Pergamon

Adv. Space Res. Vol. 18. No. 12. pp. (12)17-(12)26. 1996 CopyrIght 0 1996. PublIshed by Elsevier Science Ltd on behalf of COSPAR

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ONCOGENIC AND MUTAGENIC EFFECTS OF UV IN MAMMALIAN CELLS

T. C. Yang,* M. Mei,** K. A. George*** and L. M. Craiset

*NASA Johnson Space Center, Medical Sciences Division, Houston, Texas, U.S.A. +* South China Agricultural University. Experimental Center, Guangzhou, P. R. China *** Krug Life Sciences, Houston, Texas, U.S.A. t Lawrence Berkeley Laboratory, Life Sciences Division, Berkeley, California, U.S.A.

ABSTRACT

Ultraviolet light is present in the solar system and can cause major biological effects. The potential cytotoxic, mutagenic, and carcinogenic effects of UV have been studied at cellular and molecular level. Using cultured mouse embryonic fibroblasts (C3HlOT1/2), we investigated the induction of mutation and transformation by UV and/or X-rays. Studies were also done with normal human mammary epithelial cells for cell inactivation and mutation induction. Curvlinear dose-response curves were observed for mutation and oncogenic transformation. The interaction between UV and X-rays depends on the sequence of exposure. When UV was given following X-irradiation, there was an additive effect. When UV was given prior to X-irradiation, however, there was a synergistic effect for both cell inactivation and transformation. The basic lesion(s) important for somatic mutation and transformation remains to be determined. and the fundamental mechanism(s) of UV and ionizing radiation interaction remains to be elucidated. Copyright 0 1996. Published by Elsevier Science L!d on behalf of COSPAR

INTRODUCTION

Ultraviolet light, an important non-ionizing radiation in space, can be a major cause of human skin cancer /l/. Animal studies showed that skin carcinomas can be induced by repeated exposure of UV light /2/. For a better understanding of the fundamental mechanisms of mutagenic and oncogenic effects of UV radiation, various cultured mammalian cells have been used extensively as model systems. Experimental resuslts of earlier investigations indicated that UV acts as an initiator in the two-stage process of skin tumor development /3/. Further studies, however, demonstrated that a single exposure of 254 nm UV can cause somatic mutation and neoplastic transformation /4,5/. The mutation and transformation frequency, in general, increased with an increase of UV dose when optimal expression time was allowed. Potential lesion repair studies showed an enhancement of mutation and transformation frequency when irradiated cells were held at plateau phase for several hours /5/. Both mutation and transformation frequency, however, dropped below initial values when longer repair time was used.

In addition to UVC(200 to 280 nm), UV-B (290 to 400 nm) can induce neoplastic transformation in cultured mammalian cells /6/. The efficiency in inducing mutation and transformation was found to progressively decrease as the the short-wavelength cutoff was shifted to longer wavelengths f7/. UV radiation can also enhance oncogenic effect of ionizing radiation. Recently, a synergistic effect of UV and ionizing radiation was observed in the development of human skin cancer after scalp irradiation /8,9/. In this study, we present data for cell inactivation, mutation, and neoplastic transformation in mammalian cells in culture exposed to UV and/or ionizing radiation.

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Cell Sy&m_& Irradiaitons

The mouse embryonic fibroblasts (C3HlOT1/2; Clone 8) were grown in minimum essentiaI medium (MEM) containing 10% calf serum and antibiotics and were kept in a humidified incubator with an

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atmosphere of 95% air + 5% CO2 at 370c. Under these conditions the doubling time was about 18-20 hrs, and the confluent density of these cells was about 2~10~ cells per 35mm tissue culture dish. For radiation experiments, cells at passage 8 to 12 were used to minimim background frequency of transformation. Normal human mammary epithelial cells (H161E) from reduction mammoplasties were cultured in defined medium supplemented with growth factors and antibiotics at 370c/lO/. The doubling time was about 36 40 hrs, and cells formed monolayer when confluent.

A Philips 250 kVp X-ray unit was used for X-ray irradiation. Physical conditions of irradiation were 225 kVp, 15 mA, and a half-value layer of 1.1 mm of copper. The dose rate was calculated to be about 120 rad/min. based on measurements with a Victoreen Condenser R meter. Corrections were made for temperature, atmoshpheric pressure, and the dosimetric calibration factor. A constant factor of 0.95 was used to convert roentgen into rad. UV irradiation was done with a germcidal lamp which had a light intensity of 16 erg/cm2/sec at 254 nm wavelength. Before UV-irradiation, cells were washed once with PBS and then covered with a thin layer of PBS. Immediately after irradiation, cells were dissociated with trypsin-EDTA, diluted with growth medium, counted, and then seeded into dishes at proper density for determining survival and mutation or transformation.

