Photochemistry and Photobiology Vol. 45, No. 5 , pp. 631-636, 1987 Printed in Great Britain. All rights reserved

0031-8655/87 $03.00+0.00 Copyright 0 1987 Pergarnon Journals Ltd

COMPARISON OF UV ACTION SPECTRA FOR LETHALITY AND MUTATION IN Salmonella typ himurium

USING A BROAD BAND SOURCE AND MONOCHROMATIC RADIATIONS

JOHN CALKINS’.’*, CHRISTOPHER SELBY’,’ and HARRY G . ENOCH’.~ ‘Department of Radiation Medicine, ZGraduate Center for Toxicology and 3Kentucky Center for

Energy Research, Lexington, KY 40536-0084, USA

(Received 23 September 1986; accepted 14 October 1986)

Abstract-The UV-B region (28CL320 nm) is thought to be primarily responsible for the mutagenic, lethal, and carcinogenic effects of solar radiation. We have conducted UV-B action spectroscopy for mutagenesis and survival of Ames’ Salmonella typhimurium strain TA98 (uvrB, pKM101) using both monochromatic radiation from a dye laser and broader bandwidth radiation emitted from FS-20 sunlamps. A series of optical filters having different transmission cut-offs together with the sunlamp source provided bandwidths having successively less short wavelength components from which a “broad band” action spectrum was deduced. The two sets of action spectra differed both qualitatively and quantitatively: in comparison to the monochromatic action spectra, the “broad band” spectra showed up to a 200-fold reduced efficiency for both mutation induction and lethality by UV-B wavelengths. These results suggest a large protective effect of the background UV-A andor visible radiations which were present during the broad spectrum irradiations and which are also present in solar radiation. Additional experiments show that to the extent tested this protective effect is not due to photo- reactivation or irradiance (dose rate) effects.

INTRODUCTION

To evaluate the ecological and health effects of solar UV radiation and especially the potential conse- quences of ozone depletion it is essential to know the wavelength dependencies of biological effects. The study of the relative biological effects of the different components of sunlight has a long history. Virtually all studies of relative effectiveness as a function of wavelength (action spectra) presently available for interpretation have applied “mono- chromatic” radiations to the biological subject and then analyzed for a given biological consequence or end point (Webb, 1977). This technique of action spectroscopy has provided clear evidence regarding the behavior of each spectral component; however, it is well known that there may be interactions between the different spectral ranges of UV radi- ation components and/or visible light, especially photoreactivation (see Jagger, 1958) and other forms of synergism (Webb, 1977; Tyrrell, 1980).

While monochromatic studies provide excellent data for understanding the molecular basis of bio- logical effects of UV radiation, an accurate com- prehension of the role of sunlight as a biological factor requires knowledge of the interactions between the various radiation components which are actually arriving simultaneously on the subject organism. Analysis of the effects of ozone depletion likewise requires the broad band information; depletion of ozone will produce a small incremental

*To whom correspondence should be addressed.

change of solar UV-B radiation superimposed on a very large background of UV-A, UV-B and visible radiation.

Luckiesh (1946) and more recently Webb and Malina (1970) and many other investigators of UV radiation effects have made limited use of the broad band technique for action spectroscopy by using “cut-off” filters. A series of filters (see Fig. 1) com- bined with a broad band radiation source can be used to generate small incremental changes in wave- length in close analogy to the ozone effect. After exposing a biological test system to radiations hav- ing different bandwidths, suitable assays of bio- logical effect can be made. An action spectrum can be constructed assuming that the incremental change of biological effects (ABE) is related to the incremental change of incident radiation (AR), i.e.

BE =f(AR) (1) Webb and Malina (1970) applied this concept to mutation rate in continuous cultures using the equ- ation,

where M I is the mutation rate and II is the irradiance with filter 1 (cutting at a shorter wave- length), M 2 and I2 are the same variables with filter 2 (cutting at a longer wavelength), and is the resulting mutation rate per unit irradiance for the radiation passing through filter 1 but not filter 2.

Note that Eq. (2) was applied to a particulai situation, a continuous culture system. We make

h? 1

632 JOHN CALKINS ef al.

WAVELENGTH nm

Figure 1. The transmission of the various optical filters as a function of wavelength. The filters were Schott type WG and were 3 mm thick unless otherwise noted. Filters A-G are referred to in the text and other figures as follows: filter A, 280 nm; B, 295 nm; C, 305 nm; D, 320 nm, 1 mm; E, 320 nm, 3 mm; F, 335 nm, 1 mm; G, 335 nm, 3

mm .

the analogous extension of Eq. (1) to determine the incremental mutation frequencies (MF) produced per unit incremental irradiation ( A E ) (irradiation equals the integral of irradiance) in a situation in which two non-growing cultures are irradiated for short periods with a constant output source through filters FL and Fs cutting at long (L) and short ( S ) wavelengths.

