some further considerations on the use of repair-defective organisms as biological dosimeters for...

P L i r m bcniiurv rrnr l P h o r o h i o l o y ~ . Vol. 30. pp. 733 to 737 0 Pergamon Press Ltd 1979. Printed in Great Britain

TECHNICAL NOTE

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SOME FURTHER CONSIDERATIONS ON THE USE OF REPAIR-DEFECTIVE ORGANISMS AS BIOLOGICAL

DOSIMETERS FOR BROAD-BAND ULTRAVIOLET RADIATION SOURCES

JOHN CALKINS*? and JEANNE A. BARCELOt *Department of Radiation Medicine and tSchool of Biological Sciences, University of Kentucky,

Lexington, KY 40536. USA.

(Received 30 March 1979; accepted 6 July 1979)

I

Abstract-The extreme variation in biological effectiveness of the various components of solar ultraviolet radiation (solar UV) which reaches the earth’s surface, especially photons of wavelengths between 295 and 330 nm, makes the dosimetry of solar UV a complex and, as yet, unresolved problem. A proper weighting of the various components of solar UV would permit expression of expsoure as a single parameter (dose). Weighting could compensate for the variations in composition of solar UV which might occur during exposure or the differences in sources of UV radiations; weighting would permit comparison of exposures at various locations on the earth and extrapolation of laboratory observations to field situations where wavelength composition might be rather different. Various radiation-sensitive microorganisms have been proposed as biological dosimeters. Biological dosimeters automatically weight the subcomponents of solar UV differently than a purely physical irradiance meter. We have examined the available evidence regarding the weighting which repair-defective mutants provide in comparison with response of a number of wild-type organisms and would caution investigators that, for broad-band UV sources, especially those with significant biological actions through the range of 300-330 nm, repair-sensitive mutants may improperly weight the components, leading to errors of dosimetry and thus to possible errors of interpretation of results of solar UV exposure of wild-type organisms

Repair-defective microorganisms have been proposed as potential biological dosimeters suitable for moni- toring UV radiations (Harm, 1969; Billen and Green, 1975; Tyrrell, 1978). Biological dosimeters have many advantages for the purposes of evaluating the ecological actions of solar UV radiation. As noted by Tyrrell (1978) they may constitute a sensitive, temperature- independent, convenient and relatively inexpensive form of integrating dosimeter. In addition, biological dosimetry systems are intended to accomplish another function which is essential and not obvious. Different wavelength components of broad-beam UV sources (such as sunlight) will vary greatly in their biological effectiveness. Biological dosimeters will automatically weight the incident UV components in relation to the effectiveness of the different wavelengths. The measured result (survival of the exposed microorganisms in most cases) will depend on the summation of the incident irradiance rimes the biological effectiveness of the various incident photons. For instance, the mutant system proposed by Tyrrell (1978) would weight a 254-nm photon 1 million times greater than a 365-nm photon (see Fig. 1). Before employing biological dosimeters based on repair- defective mutants for the evaluation of the ecological action of broad-band UV sources such as sunlight on repair-competent organisms, one should consider the

appropriateness of the weighting factor for the wavelengths in question.

There is reason to believe that there may be significant qualitative differences in the injurious action of far (A < 300nm) and near (A > 300nm) UV (Harm, 1978; Eisenstark, 1971). It is not known exactly where the transition from near- to far-UV-type response occurs; however, wavelengths below 300 nm show relative effectiveness (action spectra) closely resembling the absorption spectrum of DNA (Setlow, 1974). Deviations from an efficiency proportional to the DNA absorption. spectrum become progressively greater at wavelengths greater than 300 nm. Although pyrimidine dimers contribute little to the lethal effect induced by 365-nm photons, Tyrrell (1973) has shown by direct analysis that 365-nm radiation produces some pyrimidine dimers, the principal injurious photoproduct of far-UV irradiation. Thus, it can be concluded that the transition from near- to far-UV- type lesions will occur over a substantial wavelength band and the transition will include the short-wave end of the solar spectrum reaching the earth’s surface in measurable intensity (i.e. 290-360 nm).

