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E L S E V I E R Journal of Photochemistry and Photobiology B: Biology 31 (1995) 29-34
Action spectroscopy in complex organisms: potentials and pitfalls in predicting the impact of increased environmental UVB
Betsy M. Sutherland Biology Department, Brookhaven National Laboratory, Upton. NY 11973, USA
Abstract
Evaluating the effects of broad spectrum sources, including sunlight, on humans and their environment requires knowledge both of the effects of various wavelengths of radiation in those sources on biological systems and of the kinds and magnitude of changes in those sources. Action spectroscopy - the determination of the effect of radiation on an organism as a function of wavelength - provides a major method for evaluating the effects of a source, including that of changes in its intensity and wavelength distribution. Although action spectra for UV radiation damage to higher organisms can give useful information, inappropriate analyses can lead to misleading or even incorrect results. It is thus essential to consider carefully methods of analyzing action spectra and their consequences for evaluating the effects of changes in the solar spectrum.
Keywords: Action spectra; Complex organisms; Environmental UVB
1. Introduction
Sunlight is essential for the continued survival of life on Earth. The shortest wavelength radiation in the solar spec- trum, however, poses serious problems for micro- and macro- organisms, killing and inducing mutations in simple organisms and altering growth and producing premature aging and cancers in higher organisms. Although organisms have evolved in the presence of extremely low levels of short wavelength UVB radiation (290-320 nm) present in sunlight reaching the Earth's surface, their abilities to withstand higher UVB levels resulting from stratospheric ozone depletion are not clear. Since hypothesized projections of the impact of increased UVB on individual plant and animal species range from devastation to no effect, it is critical to determine their actual susceptibility and resistance to damage by UV radia- tion.
Action spectroscopy provides a powerful means of evalu- ating the effect of narrow wave band and broad spectrum radiation on simple and complex biological systems. "Sim- ple" systems, in which little cellular or tissue material shields target DNA from incident light, include DNA in solution, in micro-organisms and in cultured cells. "Complex" biologi- cal systems, with substantial shielding by overlying cellular material, include organisms with highly UV-absorbing pig- ments or structures, including higher plants, animals and
1011-1344/95/$09.50 © 1995 Elsevier Science S.A. All rights reserved SSDI 1 0 1 1 - 1 3 4 4 ( 9 5 ) 0 7 1 6 5 - 2
humans. Although action spectra can provide important data, their design, execution and analysis - especially those for higher organisms - pose many pitfalls for the unwary.
To obtain the maximum amount of correct information from action spectra, three criteria must be met: ( 1 ) insightful experimental design, (2) accurate and precise experimental execution and (3) correct and complete analysis of the data. Jagger [1] and Coohill [2,3] have given excellent discus- sions of the principles of action spectroscopy and the re- quirements for correct design of experimental protocols. Experimental methodology, of course, will depend on the chemical or biological system of interest. The ideal assay method for action spectroscopy provides quantitative data and is sufficiently sensitive to yield accurate data over several orders of magnitude.
Even with proper experimental design and execution the methods and types of analysis chosen to treat the data deter- mine both the validity and extent of the conclusions that may be drawn. Further, in comparisons of action spectra- of great importance in evaluating the effects of various broad spec- trum sources on biological systems - incorrect analysis procedures can produce misleading or simply incorrect conclusions.
Action spectra can be compared directly or in conjunction with the spectral distribution of the source in question by calculating their effectivity spectra [4]. An effectivity spec-
30 B.M. Sutherland / Journal of Photochemistry and Photobiology B: Biology 31 (1995) 29-34
trum is calculated by multiplying the effect produced in a biological system by a specific wave band times the number of photons in that wavelength range in the broad spectrum source. Effectivity spectra allow calculation of the integrated impact for a response of a broad spectrum source, e.g. sun- light, on a biological system. Since less attention has been given to these aspects of action spectrum analysis, we shall consider several aspects of its pitfalls and potentials.
