407

Mutation Research, 62 ( 1 9 7 9 ) 4 0 7 - - 4 1 5 © Elsevier/North-Holland Biomedical Press

THE WAVELENGTH DEPENDENCE OF 8-METHOXYPSORALEN PHOTOSENSITIZATION OF RADIATION-ENHANCED REACTIVATION IN A MAMMALIAN CELL-VIRUS SYSTEM

LESLIE C. JAMES * and THOMAS P. COOHILL

Departments of Biology and Physics, Western Ken tucky University, Bowling Green, K Y 42101 (U.S.A.) (Received 18 February 1979) (Revision received 3 May 1979) (Accepted 11 May 1979)

Summary The combined effect of 8-methoxypsoralen (8-MOP) and ultraviolet (UV) radiation on the ability of an irradiated mammalian cell (CV-1) to reactivate UV-irradiated mammalian virus {Herpes simplex) was tested. Prior treatment of cells with 8-MOP was found to increase Radiation-Enhanced Reactivation (RER) at one wavelength (297 nm) in the far ultraviolet b u t not at others (240--289 nm). This same treatment induced R E R in the near UV {302--370 nm) and the visible region {380--400 nm). An action spectrum for the photosensitized induction of this cellular parameter was obtained. This action spectrum is consistent with the absorption spectrum for 8-MOP and the theory that damage to DNA is, at least in part, responsible for Radiation-Enhanced Reactivation.

The photosensitizing drug, 8-methoxypsoralen (8-MOP) is a member of a group of light-sensitive furocoumarins. Some of these c o m p o u n d s {including 8-MOP) are known to have strong skin-photosensitizing properties when used in conjunction with near-ultraviolet radiation (300--380 nm) [2,4,17]. For this reason, 8-MOP has been used in the treatment of various skin diseases [10,16, 19]. The drug is either administered topically or orally depending upon the method o f treatment [4,10]. Oral administration of 8-MOP m a y expose cells not targeted for therapy to photosensitization. Cells photosensitized b y 8-MOP plus light treatment may be subject to lethal and mutagenic effects [2,13]. This property of the drug is associated with its ability to form m o n o a d d u c t s and * School of Life Sciences, University of Nebraska-Lincoln, Lincoln, NA 68588 (U.S.A.).

408 biadducts (cross-links) with pyrimidines in DNA or RNA [17,18]. Although both DNA and RNA are affected, the predominant action is on DNA [8,17]. It is of interest, therefore, to determine the effects of 8-MOP on mammalian cells and viruses, especially viral-cell interactions known to occur in humans. Over half of all humans harbor Herpes simplex virus (HSV), usually in the latent state [12,15]. UV light alone may activate these viruses [1]. Any photosensitization of cells and/or virus in such a system could increase the likelihood of viral induction in mammalian cells. This could also increase the probability of viral or cellular mutation [21]. Therefore, we tested the ability of 8-MOPtreated cells to reactivate UV-irradiated HSV when the cells were exposed to different wavelengths of UV radiation. This cellular function is known as Radiation-Enhanced Reactivation (RER) [14]. Cells were either irradiated in the presence of 8-MOP or in the absence of 8-MOP, and RER was quantitated by plaque formation. Here we present data in the wavelength region 240--400 nm. Materials and methods The materials and methods used in this study have been previously reported [5--7] but will be described here briefly. Cell line

A permanent line of African green m o n k e y kidney cells (CV-1) was obtained from L.E. Bockstahler of the Bureau of Radiological Health, Rockville, MD. These cells were grown attached to plastic flasks or petri dishes. The growth medium used was a supplemented version of Dulbecco's Modified Eagle's Medium (DMEM) [5]. Virus assay -- p l a q u e - f o r m i n g ability (pfa)

A macroplaque strain of Herpes simplex virus type 1 was given to us by C.D. Lytle of the Bureau of Radiological Health. Cells were inoculated with virus and incubated at 37°C for 2 days in the case of unirradiated (control) virus, and for 3 days in the case of irradiated virus. Cell monolayers were stained with crystal violet and plaques were counted. E x p o s u r e s to cells and virus