A given time of exposure will produce two dif- ferent mutation frequencies (MF) (mutants per lo9 survivors). The incremental mutation frequency (AMF) would be MF, - MFL; the incremental irradiation E would be the average irradiation under filter Fs ( E s ) minus the average irradiation under FL (EL), i.e.

AE = E, - EL

The quantity we use as the endpoint in our mutation action spectrum is:

The quantity AMFIAE can be determined using various times of exposure; the average value of AMFIAE for a filter pair is the slope of the fit of individual points on a plot of M F as a function of E and can be fitted by the method of least squares.

The concepts expressed in Eqs. (1) and (2A) can be extended to survival assuming that the less lethal (longer wavelength) irradiation EL (transmitted by the long-wavelength cut-off filter FL) results in a defined fraction of survivors. i.e. S, IS,,. where S, is

the number of survivors after irradiation EL and So is the number surviving without irradiation. When an additional increment of exposure is given, AE ( A E = E , - EL), by using the shorter cut-off filters (for the same time) then the effect will be to kill an additional fraction of the survivors of EL. The incremental lethal response can be expressed as the incremental survival [AS = (SL - Ss)lSL] as a func- tion of the incremental dose ( A E ) as:

We have used irradiation obtained by the cut- off filter approach in addition to monochromatic radiations to determine action spectra for mutation and lethality over the UV-B wavelength range and made comparisons of action spectra determined by the two methods. Our results show the two tech- niques provide substantially different results. The sources of differences and how they relate to assess- ment of the environmental hazards of solar UV-B are not yet clear.

MATERIALS AND METHODS

Radiation sources. Our source of monochromatic radi- ation was a tunable dye laser (Phase R, DL 1400) operated as described in Calkins et al. (1983) and Wheeler and Calkins (1985). Dosimetry was accomplished using an elec- tronic integrating dosimeter which was calibrated for each experiment using a Scientech model 361 laser power meter (Hazle et a[. , 1984).

We used Westinghouse FS-20 fluorescent sunlamps fil- tered by gin. Pyrex as a broad-beam radiation source for the filter-derived action spectra. The absorption properties of the filters were measured using a Beckman DU spec- trophotometer and are shown in Fig. 1. The irradiance through the filters was measured with a sensitive ther- mopile which was calibrated against a Kipp & Zonen calibrated thermopile. We measured the differential energy transmission between filter pairs by balancing out the sensitive thermopile output with a battery-potentiometer system. We then switched the filter in the series and observed the small change in thermopile output with a Kiethly null detector microvoltmeter model 155. We were able to measure small changes in irradiance in a background of high irradiance.

The calculated distribution of energy and the measured incremental irradiances are indicated in Fig. 2. The area under each computed wavelength distribution corresponds to the measured incremental irradiance changes between the indicated filter combination. The sunlamp radiant out- put was held constant through the series of experiments using a variable transformer to control output and adjust for minor changes of lamp output.

Photoreactivating light was from a pair of Westinghouse 40 W F40/AGRO AGRO-LITE fluorescent lamps and was filtered with 0.001 in. Mylar film.

Organism and culture methoak. The subject organism for these experiments was the Ames strain of Salmonella fyphimurium TA98 kindly supplied by Dr. Ames’ lab- oratory. This strain is defective in excision repair, it is uvrB and recA’; it possesses plasmid pKMl0l which enhances repair through the SOS pathway. A more complete description of strain TA98 and methods of culture are given in Ames ef al. (1975) and Maron and Ames (1983).

Assay and analysis. The assay methods have been described previously (Selby et al . , 1984). Mutation fre- auencv responses were calculated based upon the increase

UV action spectra 633

1 1280

u 1 I I I I a 280 300 320 340 360

WAVELENGTH ( nm)

Figure 2. The computed incremental difference in energy transmission as a function of wavelength for pairs of filters using fluorescent sunlamp emission as the radiation source and the filter transmission data plotted in Fig. 1. The area beneath each energy distribution curve has been adjusted to be proportional to the measured incremental differences

in irradiance transmitted by pairs of filters (see text).

" I

to

'180 EXPOSURE (mid

Figure 4. Typical computed mutation (reversion) fre- quencies [calculated as described in Green and Muriel (1976)l for S. fyphimurium strain TA98 as a function of exposure through various optical filters. Additional data were omitted for clarity. Total irradiances were as indi-

cated in the caption of Fig. 3.

ity (LD,,), which was determined by least squares fitting of the dose-response data (Fig. 5). The mutagenicity endpoint we selected was the slope of the linear fit of the mutation frequency (AMF) vs radiation exposure (AE) (Fig. 6). It is clear that the mutational response was linear at some wavelengths but non-linear at others. Since bio- logical responses often change their fundamental dose-response relationships between wavelengths, there is no consistently applicable analysis for constructing an action spectrum bridging such response shifts. A number of approaches may be used and the shape of action spectra covering the UV-B range does not change greatly with the common manipulations of endpoint (see Webb, 1977). Our laser-generated action spectra for Escherichia coli B/r and wild type Saccharomyces cerevkiae (unpublished observations) are quite close in absolute sensitivity to action spectra reported by other investigators (Webb, 1977; Zolzer and Kiefer, 1983).

