Photocheiiiisrry und
Photobroloyy, 1911, Vol. 26, pp. 387-391.
Pergamon Press. Printed in Great Britain
THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL-VIRUS SYSTEM THOMAS P.COOHILL, SHARON P. MOOREand STEPHANIE DRAKE Departments of Biology and Physics, Western Kentucky University, Bowling Green, ICY 42101, U.S.A. (Received 1 April 1977; accepted 3 June 1977)
Abstract-The ability of UV-irradiated African green monkey kidney cells (CV-1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235-302nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.
INTRODUCTION
units penicillin, 0.08 g streptomycin (International Scientific Industries, Cary, IL), 100,000 units mycostatin (E. R. Capacity is defined as the ability of a cell to support Squibb, Princeton, NJ) and buffered to pH 7.5 with 0.3 g the growth of a particular virus (Benzer and Jacob, sodium bicarbonate. Cells were incubated in closed Basks 1953). This cellular function is sensitive to ultraviolet or in petri dishes in gassed (approximately 5% COz) at 37°C. (UV) radiation, irradiation of the cell usually decreas- chambers Virus assay: Plaque forming ability kfaj. A macro-plaque ing its capacity to support viral growth (Anderson, strain of HSV-1 (Nerpesuirus hominus) was given to us by 1948; Bockstahler and Lytle, 1970; Coppey, 1972; C. D. Lytle of the Bureau of Radiological Health. These Powell, 1959). In some cases an initial increase in viruses were inoculated into confluent monolayers of capacity is seen at low fluences of UV radiation, but CV-1 cells, harvested, and kept in vials at -40°C until use. For virus assay, freshly confluent monolayers of cells this is followed by a rapid decrease as the Ruence were inoculated with an appropriate viral titre in a mainis raised (Bockstahler et al., 1976; L. E. Bockstahler, tenance medium consisting of 1X MEM supplemented with 0.26 g/t L-glutamine and 2% fetal calf serum and kept personal communication). In a bacterial cell-virus system, Day and Muel at 37°C for 90min. The inoculum was then removed and (1974) argued against nucleic acid as the target mol- 4 mY of growth medium containing 0.2572 immune serum globulin (Armour Pharmaceutical, Kankakee, 1L) was ecule for UV radiation effects on capacity. They cite added to prevent non-cellular viral transfer. The cells were evidence from their study of this effect at several incubated for 2 days and then stained by adding to the wavelengths in the UV region and from the reports growth medium one drop of a 3% aqueous solution of of others involving genetic and photobiological data crystal violet in 20% ETOH and 0.8% ammonium oxalate (Carolina Biological Co., Burlington, NC). The resulting that support the exclusion of DNA as the critical mol- solution was poured off after 10niin and plaques were ecule for bacterial capacity (Harm, 1965; Fluke, 1966). counted. Virus dilutions were adjusted to give approxiHowever, Lytle et al. (1976), Lytle and Benane (1975), mately 80 plaques on unirradiated monolayers. Monochromatic exposures. To obtain sufficient exposure and Coppey and Nocentini (1976) have recently rates, a 2.5 kW high pressure mercury-xenon lamp was reported data that support DNA as the target mol- used (929B Hanovia Lamp, Newark, N.J.). This source was ecule for mammalian cell capacity of herpes virus rep- air cooled to remove ozone, and infrared radiation was lication. Accordingly, we conducted experiments in- partially removed from the beam by means of a circulating volving the UV wavelength dependence of mam- water filter. Spectral separation was achieved by means malian cell (CV-1) capacity to host the virus herpes of two 25 cm grating monochromators coupled in tandem (GM 250, Schoeffel Inst., Westwood, NJ) as shown in Fig. simplex type 1 (HSV 1). Here we present results in 1. Half band widths were measured at several wavelengths the UV region (235-365 nm). and varied from 1.5 to 6.0nm depending on the slit width ( 1 - 4 mm). The spectral purity of the monochromator output was further checked by placing a series of cut-off filters into the exit beam and checking for the presence of conMATERIALS AND METHODS taminating wavelengths. For our studies only wavelengths Cell cultures. A permanent line of African green monkey corresponding to individual lines of the mercury spectrum kidney cells (CV-1) was obtained from L. E. Bockstahler were used, and thebe were found to be spectrally pure of the Bureau of Radiological Health, Rockville, MD. within the limits of our testing. Exposure rates were These cells were maintained in a growth medium consist- measured at different times during an experiment by placing of 1X Minimum Essential Medium (Eagle’s) with the ing a calibrated UV-sensitive photodiode [Cal-UV, United following components added per liter: 10% fetal calf serum Detector Technology (UDT), Inc., Santa Monica, CAI in Reheis Chemical Co., Kankakee, IL), 0.26 g ~-glutamine, the sample position and measuring the photocurrent pro16 m/ of 50X amino acids, 8 md of lOOX vitamins, 80,000 duced by the radiation beam with a Keithley 610B elec387 P A P 2614-0
388
THOMASP. COOHILL, SHARON P. MOOREand STEPHANIE DRAKE
.
RADIATION SOURCE
.