Monochromatic exposures were obtained with the use of a 2.5-kW high-pressure mercury--xenon lamp (929 B Hanovia Lamp, Newark, NJ). Radiation from this source was passed through two grating monochromators (BM 250, Schoeffel Inst., Westwood, NJ) in order to achieve sufficient spectral purity. A diagram of this radiation source has been previously published [5]. Exposure rates were measured at least twice during any experiment by placing a calibrated UV-sensitive photodiode (Cal-UV, United Detector Technology (UDT), Santa Monica, CA) in the sample position. Cells or viruses were irradiated in horizontally oriented open petri dishes. Cell monolayers were irradiated at different wavelengths and exposure times varied from 0.3 to 90 min. Exposure rates varied from 0.5 to 6.2 W/m 2. Virus was irradiated at 265 nm with an exposure of 400 J/m 2. This exposure lowered the viral plaque-forming ability (PFA) to approximately 1--2% of control (unirradiated) virus.

409 Control (no 8-MOP) cell monolayers and virus suspensions were irradiated in Dulbecco's phosphate-buffered saline (PBS). The virus suspension was then diluted into maintenance medium and inoculated onto the cells. Cell monolayers were incubated for 1.5 h at 37°C to allow for viral adsorption and penetration. Psoralen treatment 5 mg of 8-MOP was dissolved in 0.5 ml of acetone. This solution was then added to 100 ml of magnesium and calcium-free PBS and boiled to vaporize the acetone [3]. The solution was cooled slowly, filtered, and diluted into 100 ml of Dulbecco's PBS. The final 8-MOP concentration was 25 pg/ml. Prior to radiation exposure, growth medium (DMEM) was removed from cell monolayers in 60-mm dishes. The cells were then rinsed twice in Dulbecco's PBS, 2 ml of the 8-MOP--PBS solution were added, and the cells irradiated 10 min later. The drug was removed from the cells immediately following irradiation and replaced with an appropriate virus inoculum. All handling of cells, viruses, and 8-MOP aside from the irradiation procedures, was in dimmed lighting. Results Effect o f 8-MOP on R E R in the far UV (240--297 nm) Figs. 1 and 2 show the effect of 8-MOP addition on RER at 4 wavelengths tested in the far ultraviolet. Plotted on the abscissa is the UV exposure to the cells at the indicated wavelengths. The ordinate shows the fraction of irradiated cells which retain the ability to support irradiated HSV-1 viral replication. This ability is measured by pfa. Each experiment, at every wavelength tested, was repeated at least two times. Therefore, each point represents an average of at least two values at that exposure. Error bars are a measure of standard error and are included only where the error exceeded 10% of the average value. Fig. 1 contains data from two wavelengths where 8-MOP has no effect on RER, and one wavelength where 8-MOP has a slight effect on RER. At 240 and 254 nm no increase in RER is seen. A slight enhancement of RER is seen at 270 nm. At 297 nm (Fig. 2) an effect on RER due to 8-MOP is seen, but again, this effect is small. These results imply that additional damage to the cells due to photosensitization by 8-MOP is either not occurring, or is occurring only slightly, at these wavelengths. Effect o f 8-MOP on R E R in the near UV (NUV) (302--370 nm) For the longer wavelengths tested, 8-MOP sensitization of RER is more obvious. With 302-nm radiation (Fig. 2) the effect is pronounced. Although the appearance of RER occurs at a lower exposure in the presence of 8-MOP the effect decays earlier as exposure is increased. This is what one would expect if RER depends upon cellular damage that can be sensitized by 8-MOP [14]. Figs. 3--5 illustrate wavelengths where damage accumulates in 8-MOP-treated cells at exposures where no cellular damage is apparent in control cells. Notice that wavelengths longer than 302 nm in the NUV show no effect of UV alone on

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Effect of 8-MOP on RER in the visible (380--400 nm) Although 8-MOP photosensitization of RER was present at the two visible wavelengths tested, 380 and 400 nm (Fig. 5), no peak response was obtained due to our exposure limits [7]. Again, cells treated with light alone did not reactivate virus at these wavelengths. Action spectrum for RER Table 1 is a compilation of data obtained at the 11 wavelengths presented in Figs. 1--5 and at 8 additional wavelengths. The action spectrum for the effect o f 8-MOP on RER shown in Fig. 6 was obtained from the values reported in

