Dose Response, Wavelength Dependence, and Time Course of Ultraviolet Radiation-Induced Unscheduled DMA Synthesis in Mouse Skin in Vivo1

[CANCER RESEARCH 44, 2150-2154, May 1984]

Dose Response, Wavelength Dependence, and Time Course of Ultraviolet Radiation-induced Unscheduled DMA Synthesis in Mouse Skin in ViVo1

Ken-ichi Kodama, Takatoshi Ishikawa,2 and Shozo Takayama

Department of Experimental Pathology, Cancer Institute (Japanese Foundation for Cancer Research), Kami-lkebukuro, Toshima-ku, Tokyo 170, Japan

ABSTRACT not observe dose-dependent responses. We previously devel oped a quantitative autoradiographic technique using special A new method for autoradiographic measurement of unsched forceps for measuring UDS in mouse skin after treatment with uled DNA synthesis (DOS) in the skin in vivo after treatment with various chemical carcinogens (10). To obtain dose-dependent ultraviolet light (UV) was developed. The skin of the back of ICR induction of UDS, we devised a clamping procedure to keep a mice was shaved and exposed to short-wave UV (254 nm) or carcinogen and [meffty/-3H]dThd in isotonic solution in the cuta UVAB (sunlamp, 270 to 440 nm, predominant emission at 312 neous tissue for some time. The solution seems to penetrate nm) at various doses. Immediately after irradiation, an isotonic diffusely, and [mef/jy/-3H]dThd can be efficiently incorporated aqueous solution of [mef/7y/-3H]thymidine was injected s.c. into into DNA of both epithelial and mesenchymal cells. Thus far, we a portion of the skin clamped off with ring-shaped forceps. By have been unable to obtain dose-dependent induction of UDS this method, dose-dependent DOS was clearly demonstrated as without using this special technique, forceps clamping. We have silver grains on various types of cells in the skin in response to extended this method to study UV irradiation and found that it is 254 nm UV or sunlamp UV. However, the energy values at the useful for measurement of dose-dependent UV-induced UDS. two wavelengths required to induce the same UDS level differed Moreover, this method seems to be simple, sensitive, and repro by 1 order of magnitude. These findings suggested that this ducible. In this work, we studied the time course, transmissibility, system should be useful for quantitative analysis of UV-induced and energy levels required for UDS using short-wave UV and DNA repair in individual cells of the skin in vivo. UVAB. By this method, the wavelength difference in transmissibility was studied. Autoradiographic results clearly showed that sunlamp UV could reach deeper sites in the skin than did 254 nm MATERIALS AND METHODS UV. A time course study indicated that UDS was almost complete by 48 hr after 254 nm UV but still persisted at 48 hr after sunlamp Female ICR mice (8 weeks old) weighing 24 to 30 g were obtained from Charles River Japan (Atsugi-shi, Japan). Animals were anesthetized UV. These results, together with the differences in transmissibil with sodium pentobarbital, and the skin of their backs was shaved with ity, support higher tumorigenic activity of sunlamp UV than of electric clippers. Then, the skin was exposed to short-wave UV or UVAB 254 nm UV to experimental animals. at various doses as follows. For short-wave UV radiation, mice were exposed to UV from a 15-watt germicidal lamp (Toshiba GL 15 UV lamp; INTRODUCTION the predominant emission, 254 nm, as a single sharp peak; Toshiba Co., Tokyo, Japan; dose rate, 1.2 J/sq m/sec) at a distance of 50 cm. Dose Clinical observations and epidemiological studies have sug rate was measured by a Black-ray Model J-225 UV meter (Ultraviolet gested that sunlight, especially solar UV radiation, is the principal Products, Inc., San Gabriel, CA). For UVAB irradiation, mice were ex cause of carcinomas of the human skin (6). In recent years, the posed to 3 sunlamp fluorescent tubes (Toshiba FL 20 S.E. sunlamp; Toshiba Co.), which delivered an average dose rate of 2.2 J/sq m/sec at influence of DNA repair on cutaneous carcinogenesis has re a distance of 20 cm over the wavelength range of 280 to 340 nm (this ceived much attention. The molecular mechanisms of DNA repair range included approximately 90% of the total energy output of the have been extensively studied in bacterial and cultured mam lamp). Dose rate was measured by a UVR-365 UV radiometer (Tokyo malian cells (1,11,16,17, 20), but relatively little is known about Kogaku Kikai Co., Tokyo, Japan). For simplicity, we will refer to short DNA repair in vivo, because few methods are available for its wave UV and UVAB as 254 nm UV and sunlamp UV, respectively. study. UV-induced DNA repair has been studied by direct meas Immediately after exposure, the irradiated region of the skin was clamped urement of pyrimidine dimers in the skin (2, 19). This method is off with a tongue forceps (ring shaped, 20 mm internal diameter), avoiding stretching as much as possible, and isotonic Ringer's solution (0.5 ml) specific, but it has the limitation that it cannot give information containing [mef/7y/-3H]dThd (New England Nuclear, Boston, MA; specific on the location of DNA repair within the skin. Another approach has been autoradiographic measurement of DNA repair in the activity, 82 Ci/mmol, 100 nCi/ml) was injected s.c. into the clamped off region through a fine needle. Groups of 5 animals were treated with each skin in vivo. This method should be useful for measurement of irradiation dose. After this treatment, the mice were kept at 35Â°for 1 hr DNA repair in individual cells of tissues or organs in vivo. Epstein ef al. reported UV-induced UDS3 in mouse (5) and human (7) in an incubator, and then the forceps were removed. After 3 hr, the skin. They injected [mef/)y/-3H]dThd s.c.; however, they could animals were killed, and skin was cut out and fixed in 10% neutral formaldehyde solution. The fixed skin was cut into thin strips (5 x 20 mm), embedded in paraffin, and cut into 4- to S-^m thicknesses. The 'This work was supported by Grants-in-Aid for Cancer Research from the sections were treated with 5% trichloroacetic acid solution (4Â°)for 45 Ministry of Education, Science, and Culture and the Ministry of Health and Welfare min to remove the acid-soluble fraction and then were dip covered with of Japan. NR-M2 emulsion (Konishiroku Photo Co., Tokyo, Japan) and exposed for 2 To whom requests for reprints should be addressed. 5 weeks at 5Â°.After development, the sections were lightly stained with 3 The abbreviations used are: UDS, unscheduled DNA synthesis; UVAB, UV-A (400-315 nm) plus UV-B (315-280 nm); dThd, thymidine. hematoxylin and eosin. Since the range of variability in the grain numbers Received September 26, 1983; accepted February 13, 1984. was small throughout a sample, grains were counted consecutively (in