2. Analysis of absolute action spectra
Determinations of UV effects on biological systems give data in the form of the magnitude of the chemical or biological effect per incident photon per unit area for each wavelength investigated. The biological or chemical data plotted as a
function of wavelength then comprise an absolute action spectrum.
Fig. 1A shows two absolute action spectra for UV effects on two systems, both having the same wavelength depend- ence, but one ( l l ) has 1000 times greater sensitivity to alter- ation by incident UV photons than the other (Fq). The broken curve shows a solar spectrum for a solar zenith angle of 40 ° under a stratospheric ozone column of 0.32 cm [ 5 ]. Fig. I B shows the effectivity spectra calculated from the absolute data shown in Fig. 1A. Multiplication of the absolute action spec- tra by the solar photon spectrum yields effectivity spectra that show the wavelength distribution of the total UV effect and also retain the 1000-fold difference in sensitivity.
For comparing the effects of UV on dissimilar biological systems, action spectra are frequently normalized to their value at one wavelength, resulting in a normalized action spectrum. Fig. 1C shows the result of normalizing the two
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Wavelength Wavelength (nm) Fig. t. Action spectrum and effectivity spectrum analysis of systems with the same wavelengths dependence but different UV sensitivities. (A) Action spectra for systems with high ( • ) and low (D; i/1000 that of the high sensitivity system) UV sensitivities and a solar spectrum for downward global solar flux at the surface of the Earth for a solar zenith angle of 40 ° and 0.32 cm of stratospheric ozone (broken curve) [ 5 ]. (B) Effectivity spectra computed from the data in A (product of the values in each action spectrum multiplied by the solar flux data at each wavelength). Tbese effectivity spectra clearly show that the wavelength dependences of the two systems are the same but that their sensitivities differ by ! 000. (C) Action spectra in A normalized by dividing each value by the maximum value. The nommlized spectra are superimposable. (D) Normalized effectivity spectra calculated from the data in C, showing that the wavelength dependence of the two systems is the same. However, the quantitative difference between the systems is now lost.
B.M. Sutherland/Journal of Photochemistry and Photobiology B: Btology 31 (1995) 29-34 31
spectra shown in Fig. 1A by dividing each by its maximum value. Since the two spectra in Fig. 1A have the same wave- length distribution, the resulting normalized curves are super- imposable.
If we calculate effectivity spectra from the normalized action spectra, we obtain the results shown in Fig. ID. The wavelength distributions of the original spectra are still evi- dent, but the 100-fold difference in sensitivity has now dis- appeared. This loss of quantitative information obscures the large differences between these biological systems. Such dif- ferences may be critical for correct assessment of the biolog- ical impact of various broad spectrum sources, e.g. increased solar UVB.
3. Consequences of shielding on action spectrum comparisons
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The case shown in Fig. 1 illustrates the effect of a difference in UV sensitivity in two systems that have the same wave- length dependence. However, most biological systems do not have the same wavelength dependence but differ in both magnitude and wavelength dependence. Fig. 2 shows an example of this situation. Fig. 2A shows the absolute action spectra for pyrimidine dimer induction by UV in two systems. The upper curve ( 1 ) shows data for dimer induction in unshielded DNA in solution [6] . The lower curve (r-l) shows the results for the same DNA in solution but with a hypo- thetical filter interposed between the UV lamp and the DNA. Our hypothetical filter is transparent above about 315 nm but transmits only 0.1% of all photons below 315 nm. (Note that only the overlying filter has changed, not the target DNA.) The choice of normalizing wavelength is critical for the cor- rect comparison of these spectra. I f the data are normalized at a wavelength where there is no shielding, the normalized spectra retain the same shapes as the original data shown in Fig. 2A. If, however, the data are normalized at a wavelength where there is shielding, the "shielded" DNA spectrum now shows an incorrectly high effect at the unshielded wave- lengths (Fig. 2B) !