412 TABLE 1 RADIATION-ENHANCED REACTIVATION IN C E L L S I R R A D I A T E D IN T H E P R E S E N C E A N D ABS E N C E O F 8-MOP AS M E A S U R E D BY T H E E X P O S U R E R E Q U I R E D TO GIVE A PEAK (Ep) RESPONSE Wavelength (nm)

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413 the 3rd and 4th columns o f Table 1. On the ordinate is plotted the reciprocal of exposure in j - i m 2 to produce a peak affect. Wavelength is plotted on the abscissa. Discussion The results presented in Figs. 1--5, and those listed in Table 1 show that 8-MOP addition significantly enhanced the appearance of RER at wavelengths in the range 297--400 nm. No significant photosensitization was observed for wavelengths shorter than 297 nm. The degree of photosensitization varied in the sensitized region with the peak response being above 311 nm (see Figs. 3--5 and Table 1). An effect of UV alone on RER could be seen at only two wavelengths where 8-MOP enhanced RER -- 297 and 302 nm. A comparison of the effectiveness of 8-MOP at 297 and 302 nm based on exposure-to-peak indicates that 297 nm is more effective in inducing RER than is 302 nm. However, UV alone induced a peak response at 297 nm with exposures of less than one-half of that required for the same response at 302 nm. If this difference is important, then 8-MOP photosensitization could be regarded as being more effective at 302 nm than at 297 nm. A comparison of the effects of UV alone and of 8-MOP plus UV on RER is not possible with the longer wavelengths. For wavelengths greater than 302 nm, no effect of UV alone was measurable for exposures of up to 3000 J / m 2 (see Table 1). The use of exposures greater than 3000 J / m : was prohibited due to the limited o u t p u t of our m o n o c h r o m a t o r [7]. For example, an irradiation time of 90 min per monolayer was required for an exposure of 3000 J / m : at 340 nm. The appearance of RER at these wavelengths could be assumed to be " e a r l y " due to 8-MOP + UV treatment, but n o t necessarily absent for UV treatment alone. This limitation prevents us from subtracting the capacity effect from the RER effect at all wavelengths greater than 302 nm. Hence, a true measure of photosensitization was not possible. However, relative effectiveness of the wavelengths tested could be observed. Others have reported this problem in similar studies with 8-MOP in the NUV [2,7,11]. Although the action spectrum reported in Fig. 6 differed somewhat from other published action spectra in relative wavelength effectiveness, it did define a similar photosensitive region for 8-MOP effect. For example, Freeman and Troll [11] reported an action spectrum for eye injury in guinea pigs which showed a photosensitive region (300--380 nm) similar to that shown in our action spectrum. They did n o t continue their studies at wavelengths below 300 or above 380 nm. Also Pathak [18] published an action spectrum for the erythemal response of guinea-pig skin which defined a similar photosensitive region from 300 to 400 nm. No effect of 8-MOP treatment on wavelengths less than 300 nm was observed. The present action spectrum reported in Fig. 6 is similar to the one previously reported by us for 8-MOP sensitization of mammalian cellular capacity for unirradiated HSV-1 [7]. The same photosensitive region is defined with the single exception that no effect of 297 nm was observed in the former study. Minor discrepancies between the two action spectra over the region 302--380 nm are not amenable to interpretation by us. Both spectra decrease in value for