~

0 30 60

EXPOSURE (mid Figure 3. Typical survival of S . ryphirnurium (Ames tester strain TA98) exposed to fluorescent sunlamp radiation filtered through the 280, 295 and 305 nm cut-off optical filters plotted as a function of irradiation time. The inci- dent total irradiance was 8.3 W/mZ with the 280-nm filter and the irradiances with longer cut filters were reduced as

indicated in Fig. 2.

in revertants per plate above background and the cor- responding survivors per plate using the estimation method described by Green and Muriel (1976). The endooint selected for the survival resuonse was 50% lethal-

RESULTS AND DISCUSSION

Table 1 shows the revertants per plate resulting from exposure of suspensions to filtered sunlamp radiation in one experiment. Data such as are shown in Table 1 were used to calculate induced mutation frequencies. Representative lethality and mutation frequency responses for sunlamp-filter exposed cul- tures of strain TA98 are shown in Figs. 3 and 4. From pairs of responses such as are illustrated in Figs. 3 and 4 at a given exposure time we calculated the incremental survival, Eq. (3) plotted in Fig. 5, and incremental mutation, Eq. (2A), in Fig. 6. Corresponding data for laser experiments were obtained (data not shown). Using the criteria

634 JOHN CALKINS et al.

Table 1. Revertants per plate resulting from filtered FS-20 sunlamp irradiation (average of duplicate plate counts from a single experiment)

Duration of exposure to

radiation (min) 280 nm 295 nm

Filter

305 nm 320 nm 335 nm

0 19.0(-0.2)* 18.5(-0.7) 5 198( 179) 162(143)

10 167(148) 204(184) 15 79 .O( 59.8) 163(143) 20 40 60

120 180

15.5(-3.7) 19.0(-0.2) 19.5 (0.3)

187(168) 197( 178) 138(119) 54. S(35.3) 26.0 (6.8)

80.0(60.8) 33.5( 14.3) 99.5(80.3) 28.0 (8.8)

*Values in parentheses were obtained by subtracting the number of spontaneous revertants (19.2 per plate) based on counts of 0 min plate and other control plates from other sections of this experiment where the data are not shown.

1 I I l l I I I 1 60 80 1 160 200 m 400

w

INCREMENTAL IRRADIATION J/mz Figure 5. Computed incremental survival [Eq. (3)] as a function of incremental irradiation for representative

experiments.

already noted, i.e. LDso and linear fit of mutation curves, action spectra for survival (Fig. 7) and mutation (Fig. 8) were plotted. We have plotted the filter data points (connected by dotted lines) at the wavelengths of the peak of the incremental energy difference between successive cut-off filters; the horizontal bars indicate the bandwidth at half- maximum of incremental energy difference between filters. We have also plotted (heavy lines) the broad band action spectra by locating the response points at the wavelength where one-half of the incremental biological action is contributed by the shorter-wave- length energy components and the other half is contributed by the longer wavelengths. This com- putation is made using a linear fit between the shorter-wave filter responses and the longer-wave filter resDonses as a first approximation of a proper

L 0)

> m IL

t&5“ 2000 moo INCREMENTAL IRRADIATION J/ rn2

Figure 6. Computed incremental mutation as a function of the incremental irradiation [Eq. (2A)] for representative

experiments.

biological weighting function. Since the shorter- wavelength components are much more effective than the longer, this tends to move the action spec- trum to shorter wavelengths, especially from 300 to 315 nm.

We have replotted (dashed line) the trend of the action spectra from Peak et al. (1984) which closely corresponds to the “average DNA spectrum” of Setlow (1974), as indicated in Figs. 7 and 8. For wavelengths in the 270-315-nm range, the laser but not the filter data on mutation seem to approximate

635 UV action spectra

‘ O I 1 \ \ -I \\

E ‘P, 5) 0 .{ 0 -1 1

.M)OI 1 I I I I ‘ 9 \I 270 280 290 300 310 320 330

WAVELENGTH nm

Figure 7. Representative action spectra for lethality plot- ted using the LD~,, as the endpoint. The “average DNA spectrum” of Setlow (1974), replotted from Peak et QZ. (1984) is indicated by the dashed curve. Laser data are indicated by X, means and SDs of monochromatic deter- minations from separate experiments are noted by circles and vertical bars. Lethality induced by monochromatic 295-nm irradiation followed by saturation photo- reactiviating irradiation is plotted as €4. Representative broad beam points V, are plotted at the peak of the incremental energy difference for filter sets (dotted lines); horizontal bars indicate band width at half maximum of incremental irradiances between filter sets (see text); @ and heavy lines indicate the action spectrum where the biological action point is plotted at the wavelength where half of the weighted biological action is contributed by shorter-wavelength components and half is contributed by

longer-wavelengths components (see text).

the wavelength dependence (“average DNA spec- trum”) observed with many other systems (see Peak et al., 1984).

