Biological UV dosimeters in the assessment of the biological hazard from environmental radiation

www.elsevier.nl/locate/jphotobiol J. Photochem. Photobiol. B: Biol. 53 (1999) 36–43

Biological UV dosimeters in the assessment of the biological hazard from environmental radiation a, ´ ´ ´ a, P. Grof ´ a, P. Rettberg b, G. Horneck b, Gy Ronto´ a *, A. Fekete a, S. Gaspar A. Berces a

Institute of Biophysics and Radiation Biology, Semmelweis University of Medicine, Budapest and Research Group for Biophysics, Hungarian Academy of Sciences, PO Box 263, H-1444 Budapest, Hungary b ¨ DLR, Institute of Aerospace Medicine, Radiation Biology Division, Linder Hohe, D-51170 Cologne, Germany Received 25 August 1999; accepted 13 October 1999

Abstract To determine the impact of environmental UV radiation, biological dosimeters that weight directly the incident UV components of sunlight have been developed, improved and evaluated in the frame of the BIODOS project. Four DNA-based biological dosimeters ((i) phage T7, (ii) uracil thin layer, (iii) spore dosimeter and (iv) DLR-biofilm) have been assessed from the viewpoint of their biological relevance, spectral response and quantification of their biological effectiveness. The biological dosimeters have been validated by comparing their readings with weighted spectroradiometer data, by comparison with other biological doses, as well as with the determined amounts of DNA UV photoproducts. The data presented here demonstrate that the biological dosimeters are potentially reliable field dosimeters for measuring the integrated biologically effective irradiance for DNA damage. q1999 Published by Elsevier Science S.A. All rights reserved. Keywords: Biological dosimeters; Biological hazard; DNA; UV-B radiation

1. Introduction The stratospheric ozone concentration has been measured by several methods, e.g., total ozone measurements from the Earth’s surface, satellite measurements from the Earth’s orbit (TOMS) and chemical analysis of the atmosphere with balloon measurements, in addition to spectroradiometry [1–14]. Altogether, these data indicate a slight but significant decreasing tendency of the ozone-layer thickness, superimposed by much larger seasonal changes at specific geographical locations. Recently a detailed analysis of the atmospheric changes and of the consequent UV-B radiation increase has been done [15]. It has been concluded that in spite of international efforts, the existence of the present vulnerable state of the ozone layer can be predicted for the next two decades and its recovery can be expected for the next half-century, depending on the actual composition and utilization of different CFCs [16,17]. Stratospheric ozone plays an important role in attenuating the short-wavelength components of the solar spectrum, so the consequence of a decreased ozone layer is an increased * Corresponding author. Tel./fax: q36 1 266 6656; e-mail: berces@ puskin.sote.hu

UV-B radiation level. A high level of UV-B radiation endangers the whole biosphere, therefore biological risks including human health risks have become and will also remain in the future a global problem [18]. Erythema is probably the most widely experienced form of acute solar damage to the skin. Commercially available UV meters, measuring the biological effect of radiation, have been constructed with spectral sensitivity close to the erythema action spectrum defined by the CIE. However, by using these erythemally weighted broad-band instruments to detect the long-term trends of UV-B radiation, controversial data have been obtained [12–14,19,20]. Deoxyribonucleic acid (DNA), the genetic material of cells, is the major target molecule of solar UV radiation for erythema and skin-cancer induction [21–23]. Thus, to assess the biological risks (including the risk of skin cancer) due to environmental radiation, it seems reasonable to weight the solar spectrum by the spectral sensitivity of DNA damage, which possesses high sensitivity at the short-wavelength edge of the solar spectrum [21]. Early efforts using simple biological systems for detecting solar radiation were also based on the DNA-damaging effect [24,25]. Recently several DNA-based biological dosimeters have been developed [26–32]. As part of a European Community project, in the frame of the BIODOS cooperation, four of

1011-1344/99/$ - see front matter q1999 Published by Elsevier Science S.A. All rights reserved. PII S 1 0 1 1 - 1 3 4 4 ( 9 9 ) 0 0 1 2 3 - 2

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them were improved, evaluated and applied in laboratory and/or field measurements. These dosimeters use B. subtilis spores [24,26,30], DLR-biofilm developed specifically from B. subtilis spores [31,33], bacteriophage T7 [28,34] and polycrystalline uracil as UV detectors [35]. The main goal of the present work is to summarize the essential criteria for biological UV dosimeters and to evaluate their advantages and disadvantages, especially in the case of bacteriophage T7, uracil thin-layer biodosimeters and the DLR-biofilm developed in our laboratories. The criteria have been gathered and accepted by the BIODOS partners [36] in order to establish quality control in this newly developed field of solar UV dosimetry. The necessity to utilize quality control in biological UV dosimetry is very similar to that in UV monitoring performed with physical devices [37,38]. Completing the criteria for biological dosimeters can establish principles for the intercomparison of measured biological dose values, for the transformation of the various biological doses into each other, and for the correct assessment of the biological hazard on individual and global levels as well.