4. Pyrimidine dimer induction in DNA in human skin and in solution
Our hypothetical filter with a sharp wavelength cut-off is clearly unrealistic, but similar effects occur in real situations. Two such cases are shown in Figs. 3 and 4. We will consider the effects of appropriate and inappropriate analyses in these systems.
Alterations in UVB in the solar spectrum may have major effects on human health and thus quantitating the magnitude of these effects is of major concern. A principal adverse effect of sunlight on humans is the induction of skin cancer. Pyrim- idine dimer formation in DNA in human skin has been impli- cated as an initiating event in some kinds of skin cancer [7].
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Wavelength (nm) Fig. 2. lmpact of normalizing action spectra at a wavelength at which shield- ing is significant. (A) Action spectra for production of pyrimidine dimers in DNA irradiated in dilute solution without a filter (1) and with a filter (which transmits radiation above 315 nm completely but only 0.1% of all radiation below that wavelength) interposed between the UV source and the DNA (r"l). Although the DNA is unchanged, the action spectrum is quite different, reflecting the large effect of shielding on the apparent shape of the spectrum. Normalization of these spectra at wavelengths without shielding yields shapes as shown. (B) Normalization of the spectra in A using a wavelength with shielding for the normalizing wavelength. This procedure yields incorrect spectra with apparently high values at the unshielded wave- length.
Fig. 3A shows an absolute action spectrum for induction of pyrimidine dimers in skin of healthy human volunteers (O) [8] and contrasts it with an absolute action spectrum for induction of dimers in unshielded DNA in solution ( 0 ) [6]. Fig. 3B shows the effectivity spectra calculated from the absolute action spectral data. It is clear that unshielded DNA
32 B.M. Sutherland l Journal of Photochemistry and Photobiology B: Biology 31 (1995) 29-34
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Fig. 3. Action spectra and effectivity spectra for pyrimidine dimer production in human skin in situ compared with DNA in solution. (A) Absolute action spectra for pyrimidine dimer production in DNA in solution (O) [6] and in DNA in human skin (O) [8], together with a sunlight spectrum as in Fig. IA. The skin action spectrum has a wavelength maximum at a longer wavelength owing to cellular shielding of DNA and is much less sensitive to dimer induction than unshielded DNA. (B) Effectivity spectra calculated from the absolute spectra shown in A. These spectra show the longer wavelength maximum of the skin spectrum, the lower sensitivity of skin in the UVB region and the similar sensitivities at wavelengths greater than about 320 nm. (C) Action spectra normalized at 300 nm, at which wavelength skin shows some shielding of DNA by upper cellular layers (DNA in solution (O), skin (O)), along with the same solar spectrum as in A. (D) Effectivity spectra calculated from the normalized action spectra in C. Although the wavelength maximum of the skin spectrum is shown correctly at longer wavelength than that of DNA in solution, the normalized skin spectrum shows a misleading apparent higher sensitivity at longer wavelengths (of. the original data in A).
has a maximum sensitivity at a shorter wavelength than does DNA in intact skin ( owing to shielding by upper skin layers). Furthermore, unshielded DNA is some three orders of mag- nitude higher in sensitivity to dimer induction by wavelengths in the UVB region, somewhat higher in sensitivity at wave- lengths just above 300 nm and of similar or even less sensi- tivity in the UVA region (320-400 nm).
Fig. 3C shows the same data but now normalized to the value at 302 nm, a wave band frequently used in comparisons of action spectra in different species or of different responses in the same species. Fig. 3D shows the result of calculation of the effectivity spectra from the normalized data. This method of analysis has distorted the data in two important ways. First, the difference in sensitivity is apparently
decreased, most importantly in the UVB, of critical environ- mental concern. Moreover, the data suggest an erroneous higher sensitivity of DNA in skin to longer wavelength UV radiation relative to that of DNA in solution.