The lethality response generated by laser irradiation (Fig. 7) agrees with the “average DNA spectrum” reasonably well between 280 and 310 nm but S. typhimurzum strain TA98 shows relatively high resistance at 270 nm and also high sensitivity at 315 and 330 nm. Incremental lethality determined by the filter method seems to behave much diff- erently from the monochromatic radiation response. Broad band radiation is about 100-fold less effective than monochromatic radiation and the trend of lethality from the broad band source approximates the mutation response.

We have conducted some preliminary exper- iments in an effort to explain the differences between broad band and monochromatic action spectra. For the action spectra shown in Figs. 7 and

DID d C . . C . S

I

WAVELENGTH nm

Figure 8. Representative action spectra for mutation as a function of wavelength using the slope of a linear fit of data (see text) as the mutation endpoint; points plotted

as in Fig. 7.

8, suspensions were irradiated at room temperature. Broad band (but not monochromatic) irradiations included radiation with longer, potentially photo- reactivating wavelengths which might account for the reduced sensitivities in the broad band situation. To examine the possible contribution of photo- reactivation in the broad band situation, we irradiated suspensions with sunlamp light passed through the 295-nm filter, with suspensions either at room temperature or pre-cooled suspensions irradiated in an ice bath. The low temperature did not enhance the mutagenic response, but did enhance the lethal response by 10-30% (data not shown) which is much too small to explain the 10-200-fold increased sensitivity to monochromatic radiation (Figs. 7 and 8). In addition, saturation levels of photoreactivating light only slightly reduced the lethality of monochromatic 295-nm radiation relative to the reduced effectiveness of the broad band source (Fig. 7). A second factor causing the absolute difference might be the high dose rates from the laser as compared to the low dose rates from the broad band source. We reduced the laser output to equal the irradiance of the sunlampfiltex system but observed enhanced, not reduced lethalit) at low irradiances (at 295 nm, see Figs. 9 and 10). Although we have examined the dose rate effects and photoreactivation with only one monochromatic wavelength and one wavelength band, our results leave little doubt that there are substantial dif. ferences between monochromatic and broad beam generated action spectra.

636

> 80- L r -I 7 0 - U

I- 6 0 - W -I b-0 5 0 -

4 0 -

JOHN CALKitis et al.

100 0 0

901 0

8

8

0

o o

.. 8

0

0

8 . 0

3% - .5 5 50

AVERAGE INCIDENT POWER DENSITY (w/rn2)

Figure 9. Effect of power density (irradiance) on lethality induced by monochromatic 295-nm radiation. The data shown are the responses to two different dose levels having incident irradiations of 0.23 J/m2 (H) and 0.70 J/m2 (0).

It should be noted that the usual computation of mutation frequency such as we have used is based upon assumptions that are discussed by Green and Muriel (1976). The action spectra for lethality does not involve the assumptions inherent to mutation frequency calculations and the similarity of the shapes of the mutation and survival action spectra tends to support the validity of the mutation fre- quency analysis used.

Although the studies reported here are limited in scope and the sunlampfilter system requires com- plex analysis, the broad beam technique is much closer to simulating the changes of solar UV which would reach the earth's surface if there were a depletion of the ozone layer. Our work shows that there may be serious errors in assuming that action spectra derived from monochromatic sources prop- erly represent the response of the same organism under broad band irradiation. Our observations also suggest that there are some organisms and some endpoints that do not follow the prototype action spectra pattern, and thus to properly evaluate environmental and health consequences of solar radiation, and especially the results of ozone depletion, a diversity of accurate action spectra are required.

Acknowledgements-We thank Ms. Cindy Keller and Ms. Mary Blakefield for their assistance in preparation of the manuscript, and John Wheeler for assistance with the laser. This work was supported in part by grants from the National Institute of Health (RR-1620) and the Kentucky Energy Cabinet, and cooperative agreement No. CR810294 from the United States Environmental Pro- tection 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 CR81Ct294 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.

0

8 .

0 0

0 8 ..

0 0

8

8 0

8

w

[L 2 0.L 5 5 50

AVERAGE INCIDENT POWER DENSITY (W/m2)

Figure 10. Effect of power density (irradiance) on mutation induced by mooochromatic 295-nm radiation.

Symbols are as in Fig. 9.

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