Confluent monolayer cells were exposed to various doses of radiation. After irradiation the cells were immediately treated with a solution of trypsin-EDTA and dissociated into a single-cell suspension. For each radiation experiment, two sets of MO-mm tissue culture dishes were prepared, one set to determine the fractional survival relative to unhmdiamd controls and another set to check the number of transformed foci (Figure 1). Further details on transformation assay have been reported elsewhere /l l/.

For survival information, monolayer cells, immediately after irradiation, were seeded at a density that would give about 100 survivors in each dish for each dose. For mutation determination, about 2x104 cells

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Oncogenic end Mutagenic Effects of UV (12)19

were plated into each lOO-mm dish. After 1 day incubation, cells were treated with medium with 3 mM ouabain for 2-3 weeks to select mutants. The number of mutants per survivor for each dose was calculated from the suvival data and the number of mutants scored.

RESULTS

Figures 2 and 3 show the dose-response curves for survival and ouabain-resistant muation respectively. Clearly for mouse embryonic fibroblasts (C3HlOT1/2). there was a shoulder in the survival curve, and the frequency of mutation increased curlinearly with dose. At the highest dose used, however, the mutation frequency decreased.

The dose-response curve for survival for normal human mammary epithelial cells is given in Figure 4. There is a shoulder in the survival curve. Comparing with mouse embryonic tibroblasts, human mammary epithelial cells appeared to be significantly more radiosensitive. The dose for 10% survival, for example, is about 7 seconds and 10 seconds exposure for human and mouse cells correspondingly. When the mutation frequency was plotted as a function of survival fraction, the dose-response curve was curlinear, as shown in Figure 5. At equal survival fraction, such as lo%, the mutation frequency was about 3 and 22 mutants per lo5 survivors for human and mouse cells respectively. Thus UV appears to be more mutagenic for mouse fibroblasts than for human epithelial cells.

Figures 6.7, 8, and 9 show the dose-response curves for suvival and oncogenic transformation by UV, X- rays, or both radiations. When C3HlOT1/2 cells were exposured to 200 r-ad X-rays first and then UV irradiation, the survival decrased for a given UV dose. The survival curve, however, was parallel to that of UV only (Figure 6), indicating an additive effect. Similarly an additive effect was found for oncogenic cell transformation, as shown in Figure 7. The dose-response curves for transformation were curvlinear with a decrease of transformation frequency at the highest dose used.

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When mouse embryonic cells were irradiated with 60 ergs/mm2 UV first followed by X-rays, a synergistic effect was observed for both cytotoxicity and transformation. The survival curve for UV plus X-rays showed a steeper slope than that for X-rays only, as shown in Figure 8. A linear-linear plot of the dose- response curves for transformation are shown in Figure 9. Transformation frequency increased curvlinearly with X-ray doses. The slope of transformation curve was much greater for cells exposed to UV first and followed by X-rays than that for cells irradiated by X-rays only. Figure 10 shows a semi-log plot for the same set of cell transformation data. It is clear that UV and X-rays do not act independently. Transformaiton frequency was also plotted as a function of survival fraction, as shown in Figure 11. At equal survival UV puls X-rays can be much more effective than X-ray only in casuing oncogenic cell transformation.

DISCUSSION

Solar ultraviolet light can be separated into far (200-300 nm) and near (300-380 nm) UV or into UV A (315-380 nm), UV B (280-320 nm), and UV C (200-280 nm). Epidemiological and clinical studies showed an association between UV radiation in UB-B region of the spectrum of the sun and the induction of skin cancer in humans /12,13/. Such finding prompted extensive research studies with animal and cell systems. In vitro systems using cells in tissue culture offer great advantages for determining mechanisms at cellular and molecular level, and many investigators studied the wavelengths involved in UV radiation-induced cell inactivation/l4,15,16,17,18,/, mutagenesis /15.19/, and carcinogenesis /20/. We studied the cytotoxic, mutagenic and oncogenic effects of UV in mouse fibroblasts and human epithelial cells. Our experimental results showed that a single exposure of UV can cause cell killing, mutation, and oncogenic transformation in mouse fibroblasts and can induce cell inactivation and mutation in normal human mammary epithelial cells. For both type cells, the dose-response curve for suvival is a shouldered one, and for mutation a curvlinear one. These findings in general agree with results reported earlier by other investigators. An interesting observation of our studies is that normal human mammary epithelial cells appeared to be more sensitive to UV than mouse fibroblasts in terms of cell killing, but less sensitive to UV than mouse cells in terms of mutation at equal cytotoxicity. The mechansim(s )which causes these differences between these

Oncogenic end Mutagenic Effect Of ~ (132s

two different types of cells is unknown, One of the possible mechanisms is the repair. Further comparative studies on the repair kinetics and mechanisms in these two types of cells are warranted.