2. Biological relevance One of the most important criteria for the validity of a biological dosimeter is the relevance of the photobiological/ photochemical process to the biological effects. In a great number of different biological end-effects, the key process is DNA damage due to UV radiation. DNA-damaging effects of solar UV radiation play an important role in causing human erythema, skin cancer [21,22,39–41] and damage to terrestrial and aquatic ecosystems [29,42–44]. Thus DNA-based biological dosimeters have genuine biological relevance. The basic concept of DNA-based biological dosimetry is as follows: c use of DNA-containing systems (or their components) assuming them as models of DNA damage in the chromosomes, being an essential compound of all biological systems; c quantification of the photobiological/photochemical effect induced by UV radiation;

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c extrapolation of the measured biological/photochemical effect from the simple system of a biological dosimeter to a more complex biological effect, which can include the human health effect or effects on terrestrial and aquatic ecosystems as well. DNA-based biological dosimeters, including the phage T7, uracil and spore dosimeters as well as the DLR-biofilm, fulfil the requirement to detect biologically significant photoprocesses that contribute to the biological risks of UV radiation. For quantification, the biological UV effect induced in simple biological systems has to be transferred to and evaluated in large populations. The inactivation (killing) of a phage particle as a consequence of DNA damage [28,34] is the biological effect for phage T7, which is used as an end-point in the determination of the UV dose. In the uracil dosimeter the specific polycrystalline structure of the nucleic acid bases provides a high efficiency for the photodimerization of uracil monomers [35,45]. These photoproducts are evaluated by spectrophotometry upon the decrease in specific absorption characteristics. The spore dosimeter and DLR-biofilm [26,30,31,46–49] use immobilized B. subtilis spores as detectors and the UV effect is evaluated either by measuring directly the remaining colony-forming potential of the spores after UV exposure by extracting, diluting and plating, or by measuring the total biomass of the germinated and multiplied bacteria after incubation of the whole biofilm in nutrient medium (Fig. 1).

3. Spectral response In theory, one could expect that the spectral sensitivity (action spectrum) of DNA-based dosimeters should be similar to that of DNA damage; however, the spectral responses of the simple biological systems used in our dosimeters differ slightly from each other depending on the base composition of the DNA and the kind of molecules surrounding the DNA. This difference is due partly to the fact that there are two possible mechanisms for DNA damage: (i) through direct absorption of the photon energy by the DNA; and (ii) through indirect photochemical processes between the other UVabsorbing molecules in the vicinity of the DNA and the DNA

Fig. 1. Photograph of an exposed (circular exposure areas), calibrated (rectangular exposure areas) and processed biofilm with microscopic magnifications showing the filamentous growing bacteria in areas irradiated with a highly biologically effective UV dose (left) and a dark control area (right).

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itself, and other influencing factors. In spite of these differences in the mechanisms, there are common features in the spectral responses: at the shorter wavelengths (l-300 nm) of the UV-B range, the sensitivities of DNA-based dosimeters are about three to four orders of magnitude higher than at 320 nm. For the biological dosimeters mentioned above, the action spectra have been determined earlier with monochromatic UV sources in the case of phage T7 and for uracil with a 2.5 kW Xe lamp and a Jobin–Yvon monochromator [28,34]. In addition, a comparison of the standardized MED spectrum [14] and the Robertson–Berger (RB) meter has been performed [50]. In the wavelength range 280–300 nm a higher sensitivity by one and two orders of magnitude than at 320 nm has been found for uracil and phage T7 dosimeters, respectively. Using monochromatic light for the determination of the spectral sensitivity values of the biological dosimeters and exposing them to polychromatic solar radiation, the following problems arise: 1. Are the effects of different wavelengths additive in inducing spore or phage inactivation, or uracil dimerization? 2. Is there a wavelength interaction in the UV effect in different biological systems: synergism/antagonism? Based on our earlier results [51], the phage-inactivating effect caused by different wavelengths has an additive nature. The second question can be answered in two ways: theoretically and experimentally. Both phage T7 and polycrystalline uracil do not possess metabolic activity during the irradiation, thus from the theoretical aspect, such a wavelength interaction can be excluded. From the experimental aspect, a method has been developed for the determination and mathematical description of action spectra using different UV sources with well-defined polychromatic emission spectra [52]. Using five UV lamps (Philips TL01, with and without phthalic acid filtering, Westinghouse FS20, with and without filtering, and a Xe arc solar simulator), phage T7 inactivation and uracil dimerization action spectra have been determined; both of them are described by two exponential terms of different slopes. The action spectrum of B. subtilis spore inactivation has been determined with high accuracy at nine different wavelengths in the range 254–400 nm [47] using an Okazaki Large Spectrograph (OLS). DLR-biofilm action spectra for B. subtilis spores of the strains HA101 and TKJ6312 have also been determined at 13 wavelengths between 254 nm and 400 nm with the OLS [53]. The former is a DNA-repairproficient wild type; the latter is a strain deficient in repair of DNA damage caused by UV radiation. Using this powerful UV source, the extension of the action spectrum of the repairproficient strain to the UV-A range (l-350 nm) became possible. As the spores have no metabolic activity in their dried form in the spore dosimeter and in the DLR-biofilm as well, the possibility of wavelength interactions can be excluded on a theoretical basis.