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one direction) on 200 basal cells started from a randomly selected point. Table 1 The background grains on control sections were counted in the same Difference in transmissibility of 254 nm UV and sunlamp UV way. The transmissibilities of 254 nm UV and sunlamp UV were compared Samples were obtained from groups of mice treated with 254 nm UV (30 J/sq as follows. Autoradiographs, showing similar numbers of grains on the m) and sunlamp UV (1300 J/sq m). surface epithelial cells from mice treated with 254 nm UV and sunlamp No. of grains/nucleus UV, were chosen on the basis of results on dose-response curves. Since Epidermal cells Hair follicle cells the animals exposed to 254 nm UV (30 J/sq m) and sunlamp UV (1300 2.4 Â±2.6a J/sq m) had approximately similar numbers of grains on the surface 254nmSunlamp1 + 0.1 13.3 Â±0.82.8 5.2 Â±0.5 epithelium, grains were counted on 200 hair follicle cells in each sample. a Mean Â±S.D. for 3 or 5 mice. For convenience, all the hair follicle cells were included in grain counts. To study the photoprotective role of hair, groups of mice with shaved skin and control mice with hair were irradiated with 254 nm UV (30 J/sq Table 2 m) and with sunlamp UV (400 J/sq m), respectively. Subsequent auto- Photoprotective role of hair against UV radiographic procedures were as described above. Mice were exposed to 254 nm UV (30 J/sq m) or sunlamp UV (400 J/sq m). For study of the time course of UDS, mice were exposed to 254 nm No. of grains/nucleus UV (30 J/sq m) and sunlamp UV (1300 J/sq m). One hr before sacrifice, 5 mice each were given injections of [meifty/-3H]dThd. Skins samples Shaved With hair 12.4 Â±2.6a 4.0 + 1.2(32)" were obtained 1,2,4, 8, 24, and 48 hr after treatment. 254 nm Sunlamp 9.0 Â±1.2 2.7 + 0.2 (30) Mean Â±S.D. for 5 mice. RESULTS 6 Numbers in parentheses, percentage of reduction. We observed silver grains indicative of UDS on the nuclei of epithelial cells in groups treated with 254 nm UV (Fig. 1) and with the dose above 30 J/sq m of 254 nm UV but continued to rise sunlamp UV (Fig. 2). The range of variability in the intensity of with sunlamp UV up to 3900 J/sq m. No further increase was grains on the nuclei of epithelial cells in each section was small, observed in response to 254 nm and sunlamp UV at 600 and and only background numbers of grains (1.5 to 1.8 grains/cell) 7800 J/sq m, respectively. We also observed silver grains on were seen on the nuclei of control animals (Fig. 3). The mean hair follicle cells and dermal fibroblastic cells. The number of grain counts and standard deviation are given in Chart 1. The grains on these cells decreased gradually with the increase in dose-response curves with 254 nm UV and sunlamp UV had the distance of the cells from the surface, but in general hair some differences in the energy values required to induce the follicle cells had more grains than did fibroblastic cells at the same UDS levels by 1 order of magnitude (approximately 4 x same anatomical level. Significant differences were seen in the 10). The number of silver grains on epithelial cells leveled off with number of grains on the nuclei of hair follicle cells induced by UV at the 2 wavelengths; more grains were observed after sunlamp UV irradiation than after 254 nm UV irradiation at similar UDS A : 254 nm UV levels in the surface epithelium (Table 1). 20 We also investigated the protective role of hair against UV. Mice with hair were exposed to 254 nm UV (30 J/sq m) and to sunlamp UV (400 J/sq m). Skin with hair showed much less UDS (about 30%) than did shaved skin at the same UV dose (Table 10- 2). By conversion of the grain values into energy values in dose- response curves (Chart 1) (4.0 grains/nucleus corresponds to 4 J/sq m in 254 nm UV and 2.7 grains/nucleus to 25 J/sq m in sunlamp UV), the protection efficiency of hair was calculated as 20 40 87 and 94% with 254 nm and sunlamp UV, respectively. J/m2 eo eoo Time course studies of UDS (Chart 2) indicate that repair synthesis was completed within 48 hr after 254 nm UV but was still observed at 48 hr after sunlamp UV, considered from their 8 : Sunlamp UV background level. After sunlamp UV, the repair synthesis level did not change for the first 2 hr and then decreased rapidly until 20. 10 hr and slowly until 48 hr; after 254 nm UV, repair synthesis decreased rapidly until 8 hr and slowly until 48 hr.