There are at present only a very limited number of published action spectra of wild-type organisms. Repair-defective forms of bacteria have received more extensive study regarding action spectra; such

733

734 JOHN CALKINS and JEANNE A. BARCELO

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I d 260 280 300 320 340 ?m

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Figure 1. A compendium of action spectra for UV-induced lethality assembled from various published action spectra of diverse microorganisms. All spectra include observations of response to the two mercury lines 254 nm (far UV) and 365 nm (near UV). Results have been normalized to the response at 254 nm; where survival curves were available the 90%-lethal effects were utilized as the criterion of biological response; if survival data was not reported, action spectra were included as published. The indicated observations have been replotted from the following sources as noted by the numbers: ( 1 ) Webb er a/., 1978; (2) McAulay and Taylor, 1939; (3) Danpure and Tyrrell, 1976; (4) Peak, 1970; (5) Luckeish, 1946; (6) Tyrrell, 1978; (7) Webb and Lorenz, 1970; (8) Tyrrell, 1976; (9) Webb and Brown, 1976; (10) Mackay et a/., 1976; (11) Tyrrell, 1978. Action spectra of very sensitive repair-defective organisms are noted and are similar to each other while clearly different in relative response compared to the remaining organisms which are presumed to be wild-type in radiation response (E. coli B/r and its derivative WP-2 have been proposed by Adler (1966) as showing wild-type response). Since normalization was to the 254 nm response, the large deviations appear in the near UV ( A > 300 nm); however, if normalization had been to 365 nm the deviations would appear in the far (A .= 300 nm) response. It is not possible to distinguish through this plot if the radiation-sensitive organisms are excessively sensitive to far UV or abnormally resistant to near UV; however, it is clear that the action spectra of the very

sensitive mutants are not sensitized equally for near and far UV.

Technical Note 735

organisms are of intrinsic interest and are also easier to study since the requirement for very high irra- diances required to kill wild-type organisms with near UV is reduced in the repair-defective mutants. A repair-defective mutant which demonstrated the same action spectrum as wild-type organisms might be an extremely useful biological dosimeter for organisms showing the wild-type response. Unfortunately, it appears that the currently available repair-defective mutants deviate in response from the wild-type action spectra and should only be used recognizing the dosimetry errors which could arise from improper spectral response. It is known that there may be ‘synergistic’ interactions between UV-A (2 > 320nm) and UV-B (E. = 32CL280nm) components present in sunlight; repair-defective organisms show a reduced synergism (see Webb, 1977, for an extensive review). The principal modification of solar UV-B reaching the earth’s surface arises from the difference in the amount of ozone which sunlight must traverse to reach the earth’s surface, a factor which varies greatly with time of day, latitude and season. The greater the ozone penetrated, the less will be the UV-B compared to UV-A since the UV-B components are more strongly absorbed by ozone. Action spectra can be determined by using optical filters to remove various short-wave components from sunlight and then observing the reduced biological effectiveness of the remaining solar radiation. Such a method used by Luckiesh (1946) simulates the action of ozone and compensates for the synergistic action of near- and far-UV and may be the most desirable method of determining action spectra for purposes of photoeco- logy.

We have assembled the action spectra of the most radiation-sensitive bacterial mutants and all action spectra available to us of wild-type organisms which include both the mercury lines 254 and 365nm (Fig. 1). These two lines appear to elicit the far and near UV responses, respectively. We have normalized responses to 254 nm. The relative effectiveness of different wavelengths cannot always be expressed as a single number; if the dose-response kinetics of two radiations differ, then the relative effectiveness will depend on the level of response chosen for comparison. Hollaender (1943) noted that near UV tends to produce sigmoidal responses while far UV more often produces exponential dose-response curves. We have compared the 90% lethal doses when possible, as sug- gested by Webb and Brown (1976). When only action spectra were published we accepted the author’s values. We also note a few determinations of lethality of 254 nm compared with the response to 365 nm for the same organisms; although these do not constitute complete action spectra, they show the relative effectiveness of near and far UV. It is clear from Fig. 1 that the repair-defective mutants do not simulate the wild-type response throughout the spectral region critical for sunlight dosimetry.

We have averaged the response of the three repair-

defective organisms and compared the various indivi- dual action spectra to the weighting which would result from the use of a repair-defective organism as a biological dosimeter (Fig. 2). The response of repair- defective organisms appears consistent with the wild- type response from 254300 nm. However, above 300 nm the relative responses of repair-defective organisms, although closely resembling each other as noted by Tyrrell(1978), progressively deviate from the wild-type response. The overall trepd is for increasing deviation between 300 and 334 nm, whereas a number of wild-type organisms seem to differ from the repair- defective mutants by a relatively constant factor at 334 and 365 nm. More accurate and extensive action spectra are needed to determine if the deviation is generally constant or continues to increase as indicated for some organisms. It is clear from Figs. 1 and 2 that the radiation-sensitive mutants have been sensitized much more to far UV than to near UV and would thus tend to overweight the far UV components of mixed radiations. The sensitive mutants presently available may be adequate for biological dosimetry of monochromatic radiations and for radiations entirely below 300 nm or above 330 nm, but it is clear that they are not suitable for inferring responses of wild-type organisms exposed to broad- band UV including appreciable energy in the 300-330-nm band. Since radiation in the 3W330 nm accounts for much of the injurious action of sunlight and is also the most variable in intensity, the presently available mutants do not appear to provide an accurate system for sunlight dosimetry.