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4. Quantification of the biological effectiveness The biologically effective dose (BED) is defined according to the CIE recommendations [14] and adapted for the practical purposes of dosimetry as follows: BEDstSE(l)S(l)Dl

(1)

where E(l) is the spectral irradiance/fluence rate and S(l) is the spectral sensitivity of the biological effect in question. This quantity can be expressed either in absolute values (e.g., a cross-section-like quantity in m2 Jy1) or in relative ones. In the latter case S(l) equals unity at a specific wavelength and the biological dose is expressed, e.g., in (J my2)eff, in which case an indication of the reference wavelength is essential. This procedure was applied to the results obtained by the BIODOS partners for both the DLR-biofilm [31] and the B. subtilis spore dosimeter in solution [30]; the reference wavelength is 254 nm. In the case of the biofilm dosimeter, the optical density of the exposed and processed biofilm is compared with calibration areas on each biofilm that have been irradiated with a standard UV lamp in the laboratory. As a result, biologically effective doses were calculated as equivalent doses of UV-C (254 nm) resulting in the same biological effect [49]. When absolute S(l) values are used, appropriate dose units are required. For phage T7 inactivation it was shown that the BED can be given as Gln (n/n 0)Gs tSE(l) S(l) Dl

(2)

where n/n0 is the survival rate of phage particles after an exposure time of t; n and n0 can be determined, e.g., by a microbiological method. Eq. (2) demonstrates an important advantage of biological dosimeters: their ability to weight directly the fluence/irradiance, so that the measured effect is independent of the accuracy of the action-spectrum determination. However, the correct action spectrum is required for a correct comparison of the readings of the biological dosimeters with each other and with the spectroradiometric data. According to our earlier results [54], the term Nln (n/n0)N in Eq. (2) corresponds to the average amount of UV damage in one phage particle. Consequently the unit dose for phage T7 is defined by a survival rate of ey1 or, in other words, on average one lethal damage/phage particle. In the uracil dosimeter the decrease of the absorption as a function of the irradiation dose follows an exponential law up to a certain saturation value, thus the unity of the biologically effective dose (HU) is the decrease of the absorption to the e-th part of the original one. The B. subtilis spore dosimeter evaluates the inactivation of the spores (in dried form) by UV radiation, which is also exponential. The spore inactivation dose unit (ID) is defined as the reduction of the survival rate of the spores to ey1 [46].

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Fig. 2. Different measuring arrangements for phage T7 field dosimetry: (a) silica cell at the top of a tube with a sun-tracker, measuring in 4p geometry; (b) flat silica cell with a black background; (c) silica cell with rotating shadow bands covered according to the cosine law.

5. Validation of the biological dosimeters Biological dosimeters can be validated by comparing the biological dose values: c with biological doses obtained by measuring the solar radiation by spectroradiometry and weighting with the action spectrum of the biological effect applied for dosimetry, c with other biological doses, c with photoproducts (qualitatively and quantitatively) induced by UV radiation in the dosimetric system. In the following the results of comparisons with the phage T7 biological dosimeter will be presented according to the aforementioned three aspects. 5.1. Comparison with spectroradiometer measurements For this comparison the angular sensitivity of the phage T7 dosimeter in different arrangements has been determined. Generally, spectroradiometers (e.g., Optronic 754 or Brewer) are constructed to have an angular response that follows the cosine law. The phage T7 dosimeter has been used in three different measuring arrangements (a, b, c) presented in Fig. 2. The silica cell in Fig. 2(a) measures the biological effect in 4p geometry, while those in (b) and (c) (in a flat cell embedded in a black background and a spectrophotometer cell covered by rotating shadow bands, respectively) follow the cosine law. Fig. 3 demonstrates the difference between the cumulated doses measured in 4p geometry (curve a) and in the flat cell (curve b) at Nea Michaniona (Greece), on a fine sunny day, 21 July, 1997. The time evolution of the T7 doses goes forward parallel from early morning to sunset in the two different measuring conditions, deviating from each other by a factor of about two. The value of this factor corresponds to the expectations according to the difference in detectors with a 4p or cosine