10- DISCUSSION We have reported a procedure for detecting UDS in mouse skin with chemical carcinogens in vivo (10). This procedure was extended to detect DNA repair synthesis induced by UV irradia 800 2400 4000 8000 tion. J/m It is well known that 254 nm UV effectively induces pyrimidine Chart 1. Dose response to UV of UDS in epithelial cells of mouse skin exposed dimer in DNA strands in vitro (3, 15, 17) and in vivo (2, 12) but to 254 nm UV (A) and sunlamp UV (B). Numbers of grains per nucleus are plotted against the energy dose. Numbers of grains per nucleus are averages for 5 mice. has low tumorigenicity (21). On the other hand, sunlamp UV is Bars, S.D. known to be more tumorigenic to experimental animals (8, 18),

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A : 254 on UV UV, although in general there were fewer grains on these cells than on surface epithelial cells. It is interesting that the grain levels induced on follicle cells by 254 nm UV and sunlamp UV were very different when the surface epidermal cells had similar numbers of grains. These differences can be explained by wave length differences in transmissibility. A study on transmission spectra of human epidermal specimens revealed that the whole epidermis can transmit about 5 to 8% at 250 nm, 10 to 15% at 300 nm, and 50 to 55% at 400 nm (13). Our autoradiographic method clearly demonstrated in vivo that sunlamp UV can reach into deeper sites than could 254 nm UV of hair follicle cells. Differences in the DNA repair ability of epithelial and fibroblastic cells treated with various chemical carcinogens were reported previously (10). We confirmed that hair follicle cells are more proficient in UV-induced DNA repair than were dermal fibroblastic cells. In this study, we examined whether hair protects DNA of the skin against UV. Our results showed that hair serves as an effective sunscreen against UV, as might be expected. Our results showed that hair blocked about 90% of the UV light energy. Defense mechanisms of the human skin against UV are of considerable interest, since there are relatively few other hairless mammals (4). In time course studies at 2 different UV wavelengths, a similar time course was observed, but some differences were seen at 48 hr after exposure. In both cases, DNA repair synthesis was induced immediately after irradiation and continued for 48 hr. However, after 48 hr, grains on nuclei had decreased to almost the background level in the case of 254 nm UV, whereas they were still about 3 times the back Chart 2. Time course of UDS in epithelialcells after exposure to 254 nm UV (30 J/sq m) (A)and sunlamp UV (1300 J/sq m) (B). Numbers of grains per nucleus are ground level in the case of sunlamp UV. These results, together plotted against the time (hr). , background level of UDS. Numbers of grains with those showing the higher transmissibility of sunlamp UV, per nucleus are averages for 4 or 5 mice. Bars, S.D. support higher tumorigenicity of sunlamp UV than of 254 nm UV although its main peak (312 nm UV) has been shown to have a to experimental animals. low ability to induce dimer formation in DNA strands (9,14). We The present study showed significant differences in DNA repair observed dose-dependent induction of UDS by UV radiation at synthesis in mouse skin after 254 nm and sunlamp UV. These differences may explain the wavelength dependency of