Our experimental results also showed that UV and X-rays can interact in inducing oncogenic cell transformation, i.e., they do not act independently. These two types of radiations can act additively when X-rays are given first followed by UV irradiation and can act synergistically when the exposure sequence is reversed. Similar results were found and reported by other investigators using different cell system /21/. The synergistic effect of UV is of great interest. An ehancement of mutation frequency was found when confluent C3HlOT1/2 cells were exposed to UV and then allowed to repair /5/, which indicated that UV may induce error-prone repair. When cells are exposed to UV first followed by X-rays, UV may induce error-prone repair which will convert more potentially oncogenic lesions produced by X-rays into irreparable oncogenic damages. These results, thus, provide further cellular basis for the observation that UV and ionizing radiaiton can enhance carcinogenesis /8,9/.

The fact that UV and X-rays can interact in inducing oncogenic transformation suggests that common lesions are produced by both types of radiations. Studies with monochromatic UV light showed a correlation between DNA absorbtion spectrum and oncogenic transformation /20/, and DNA was suggested as the primary target for neoplastic cell transformation. Other investigations indicated that the mechanism of cell killing may be different from that of mutation and transformation /7/. Analyses of dam from studies with ionizing radiaiton suggested that double strand DNA breaks are important lesions in oncogenic transformatiorw‘22/. Although DNA dimers are major products of UV irradiation, double strand DNA breaks have been detected in W irradiated mammalian cells. Since UV does not seem to induce double strand DNA breaks effectively, it is logical to expect that UV will not be as effective as ionizing radiaiton in causing oncogenic transformation. Interestingly, at equal cytotoxicity, W light has been found to be less effective than ionizing radiation in inducing oncogenic transformation /‘23/. The mechanism(s) of W oncogenesis, however, remains to be elucidated.

It is worth to notice that C3HlOT1/2 mouse fibroblasts transformed by ultraviolet radiation are antigenically similar to those from skin cancers produced in mice by repeated exposure to ultraviolet radiation. Both types of tumor cells grew preferentially in ultraviolet-irradiated syngeneic mice relative to untreated animals, and both were recognized by ultraviolet radiation-induced tumor-specific suppressor lymphocytes /24/. This particular finding strongly suggests that cultured cell systems can be highly useful for oncogenesis studies and can provide results relevant to tissue in organism.

ACKNOWLEDGEMENTS

We thank Dr. M. R. Stampfer for providing the normal human mammary epithehal cells and her valuable suggestions. This study was supported by NASA’s Space Radiation Health Program.

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2. J. F. Epstein and K. Fukuyama, Ultraviolet light carcinogenesis. In: The Biological Effects of Uhraviolet Radiation -- With Emphasis on the Skin (F. Urbach, Ed.), Pergamon, New York, 1969

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4. G. L. Chart and J. B. Little, Induction of oncogenic transformation in vitro by ultraviolet. Nature (London) 264:442444 (1976)

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16. G. J. Kantor, J. C. Sutherland, and R. B. Setlow, Action spectra for killing non-dividing normal human and xeroderma pigmentosum cells. Photochem. Photobiol. 31:459-464 (1980)

17. R. H. Rothman and R. B. Setlow, An action spectrum for cell killing and pyrimidine dimer formation in Chinese hamster V-79 cells. Photochem. Photobiol. 29:57-61 (1979)

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19. S. R. Eldridge and M. N. Gould, Specific locus mutagenesis of human mammary epithelial cells by ultraviolet radiation. Int J. Radiat. Biol. 59:807-814 (1991)

20. J. Doniger, E. D. Jacobson, K. Krell, and J. A. DiPaolo, Ultraviolet light action spectra for neoplastic transformation and lethality of Syrian hamster embryo cells correlate with spectrum for pyrimidine dimer formation in cellular DNA. Proc. Natl. Acad. Sci. U.S.A. 78:2378-2382 (1981)

21. J. A. DiPaolo and P. J. Donovan, In vitro morphologic transformation of Syrian hamster cells by UV irradiation is enhanced by X-irradiation and unaffected by chemical carcinogens. Int. J. Radiat. Biol. 30:41-54 (1976)

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24. M. S. Fisher, M. L. Kripke, and G. L. Chan, Antigenic similarity between cells transformed by ultraviolet radiation in vitro and in vivo. Science 223;593-594 (1984)