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law angular sensitivity. From this result it follows that the reading of the phage T7 dosimeter and the weighted spectroradiometer data can be directly compared in the cosine arrangement. Fig. 4 presents a comparison of the time evolution of phage T7 dose in the flat cell (see Fig. 2(b)) and in the cell covered according to the cosine law (Fig. 2(c)). The two measuring procedures were similar to those presented in Fig. 3, i.e., the time evolution of the cumulated doses was determined on 20 July, 1997, in Greece (Nea Michaniona) on a fine sunny day. No significant difference has been found between the results obtained in the two different measuring conditions. Similar comparative measurements were performed on 16 and 17 July, 1997 as well, and a 20% ("10%) deviation in the results of the two types of arrangements has been found. For comparison the spectroradiometer data are also presented (Fig. 3), weighted by the spectral sensitivity of the phage T7 inactivation. The spectroradiometer data were provided by Dr D. Gillotay and D. Bolsee (Institut d’Aeronomie Spatiale de Belgique, Brussels). The two curves, obtained by

Fig. 3. Time evolution of the phage T7 dose from early morning to sunset on a nice sunny day at Nea Michaniona, 21 July, 1997, measured in 4p geometry (curve a) and in a flat silica cell with dark background (curve b). Bars indicate the measuring errors. Comparison with the spectroradiometric data, weighted by the spectral sensitivity of T7 phage inactivation (full line).

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Fig. 4. Time evolution of the phage T7 dose from early morning to sunset on a nice sunny day at Nea Michaniona, 20 July, 1997, measured with flat cells (triangles) and with shadowing by the cosine law (squares). No significant difference can be shown between the two measuring results; the weighted spectroradiometric data (full line) also fit well.

direct inactivation measurement in the flat cell filled with phage T7 and by weighted spectroradiometry, fit quite well. This fitting validates the phage T7 biological dosimeter. Similarly, a good agreement between weighted spectroradiometer data (Brewer) and B. subtilis spore inactivation dose has been reported by Munakata et al. [47]. Fig. 5 shows an example of similar results obtained with the DLR-biofilm as diurnal biologically weighted UV profile and as cumulative biologically weighted UV dose. 5.2. Comparison with other biological doses Comparison of two different biological doses (i.e., BED1 and BED2, respectively) can be performed by calculating a quotient f: fs

E(l)S1(l) E(l)S 2(l)

(3)

The measured biological doses can be transformed into each other by Eq. (3), provided that the measurements have been performed at identical spectral distribution of the irradiance/fluence rate. Applying biological dosimeters for outdoor measurements, this precondition can be realized in the same geographical location for the same weather condition at the same time of the year. The measuring results to be

Fig. 5. Diurnal profile of biologically effective UV and cumulative biologically effective UV doses measured with the DLR-biofilm at Nea Michaniona, Greece, 21 July, 1997.

compared can be related for a short period (a few days), but in this case they have to be obtained under clear-sky weather conditions. However, they can be related for a longer period as well (e.g., annual profile determination). In this case the average of the measured biologically effective dose is taken into account. Both cases are demonstrated in Table 1, where an average of a long-term measurement in three different stations of the Hungarian Meteorology Service (47–488 N) as well as three short-term measurements with phage T7 (in 4p geometry) and erythemally weighted broad-band radiometer (MED) results are compared. The latter are for about the same summer days but in various geographical locations: in Abisko (688 N, 198 E, Sweden), in the Hungarian National ¨ (488 N, 208 E) and in Nea Michaniona (418 N, Park, Bukk 238 E, Greece). In rows 2–4 the MED and phage T7 doses are compared in short-term measurements at various geographical latitudes (from 418 N to 688 N). Going from north to south, a decrease of about 70% can be found in the MED/HT7 quotient. The reason for this difference may be that the shorter-wavelength components of the solar radiation are present in higher proportion at the latitude 418 N than at 688 N. Moreover, the

Table 1 Comparison of biologically effective doses (BEDs) with phage T7 dose Geographical location