photo- different wavelengths in our mouse skin system in vivo. The dose-response curves for 254 nm UV and sunlamp UV differed carcinogenesis. in energy level by 1 order of magnitude; much higher energy was required with sunlamp UV than with 254 nm UV to induce the same grain levels. This fact is in the main consistent with the REFERENCES previous work which gave estimates of dimer yields at 313 nm 1. Bowden, G. T., Giesselback, B., and Fusening,N. E. Postreplicationrepair of UV and 265 nm UV (9, 14). Marinaran and Cerutti (9) studied DNA in ultraviolet light induced normal and malignantly transformed mouse action spectra for pyrimidine dimer and monometric, ring-satu epidermalcell cultures. Cancer Res., 38: 2709-2718,1978. 2. Bowden, G. T., Trosko, J. E., Shaps, B. G., and Boutwell, R. K. Excision of rated products of thymine; the latter products represent minor pyrimidine dimers from epidermal DNA and nonsemiconservative epidermal lesions relative to pyrimidine dimer in far UV but major lesions in DNA synthesis following ultraviolet irradiationof mouse skin. Cancer Res., 35: near UV. They indicated a factor of 6.8 x 10~3 difference in the 3599-3607,1975. 3. Brash, D. E., and Haseltine, W. A. UV-induced mutation hotspots occur at ability of 313 nm UV and 265 nm UV to form dimers. Similarly, DNA damage hotspots. Nature (Lond.),298: 189-192,1982. Rothman and Setlow (14) reported that the amount of dimer 4. Daniels,F.. Jr., and Johnson, B. Normal, physiologic and pathologic effects of formed at 313 nm UV relative to 265 nm UV is 6.1 x 1(T4. solar radiation on the skin. In: T. B. Fitzpatrick, M. A. Pathak, L. C. Harter, M. However, our results indicate only a factor of 2.5 x 10~2 differ Seiji, and A. Kukita (eds.). Sunlight and Man, pp. 117-130. Tokyo: University of Tokyo Press, 1974. ence in the ability of sunlamp UV (peak at 312 nm) and 254 nm 5. Epstein,J. H. Ultravioletcarcinogenesis.In: A. C. Giese(ed.),Photophysiology, Vol. 5, pp. 235-273. New York: Academic Press, Inc., 1978. UV to induce UDS in surface epidermal cells. Although it has not 6. Epstein, J. H. Photocarcinogenesis:a review. Nati. Cancer Inst. Monogr., 50: been demonstrated that DNA lesions other than pyrimidine dimer 13-25,1982. are involved in UDS measurement, it seems quite possible that 7. Epstein, J. H., Fukuyama, K., Read, W. B., and Epstein, W. L. Defect in DNA synthesis in skin of patients with xeroderma pigmentosum demonstrated in our UDS assay is detecting the repair of these nondimeric vivo. Science (Wash. DC), Ã•68:1477-1478,1970. products. However, we cannot exclude another possibility that 8. Freeman, R. G. Data on the action spectrum for ultraviolet carcinogenesis.J. Nati. Cancer Inst., 55: 1119-1122, 1975. the relatively lower wavelength of UV fluence contained in the 9. Hariharan,P. V., and Cerutti, P. A. Formation of products of the 5,6-dihydrox- sunlamp might play a major role in UDS measurement on the ydihydrothyminetype by ultraviolet light in HeLacells. Biochemistry, 72: 2791- basis of dimer excision. Further studies with monochromatic UV 2795,1977. 10. Ishikawa, T., Kodama. K., Ide, F., and Takayama, S. Demonstrationof in vivo should clarify this point. DNA repair synthesis was also detected DNA repair synthesis in mouse skin exposed to various chemicalcarcinogens. in hair follicle cells in response to both 254 nm UV and sunlamp Cancer Res., 42: 5216-5221, 1982.

