection Agency.

Although the research described in this article has been funded wholly or in part by the United States Environ- mental Protection Agency under assistance agreement number CR810294 to John Calkins, it has not been sub- jected to the agency’s required peer and administrative review, and, therefore, does not necessarily reflect the view of the agency and no official endorsement should be inferred.

REFERENCES Bockstahler, L. E. and C. D. Lytle (1970) Ultraviolet

enhanced reactivation of a mammalian virus. Biochem. Biophys. Res. Commun. 41, 184-189.

Caldwell, M. M. (1982) Some thoughts on UV action spectra. In The Role of Solar Ultraviolet Radiation in Marine Ecosystems. (Edited by J. Calkins), pp. 151-159. Plenum Press, New York.

Calkins, J. (1964) A correction for multiple scattering in absorption measurements. Appl. Optics 3, 98S984.

Calkins, J. (1965) A determination of the absorption and scattering of visible and ultraviolet light by six species of protozoans. Appl. Spectrosc. 19, 15-17.

Calkins, J. (1967a) An unusual form of response in x- irradiated protozoa and a hypothesis as to its origin. Int. J . Radiat. Biol. 12, 297-301.

Calkins, J. (1967b) Similarities in the radiation response of Escherichia coli and Tetrahyrnena pyriformis. Int. J . Radiat. Biol. 13, 283-288.

Calkins, J. (1968) The variation of radiation sensitivity of bacteria-fed Tetrahymena pyriformis during the growth cycle and factors related to its origin. Photo- chem. Photobiol. 8, 115129.

Calkins, J. and J. A. Barcelo (1979) Some further con- sideration on the use of repair-defective organisms as biological dosimeters for broad band ultraviolet radi- ation sources. Photochem. Photobiol. 30, 733-737.

Calkins, J., E. Colley, J. Hazle and M. A. Han- nan (1983) A dye laser source of monochromatic UV-B and UV-C radiations for biological action spec- troscopy. Photochem. Photobiol. 37, 669-674.

Calkins, J. and W. Todd (1969) Evidence for a trig- gered or activated repair system in Saccharomyces. h i . J . Radiol. Biol. 14, 487491.

Gates, F. L. (1930) A study of the bactericidal action of ultraviolet light. J . Gem Physiol. 14, 31-42.

Giese, A. C. (1945) The ultraviolet action spectrum for retardation of cell division of Paramecium. J . Cell. Comp. Physiol. 26, 47-55.

Giese, A. C. (1953) Protozoa in photobiological research. Physiol. Zoo. 26, 1-22.

Giese, A. C. and P. A. Leighton (1935) Quantitative studies of the photolethal effects of quartz ultra-violet radiation on Paramecium. J . Gen. Physiol. 18, 557-571.

An Hazle, J. D., J . Calkins and I. P. Stapp (1984)

integrating system for measuring energy delivered by a flashlamp-driven dye laser ultraviolet radiation source. Phys. Med. Biol. 29, 449-454.

Jagger, J . , W. C. Wise and R. S. Safford (1964) Delay in growth and division induced by near ultraviolet radi- ation in Escherichia coli B and its role in pho- toprotection and liquid holding recovery. Photochem. Photobiol. 3, 11-24.

Kantor, G. J. (1985) Effects of sunlight on mammalian cells. Photochem. Photobiol. 41, 741-746.

Kimball, R. F. and N. G. Gaither (1951) The influence of light upon the action of ultraviolet on Paramecium aurelia. J . Cell. Comp. Physiol. 37, 21 1-233.

Litchfield, J. T. and F. Wilcoxon (1949) A simplified method of evaluating dose-effect experiments. J . Phar- macol. Exptl. Therap. 96, 99-113.

Loofbourow, J. R. (1948) Effects of ultraviolet radi- ation on cells. Growth Symposia 12, 75-149.

Luckiesh, M. (1946) Applications of Germicidal and Infrared Energy. Van Nostrand, New York.

Mackay, D., A. Eisenstark, R. B. Webb and M. S. Brown (1976) Action spectra for lethality in recom- binationless strains of Salmonella typhimurium and Escherichia coli. Photochem. Photobiol. 24, 337-343.

McAulay, A. L. and M. C. Taylor (1939) Lethal and quasi-lethal effects produced by monochromatic ultra- violet irradiation. J . Exp. Biol. 16, 474-482.

Morowitz, H. J. (1950) Absorption effects in volume irradiation of microorganisms. Science 111, 229-230.

Murphy, T. M., H. C . Hurrell and T. L. Saski (1985) Wavelength dependence of ultraviolet radiation induced mortality and K‘ efflux in cultured cells of Rosa damascena. Photochem. Photobiol. 42, 281-286.

Parry, E. M. and J . M. Parry (1973) Genetic analysis of UV inactivation, recovery and regulatory phenomena in a strain of the yeast Saccharomyces cerevisiae. Molec. Gen. Genet. 124, 117-133.

Peak, M. J . , J. G. Peak, M. P. Moehrins and R. B. Webb (1984) Ultraviolet action spectra for DNA dimer induction lethality, and mutagenesis in Esch- erichia coli with emphasis on the UV-B region. Photo- chem. Photobiol. 40, 613-620.

Peters, J . and J. Jagger (1981) Inducible repair of near- UV radiation lethal damage in E. coli. Nature 289, 194195.

Rupert, C. S. (1982) Photobiological dosimetry of environmental ultraviolet radiation. In The Role ofsolar Ultraviolet Radiation in Marine Ecosystems (Edited by J. Calkins), pp. 131-150. Plenum Press, New York.