5. Comparisons of action spectra with large differences at the normalizing wavelength
Calculation of effectivity spectra from normalized action spectra can be even more misleading in a biological system with higher shielding at the wavelength chosen for normali- zation. Fig. 4 shows such a case, namely a comparison ot pyrimidine dimer formation in alfalfa seedlings [9] and in
B.M. Sutherland / Journal of Photochemistry and Photobiology B: Biology 31 (1995) 29-34 33
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Fig. 4. Action spectra and effectivity spectra for pyrimidine dimer production in alfalfa seedlings compared with DNA in solution. (A) Absolute action spectra for pyrimidine dimer production in DNA in solution ( • ) [6] and in DNA in UV-irradiated intact alfalfa seedlings (A) [9], together with a sunlight spectrum as in Fig. 1A. The alfalfa action spectrum has a wavelength maximum at a longer wavelength owing to cellular shielding of DNA and shows significant DNA shielding at 302 rim. (B) Effectivity spectra calculated from the absolute spectra shown in A. These spectra show the longer wavelength maximum of the alfalfa spectrum, the lower sensitivity of DNA in alfalfa seedlings in the UVB region and the similar sensitivities at wavelengths greater than about 320 nm. (C) Action spectra normalized at 302 nm, at which wavelength alfalfa seedlings show considerable shielding of DNA by upper cellular layers or pigments ( DNA in solution ( • ) , alfalfa ( A ) ), along with the same solar spectrum as in A. (D) Effectivity spectra calculated from the normalized action spectra in C. Although the wavelength maximum of the alfalfa spectrum is shown correctly at longer wavelength than that of DNA in solution, the normalized alfalfa spectrum show less sensitivity in the UVB and an apparent, incorrect, higher sensitivity of greater than a factor of 10 than in DNA in solution at longer wavelengths (cf. the original data in A).
DNA in solution [6]. Fig. 4A contains the absolute action spectrum for dimer induction in alfalfa seedlings (A) and the absolute spectrum for dimer induction in unshielded DNA in solution (A) , along with the solar spectrum [5] shown in the previous figures. Calculation of the effectivity spectra from the absolute spectra yields the curves shown in Fig. 4B. These results clearly indicate that DNA in alfalfa is highly shielded in the UVB region in comparison with DNA in solution. The longer wavelength data suggest approximately equal sensitivity of the two systems, although the alfalfa data at 334 nm suggest possible higher UVA sensitivity than in isolated DNA. Clearly additional data would be required to determine the validity of this possibility.
The absolute data in Fig. 4A are qualitatively quite differ- ent at 302 nm. Fig. 4C shows the result of normalizing the data to their values at this wavelength (DNA in solution, A; DNA in alfalfa seedlings, A). Fig. 4D shows the result of using the normalized data to calculate effectivity spectra. Like the normalized human skin effectivity spectra, the normalized spectra for alfalfa and for unshielded DNA appear (incor- rectly) similar in the UVB region. More importantly, how- ever, the longer wavelength UV data for alfalfa are clearly misleading: they show a consistent, apparent order-of-mag- nitude higher sensitivity than unshielded DNA! This com- parison clearly illustrates the erroneous result which can be obtained by (1) normalizing the data at a wavelength at
34 B.M. Sutherland / Journal of Photochemistry and Photobiology B: Biology 31 (1995) 29-34
which shielding by cellular pigments or structures occurs and (2) using such normalized data to calculate normalized effec - tivity spectra.
the evaluation of UV effects on simple and complex organ- isms and ecosystems and it is important to develop approaches for obtaining the maximum amount of correct information from these complex and demanding experiments.
6. C o n c l u s i o n s a n d s o l u t i o n s
What are the solutions to this complex problem? It is clear that the same solution will not be applicable to every spec- trum. Certainly an ideal spectrum would retain both the quan- titative and qualitative information of the data: the magnitude of the effect produced per photon and its wavelength distri- bution. Frequently quantitative aspects are lost and this infor- mation can be critical. If a UV effect requires 9 MED of UV in an acute dose, it is likely to be of less importance than one which requires 1 MED or even an accumulated dose of 9 M E D - since 4--5 MED can produce severe skin peeling, 9 MED would correspond to a huge dose and humans would not be likely to subject themselves to those conditions.