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11. Kantor, G. J., and Setlow, R. B. Rate and extent of DNA repair in nondividing 231, 1964. human diploid fibroblasts. Cancer Res., 41: 819-825,1981. 17. Smith, K. C. The cellular repair of radiation damage. In: T. B. Fitzpatrick, M. 12. Ley, R. D., Sedita, B. A., Grube, D. D., and Fry, R. J. M. Induction and A. Pathak, L. C. Harber, M. Seiji, and A. Kukita (eds.). Sunlight and Man, pp. persistence of pyrimidine dinners in the epidermal DNA of two strains of hairless 67-77. Tokyo: University of Tokyo Press, 1974. mice. Cancer Res., 37: 3243-3248,1977. 18. Strickland, P. T., Bums, F. J., and Albert, R. E. Induction of skin tumors in the 13. Pathak, M. A., and Fitzpatrick, T. B. The rote of natural photoprotective agents rat by single exposure to ultraviolet radiation. Photochem. Photobiol., 30: 683- in human skin. In: T. B. Fitzpatrick, M. A. Pathak, L. C. Harber, M. Seiji, and 688, 1978. A. Kukita (eds.), Sunlight and Man, pp. 725-750. Tokyo: University of Tokyo 19. Sutherland, B. M., Harber, L. C., and Kochevar, I. E. Pyrimidine dimer formation Press, 1979. and repair in human skin. Cancer Res., 40: 3181-3185,1980. 14. Rothman, R. H., and Setlow, R. B. An action spectrum for cell killing and 20. Taichman, L. B., and Setlow, R. B. Repair of ultraviolet light damage to the pyrimidine dimer formation in Chinese hamster V-79 cells. Photochem. Pho- DNA of cultured human epidemial keratinocytes and fibroblasts. J. Invest. tobiol., 29: 57-61, 1979. Dermatol., 73:217-219,1979. 15. Setlow, R. B. Cyclobutane-type pyrimidine dimer in polypeptktes. Science 21. Urbach, F., Epstein, J. H., and Forbes, P. D. Ultraviolet carcinogenesis: (Wash. DC), Ã•53:379-386,1966. experimental, global and genetic aspects. In: T. B. Fitzpatrick, M. A. Pathak, 16. Setlow, R. B., and Carrier, W. L. The disappearance of thymine dimers from L. C. Harber, M. Seiji, and A. Kukita (eds.), Sunlight and Man, pp. 259-283. DNA: an error-correcting mechanism. Proc. Nati. Acad. Sci. USA, 57: 226- Tokyo: University of Tokyo Press, 1974.

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Fig. 1. Autoradiograph showing silver grains on the nuclei of epithelial cells, hair follicle cells, and dermal fibroblastic cells indicative of UDS after exposure to 254 nm UV (30 J/sq m). All mouse skins were treated with [mefhy/-3H]dThd (100 Â»iCi/ml).Cells with heavily labeled nuclei were in S phase during treatment. Lightly stained with H&E, x 1200. Fig. 2. Autoradiograph showing silver grains on the nuclei of epithelial cells, hair follicle cells, and dermal fibroblastic cells indicative of UDS after exposure to sunlamp UV (1300 J/sq m). All mouse skins were treated with [mef/7y/-3H]dThd (100 (jCi/ml). Cells with heavily labeled nuclei were in S phase during treatment. Lightly stained with H&E, x 1200. Fig. 3. Control autoradiograph showing few background grains. Mouse skin was treated with [meffiy/-3H]dThd (100 Â»iCi/ml).Cells with heavily labeled nuclei were in S phase during treatment. Lightly stained with H & E, x 1200.

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Ken-ichi Kodama, Takatoshi Ishikawa and Shozo Takayama

Cancer Res 1984;44:2150-2154.

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