Setlow, R. B. (1974) The wavelengths in sunlight effec- tive in producing skin cancer: A Theoretical Analysis. Proc. Natl. Acad. Sci. USA 71, 336S3366.

Sutherland, J. C. and K. P. Griffin (1981) Absorption spectrum of DNA for wavelengths greater than 300 nm. Radiat. Res. 86, 39W09.

Tyrrell, R. M. (1973) Induction of pyrimidine dimers in bacterial DNA by 365 nm radiation. Photochem. Photobiol. 17, 69-73.

Webb, R. B. (1977) Lethal and mutagenic effects of near-ultraviolet radiation. In Photochemical and Pho- tobiological Reviews (Edited by K. C. Smith), pp. 169-261. Plenum Press, New York.

Wheeler, J. and J. Calkins (1985) Dyes and dye mix- tures useful for generation of UV in a flashlamp driven tunable dye laser. Photochem. Photobiol. 42, 331-334.

Monochromatic ultraviolet action spectra and quantum yields for inac- tivation of TI and T2 Escherichia coli bacteriophages. J. Bacteriol. 68, 210-215.

Zelle, M. R. and A. Hollaender (1955) Effects of radi- ation on bacteria. In Radiation Biology, Vol. 2 (Edited by A. Hollaender) pp. 365-430, McGraw-Hill, New York.

Zelle, M. R. and A. Hollaender (1954)

398 JOHN CAI MNS et a[.

Zolzer, H. and J. Kiefer (1983) Wavelength depen- dence of inactivation and mutagenesis in haploid yeast cells of different sensitivities. Photochem. Phorobiol. 37, 39-48.

APPENDIX

The determination of the true absorption of Tetrahymena pyriformis at selected UV and visible wavelengths ( B y John Calkins)

Terrahymena, as most other living organisms, undergo changes in size and organization which are dependent on many factors such as temperature, oxygen, etc. In particular, the length of time they have been growing in the medium modifies form. At the end of “lag” phase the animals are very large, and almost spherical; they become smaller and more oval after a few divisions and are quite small and elongated in stationary phase. Changes in mor- phology are paralleled by changes in response to UV and X-rays (Calkins, 1967, 1968) and would also be expected to change the absorption of non-ionizing radiation.

The “apparent” absorption parameters of an animal such as Tetrahymena can be measured by suspending the animal in a non-absorbing medium such as a “balanced salt solution” and observing the optical transmission of the suspension in a spectrophotometer. The more difficult problem is determining the fraction of energy lost from the incident beam by scattering relative to the true absorp- tion in the animals. Methods for separating scatterinp.

100

. lo 400 . 70 .

\ \

100

90

z 00 0 0 v) 9 70 I v) z

60

$ FI 50 10,000 20,000 30,000

CELLS/cm3 Figure A l . Measurements of the optical transmission of suspensions of Terrahyrnena at various cell concentrations and wavelengths. Panel A: Note that at higher cell con- centrations (45 000 cm-’) there is a deviation from Beer’s law thus cross sections must be determined at nondeviating lower concentrations. Panel B: Typical data; although linear the plots fail to pass through 100% transmission at zero concentration because of ‘‘leakage’’ (see text). The measured cross section is the slope of the fitted line, i.e. reciprocal of the concentration producing l/e (37%)

transmission.

from true absorption by measuring scattered irradiance at various angles and computing the total scattered irradiance in this organism have been described by Calkins, 1964, 1965. The same suspensions used for scattering analysis (see Calkins, 1965 for technical details) were also used for total cross section measurements. It would be easier and more accurate to measure transmission and scattering of suspension containing many animals which would absorb or scatter most of the incident radiation. However, in excessively dense suspensions there is multiple scattering indicated by optical properties which fail to follow Beer’s law; Fig. 1A shows that concentrations of 4.5 000 animals cm-3 and greater are too dense. The deviations from Beer’s law are, as expected, greater in the visible range where scattering predominates over absorption, than in the UV-C where photons are lost from the beam primarily by absorption.

A second experimental factor which must be considered is absorbing materials in the suspending medium. When suspended in a balanced salt solution Tetrahymena “leak” UV-C and UV-B absorbing materials into the suspending medium. This leakage could not be prevented but sta- bilized after a short suspension time. Figure 1B shows the type data which were analyzed for computation of total absorption cross section. The slope of the logarithm of measured transmission vs cell concentration data were fitted by least squares method, not including the zero concentration value (pure balanced salt solution). All fit- ted slopes had a correlation coefficient in excess of 0.99. Figure 2 shows computed total cross sections and the “pure absorption” cross section plotted as a function of wavelength. The “pure absorption” cross sections were computed using the scattering data from Calkins (1965). The two ‘.total” cross section plots reflect differences in the state of subject animals; the mean of the two responses was used to compute a representative “pure absorption” cross section of log phase Tetrahyrnena.

2GoGr \

1 1200-

I= 1000-

v) 800-

600-

z 0

u W 0)

v) 0

400 -

2ooL 220 240 260 a L L L L 280 300 320 340 400 500

450 600 WAVELENGTH (nrn)

Figure A2. A plot of the cross section of Tetrahymena as a function of wavelength. The different symbols indicate two determinations under somewhat different physio- logical conditions when the animals were placed into the “balanced salt solution” for optical measurements. A mean or typical cross section is indicated by the dashed line. The lower solid line indicates the true absorption cross section (corrected for scattering) using the data and methods described in Calkins (1964 and 1965). The geo- metrical cross section of this animal would average about

2200 um’.