Even qualitative information can be lost or - even worse - produce misleading results upon analysis by treatments which would seem reasonable upon first glance. Two exam- ples illustrate this problem: pyrimidine dimer formation in human skin (Fig. 3A) and in intact alfalfa seedlings (Fig. 4A) vs. isolated DNA. Normalization of the action spectra at a wavelength at which there is shielding or absorption of incident UV (see Figs. 3C and 4C) produces spectra which change the apparent effectiveness of different UV wave- lengths and which give effectivity spectra with significantly different implications for consequences of ozone depletion.
One solution is to compare only the original, non-normal- ized, action spectra. One complication is the requirement for graphs which span even more orders of magnitude than are required for a single absolute spectrum (see Fig. 4B for exam- pie). A second is that the unwary might conclude that bio- logical responses requiring a large exposure are necessarily less important than those requiting a low exposure, which is certainly not the case. It might be quite useful in cases of complex biological responses, e.g. cancer production in a mouse, to include in the same graph the action spectrum (non- normalized) for erythema, peeling or some other measure of biological response.
It is clear that action spectrum analysis can be difficult and it is easy to obtain unexpected or even misleading results by some treatments. It is also clear that this is a critical field for
A c k n o w l e d g m e n t s
I thank the many collaborators- Ms. Ann Emrick and Drs. S.E. Freeman, H. Hacham, R.W. Gange, D.J. Maytum, J.C. Sutherland and F.E. Quaite - who have made the action spectra discussed in this review a reality and John Sutherland for reading the manuscript and for cogent suggestions. This research is supported by the National Cancer Institute of the National Institutes of Health, by the US Department of Agri- culture and by the Office of Health and Environmental Research of the US Department of Energy.
R e f e r e n c e s
[ 1 ] J. Jagger, in A. Hollaender (ed.), Introduction to Research in Ultraviolet Photobiology, Prentice-Hall, Englewood Cliffs, N J, 1967.
[2] T.P. Coohiil, Action spectra again?, Photochem. Photobiol., 54 ( 1991 ) 859-870.
[ 3 ] T.P. Coohill, Action spectra revisited, J. Photochem. Photobiol. B: Biol., 13 (1992) 95-100.
[4l R.B. Setlow, The wavelengths in sunlight effective in producing skin cancer: a theoretical analysis, Proc. Natl. Acad. Sci. USA, 71 (1974) 3363--3366.
[5] E.P. Shettle, M.L. Nack and A.E.S. Green, Multiple scattering and influence of clouds, haze, and smog on the middle UV reaching the ground, in D.S. Nachtwey, M.M. Caldwell and R.H. Biggs (eds.), Impacts of Climatic Change on the Biosphere, Part 1, Rep. DOT TST- 76-55, National Technical Information Service, Spinglfleld, VA, 1975, pp. 2-38-2.-49.
[6] A. Emrick and J.C. Sutherland, Action spectrum for pyrimidine dimer formation in T7 bacteriophage DNA, Photochem. Photobiol., 49 (1989) 35S.
[7] D.E. Brash, J.A. Rudolph, J.A. Simon, A. Lin, G.J. McKenna, H.P. Baden, A.J. Halperin and J. Ponten, A role for sunlight in skin cancer: UV-induced p53 mutations in squamous cell carcinoma, Proc. Natl. Acad. Sci. USA, 88 (1991) 10 124-10 128.
[8] S.E. Freeman, H. Hacham, R.W. Gange, D. Maytum, J.C. Sutherland and B.M. Sutherland, Wavelength dependence of pyrimidine dimer formation in DNA of human skin irradiated in situ, Proc. Natl. Acad. Sci. USA, 86 (1989) 5605-5609.
[9] F.E. Quaite, BM. Sutherland and J.C. Sutherland, Action spectrum for DNA damage in alfalfa lowers predicted impact of ozone depletion, Nature, 358 (1992) 576-578.
