[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 sun- lamp 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 35°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 (4°)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 5°.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 2150 CANCER RESEARCH VOL. 44 Downloaded from cancerres.aacrjournals.org on September 24, 2021. © 1984 American Association for Cancer Research. UV-induced DNA Repair Synthesis in Mouse Skin 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 ±2.6a J/sq m) had approximately similar numbers of grains on the surface 254nmSunlamp1 + 0.1 13.3 ±0.82.8 5.2 ±0.5 epithelium, grains were counted on 200 hair follicle cells in each sample. a Mean ±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 ±2.6a 4.0 + 1.2(32)" were obtained 1,2,4, 8, 24, and 48 hr after treatment. 254 nm Sunlamp 9.0 ±1.2 2.7 + 0.2 (30) Mean ±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.