Measuring period

BED to be compared

BED/HT7

1. Hungary 2. Abisko ¨ National Park 3. Bukk 4. Nea Michaniona 5. Hungary ¨ National Park 6. Bukk 7. Nea Michaniona ¨ National Park 8. Bukk ¨ National Park 9. Bukk

average in summer, 1994 23 June, 1998 18–21 August, 1996 16–21 July, 1997 average in May–September 17–21 August, 1996 16–20 July, 1997 17–21 August, 1996 18–21 August,1996

MED MED MED MED HU HU HU ID biofilm unit

0.58"0.08 MED/HT7 0.79 MED/HT7 0.53 MED/HT7 0.46 MED/HT7 8.7=10y4"1.3=10y4 HU/HT7 1.25=10y3-0.13=10y3 HU/HT7 1.3=10y3"0.3=10y3 HU/HT7 4.0"0.8 ID/HT7 9"1.8 biofilm unit/HT7

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spectral sensitivity of phage T7 killing is higher for these shorter components, while erythema induction possesses a lower relative sensitivity for these wavelengths. Thus the change in the quotient from north to south reflects the change of the spectral composition of the solar radiation. Comparing the MED/HT7 data in the first and third rows of Table 1, the slight difference between the two values can be similarly interpreted. The increased value of T7 dose related to MED (row 3) is caused by the presence of a higher proportion of short-wavelength components in the solar light in mid-summer days, while the average of the solar spectral composition during the whole year contains a smaller amount of short wavelengths. In the rows 6 and 7 the uracil doses and phage T7 doses are compared in two various places, in Hungary and Greece. The difference in the HU/HT7 quotients is lower as in the case of MED/HT7 because of the smaller difference in the sensitivities of the two dosimeters for the shorter wavelengths. The increase in the short-wavelength components has been demonstrated recently [55] by comparing the measured results obtained with biological dosimeters of different spectral sensitivities. Rows 5 and 6 compare HU/HT7 data obtained practically at similar geographical locations (47–488 N) but on one hand only on a few summer (August) days (row 6), and on the other as an average of the whole summer (May–September). The difference is small but significant, which can also be interpreted by the differences in the spectral composition of the solar light in the different periods. 5.3. Comparison with UV photoproducts The biological dosimetry systems can be validated by detecting and quantifying the photoproducts induced by solar UV radiation. It was demonstrated that the major photoproducts due to UV-C and short-wavelength UV-B radiation are the bipyrimidine adducts, cyclobutane dimers (CPDs) and (6–4) adducts [(6–4) PDs]. With an increasing proportion of longer-wavelength components in the radiation, the proportion of bipyrimidine adducts decreases and the contribution of the other photoproducts increases [56,57]. Fig. 6 presents the formation kinetics of the amount of fully assessed CPDs and (6–4) PDs induced by solar light as a function of the HT7 dose. On average 0.4, 0.8 and 1.6 [CPDsq(6– 4)PDs]/phage belong to the dosimeter readings 2, 4 and 8 T7 dose units respectively. Taking into account the repair efficiency of host bacteria [54], these dose values correspond without host-cell repair to 4, 8 and 16 lesions/phage particle, respectively; thus the relation of the number of determined bipyrimidines to the total number of lethal lesions is about 10%. 6. Conclusions DNA-based biological dosimeters fulfil the criteria required for correct measurements. As it has been demon-

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Fig. 6. Increase of the number of bipyrimidine photoproducts {CPDsq(6– 4)PDs} in a phage particle induced by solar radiation as a function of the phage T7 dose, i.e., of the total number of damaged sites in the particle.

strated, these dosimeters can be used for evaluation of the global UV exposure of terrestrial ecosystems [27,28,31, 46,48,49,55]. In addition, they can be applied for assessment of the exposure of aquatic ecosystems in underwater measurements [29,32,34,44] as well. These types of biological dosimeters have been and can be used also for testing the efficiency of water disinfection [30]. Moreover, both DLRbiofilm and spore dosimeters have been successfully applied in personal dosimetry [53,58,60] for assessing the UV exposure of individuals. A new, promising application of DNA-based dosimeters might be the measurement of extraterrestrial solar radiation [49,59].

Acknowledgements This research was sponsored by grants from the European Community (BIODOS, ENV4-CT95-0044; UVB CELLS, ENV4-CT95-0174; OMFB, EU-96-C6-005; PHARE, HU9305-02/1084), the European Community Research and Development Grant No. EV5VCT910034, and by a grant from the Hungarian Ministry of Welfare (ETT, T-08 328/ 93) by the Hungarian Academy of Sciences supporting the MTA–SOTE Research Group for Biophysics.

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