414 wavelengths longer than 340 nm. For the wavelength regions below 287 nm, both spectra are consistent with those we have reported for UV effects on cellular capacity [5] and R E R [6] for cells not treated with any photosensitizing drug. Finally, the action spectrum presented here is similar to the published action spectrum for 8-MOP binding to native DNA [17]. Both action spectra differ from the 8-MOP absorption spectra [2] mainly in the region beyond 360 nm. While the 8-MOP absorption spectrum shows little absorption b e y o n d 360 nm, these action spectra continue to indicate an effect up to 400 nm. These two action spectra define a photosensitive region almost identical to that region defined by the 8-MOP absorption spectrum from 270 to 360 nm. Both action spectra show a significant response at 334 nm which is the peak for the 8-MOP--RER action spectrum. The peak value for the 8-MOP--DNA action spectrum is at 312 nm. The similarities between the action spectrum presented here, the action spectrum for 8-MOP--DNA, and the absorption spectrum for 8-MOP indicate that damage to cellular DNA may be involved in the enhancement of R E R by 8-MOP + UV. The wavelengths used in this study do not normally induce R E R at these exposures. However, with the addition of 8-MOP to the system, enough damage to the cells is apparently caused for the induction of RER. Igali et al. [13], working with E. coli, reported a mutagenic effect of these wavelengths when cells are irradiated in the presence of 8-MOP. Igali [13] and Baden [3] proposed a mechanism for mutagenesis by 8-MOP plus light that was similar to that for far UV damage alone. Recently, Das Gupta and Summers [9] have reported that R E R is linked to mutagenesis in an HSV--mammalian cell system. Mutagenesis, prophage induction and R E R are all observed in the far UV [20]. They may be interrelated in that all of these cellular occurrences could be the result of error-prone repair functions [20,21]. Error-prone repair is induced by agents which damage DNA or halt its replication [9]. Presumably, this means of repair could also be induced in response to the photoproducts produced by 8-MOP plus UV-radiation treatment. If 8-MOP plus NUV or visible light is an effective inducer of error-prone repair (as evidenced by the appearance of R E R at these wavelengths) then this treatment might also be mutagenic to mammalian cells. We do not exclude the possibility that other models exist that can explain the responses reported here. These are not limited to an error-prone repair system. They include, b u t are not limited to, induced error-free repair, or the rescue of damaged virus by recombinational events. The limited data available for mammalian systems prevents us from offering a firmer analysis of our data.

Acknowledgements We would like to thank B o b b y Cobb and Richard Detsch for their help in performing these experiments. We would also like to thank L.E. Bockstahler of the Bureau of Radiological Health (Rockville, MD), for many helpful discussions. All the reported work was supported by FDA contract No. 223-74-6067.

415

References 1 A c t o n , J., L. K u c e r a , P. M y r v i k a n d R. Weiser, in: F u n d a m e n t a l s o f M e d i c a l V i r o l o g y , L e a a n d Febiger, Philadelphia, 1976. 2 A s h w o o d - S m i t h , M . J . , a n d E. G r a n t , E f f e c t o f t e m p e r a t u r e o n d o s e - d e p e n d e n t c h a n g e s in s e d i m e n t a t i o n c h a r a c t e r i s t i c s o f b a c t e r i a l D N A p r o d u c e d , in vivo, b y n e a r u l t r a v i o l e t i r r a d i a t i o n a n d 8 - m e t h o x y P s o r a l e n , C r y o b i o l . , 11 ( 1 9 7 4 ) 1 6 0 - - 1 6 9 . 3 B a d e n , H . P . , J.M. P a r r i n g t o n , J . D . A . D e l h a r t y a n d M.A. P a t h a k , D N A s y n t h e s i s in n o r m a l a n d x e r o derma pigmentosum fibroblasts following treatment with 8-methoxypsoralen and long-wave ultraviolet light, B i o c h i m . B i o p h y s . A c t a , 2 6 2 ( 1 9 7 2 ) 2 4 7 - - 2 5 5 . 4 B u c k , H.W., I.A. M a g n u s a n d A . D . 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M o u l e , I n d u c e d r e a c t i v a t i o n o f U V - d a m a g e d p h a g e k i n E. coli K 1 2 h o s t cells t r e a t e d w i t h a f l a t o x i n B 1 m e t a b o l i t e s , M u t a t i o n R e s . , 4 2 ( 1 9 7 7 ) 2 0 5 - - 2 1 5 . 21 W i t k i n , E.M., U l t r a v i o l e t m u t a g e n e s i s a n d i n d u c i b l e D N A r e p a i r in E s c h e r i c h i a coli, B a c t e r i o l . R e v . , 4 0 (1976) 869--907.