The dosimetry of sunlight for biological and ecological (including human) purposes poses many diffi- cult problems which are as yet unresolved. Simply stating the amount of energy incident within a wavelength band is clearly inadequate whenever the biological effectiveness of different wavelengths vary sig- nificantly within the band. Sunlight is so variable it becomes an enormous problem to determine and specify the variations of irradiance in numerous narrow wavelength bands. Thus, a weighting system seems the only practical solution. We note that the SI system includes several biologically weighted units (the sievert for mixed LET ionizing radiation, the lux and tolbert for visual-efficiency-weighted visible light). The diversity of responses illustrated in Fig. 1 can be inter- preted in several different ways. It may be impossible ’to select a single action spectrum as the ‘typical’ or ‘standard for all organisms. The concept of ‘biologically effective dose’ might have to be restricted to a single species or small group of species which respond in a sufficiently similar manner. Since the number of action spectra is so limited and they are of varying quality, it is also possible that in the future more accurate determinations of relative response will con- verge to some ‘standard response’ adequate for gen- eral ecological purposes. There would doubtless be deviations from such a standard which would require further consideration when extensive studies on a

736 JOHN CALKINS and JEANNE A. BARCELO

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REPAIR DEFECTIVE MUTANTS

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i . I L . 1 " I ' 1 " ' 1 " L 240 260 280 300 320 340 3m

WAVELENGTH (nm)

Figure 2. Relative responses of the various organisms plotted in Fig. 1 compared with the average response of the three sensitive mutant organisms. If a 'typical' radiation-sensitive mutant was used as a biological dosimeter then it would produce weighting errors as indicated in this figure. Weighting is again normalized to 254 nm. Radiation-sensitive organisms can be calibrated to serve as biological dosimeters for monochromatic radiations even if they vary considerably from wild-type action spectra. However, the use of mutants for biological dosimeters for broad-band radiation sources requires con- stancy of weighting relative to the response of the system under investigation. Dosimetry for the various wild-type organisms could not utilize the mutants if both near and far UV were contributing signifi- cantly to the biological actions. Solar UV action frequently includes contributions from 300-340 nm and

would be improperly measured by organisms responding with the mutant action spectra.

given organism were planned. If it is possible to express the biological action of solar UV as a single dose parameter, it is clear that many wild-type action spectra should be available and a consensus should be

reached as to which 'standard' or group of standard curves best fits the needs as a system for expression of exposure to solar UV.

REFERENCES

Adler. H . 1. (1966) Adu. Radiat. Biol. 2, 167-191. Billen, D. and A. E. S. Green (1975) Photochem. Photobiol. 21, 449451. Danpure, H. J. and R. M. Tyrrell (1976) Photochem. Phorobiol. 23, 171-177. Eisenstark, A. (1971) Adu. Genet. 16, 167-198. Harm, W. (1969) Radiat. Res. 40, 63-69. Harm, W. (1978) Mutat. Res. 51, 301-310. Hollaender, A. (1943) J. Baceteriol. 46, 531-541. Luckeish, M.

Mackay, D., A. Eisenstark, R. B. Webb and M. S. Brown (1976) Photochem. Photobiol. 24, 337-343. McAulay, A. L. and M. C. Taylor Peak, M. J. (1970) Photochem. Photobiol. 12, 1-8. Setlow, R. B. Tyrrell, R. M. (1976) Photochem. Photobiol. 24, 345-351.

(1946) Application of Germicidal, Erythema1 and Infrared Energy. Van Nostrand, New York.

(1939) J. Exp. Biol. 16,474482.

(1974) Proc. Natl. Acad. Sci. U.S.A. 71, 3363-3366.

Technical Note 737

Tyrrell, R. M. (1978) Photochem. Photobiol. 27, 571-579. Tyrrell, R. M. and M. J. Peak (1978) J . Bacreriol. 136, 437440. Webb, R. B. (1977) In Photochemical and Phorohiological Reviews (Edited by K. C. Smith), Vol. 11, pp.

Webb, R. B. and J. R. Lorentz (1970) Photochem. Photobiol. 12, 283-289. Webb, R . B. and M. S. Brown (1976) Photochem. Photobiol. 24, 425432. Webb, R. B., M. S. Brown and R . M. Tyrrell (1978) Radiat. Res. 74, 298-311.

169-261. Plenum Press, New York.

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