DOUBLE MONOCHROMATOR
OU PUQTODIODE
Figure 1. Source and dispersion system for the production of monochromatic radiation beams (see Materials and Methods). trometer (Keithley Inst., Cleveland, OH). The conversion factor for converting photocurrent output to energy fluence rate at each wavelength was obtained by combining a relative spectral response curve with the absolute calibrations at 250, 320 and 360nm supplied by UDT. These values were reliable within an accuracy of 2% (UDT, personal communication). Cells were irradiated in horizontally oriented open petri dishes. The UV beam was deflected downward onto the dishes by means of a 45-degree frontsurfaced mirror. Dishes were rotated at 0.5 rps to compensate for possible inhomogeneities in the radiation beam. Exposure times varied from 1 to 120s. Exposure rates .varied from 0.05 to 21 W/m2 (Table 1). Reciprocity of time and intensity was tested over a factor of at least three at each wavelength and no dependence of capacity on intensity was observed. Before exposure to radiation the growth medium was removed from confluent monolayers in plastic petri dishes. These monolayers were then rinsed twice in Dulbecco's phosphate buffered saline (PBS) to remove UV-absorbing components in the medium. The cell layers were kept moist during irradiation by the presence of 2m/ PBS. Immediately after irradiation the PBS was removed, and the cells were inoculated with unirradiated virus in maintenance medium.
RESULTS
Figures 2-7 show the effect on cell capacity of U V radiation of different wavelengths (235-365 nm). On the ordinate is plotted the ability of these irradiated cells to support viral growth (capacity). The abscissa shows the fluence of radiation of the stated wavelength. Each datum point in these figures represents the average value for points of the same fluence obtained from a minimum of three separate experiments a t each wavelength. During any experiment 10 monolayers were assayed at each fluence, and each monolayer contained approximately 20-100 plaques, therefore, each average value corresponds to 600-3000 plaques. Error bars, corresponding to a measure of the standard error, were added only if the error exceeded 10% of the average value. It is evident from Figs. 2-6 that the shapes of all the exposure response curves are similar and exhibit an initial flat shoulder the extent of which depends on the wavelength being tested. After the initial
Table 1. Fluence rate ranges for monochromatic exposures and cell capacity responses to the F,, level Wavelength (nm)
Fluence rate range* (w m-2)
Capacity (J- m2)
Quantum corrected (J-' m2)
235 238 240 245 248 254 260 265 210 275 280 289 297 302
0.50-2.00 0.60-3.50 1.10-3.90 1.30-5.10 1.25-5.40 0.45-1.45 0.05-0.20 0.30-1.10 0.60-2.00 0.75-2.60 0.90-3.70 1.05-3.50 6.00-19.00 5.W21.00
0.0085 0.0091 0.0103 0.0149 0.0176 0.0236 0.364 0.330 0.313 0.272 0.0216 0.0116 0.0017 0.0005
0.0109 0.0115 0.0130 0.0184 0.02 14 0.0281 0.423 0.376 0.350 0.0299 0.0233 0.0121 0.0017 0.0005
l/tilO
W l O
* Upper fluence rate limited to monochromator output at that wavelength.
Wavelength dependence of cell capacity for virus growth
389
1.0
0.5
0.1
LL 0
I
0
I
I
1
I
20
40
60
80
I
I
100
120
FLUENCE (M) Figure 2. Figures 2-7. The effect of radiation of different wavelengths on the capacity of CV-1 cells to support the growth of HSV 1.
shoulder there is an approximate exponential decrease in capacity to at least that fluence which lowers the cell's capacity to 10% (Flo)of the control.(unirra: diated) value. To minimize slight differences (less than 1004) in daily cellular response to UV radiation each experiment was repeated at least three times. In addition, since age-related changes in cellular response were observed, data were limited to that obtained with cells passaged no more than 18 times. As a control in each experiment a group of monolayers .was exposed to 280nm light of an amount sufficient to give an Flo value. This standard response to 280nm
0
20
40
60
FLUENCE Figure 3.
80
(JA)
100
I
0
I
10
I
20
I
30
FLUENCE Figure 4.
I
40
I
50
(J,)
light exposure confirmed that the cells were respwding in a similar manner during each experiment. Experiments conducted at wavelengths of 313, 334 and 365 nm were not at exposures sufficient to obtain an F,, (Fig. 7). At these wavelengths, exposures of 100 times as much radiation (3000 J/mz) as required for an Flo at 260 nm (28 J/m') were not sufficient to lower capacity by this amount. After a value for an Flo at each wavelength was determined, the action spectrum for capacity was plotted (Fig. 8) as the reciprocal of the F , , value against wavelength (Jagger, 1967). Since it is usually assumed that the photochemical changes occurring in biological systems are due to photon absorption rather than to the total energy of exposure, these F,, values were quantum corrected by the method of Jag-
0
10
20
30
FLUENCE Figure. 5.
40
(JJ)
50
THOMAS P.COOHILL, SHARON P. MOOREand STEPHANIE DRAKE
390
.04-
,03-
2 -
n L/
2
.02-
.Ol-
-
0I
1 0
I
400
660
FLUENCE
I
I
I
I
1200 1600 2000 2400
(m)
Figure 6 . ger (p. 83, 1967) (Table 1). For comparison, an absorption spectrum for DNA (Jagger, 1967) is included in Fig. 8 as a dashed line.
I
I
I
I
I
I
1
230 240 250 260 270 280 290 300
Figure 8. An action spectrum for cellular capacity plotted as the reciprocal of the F,, value for each wavelength (from Figs. 2-6). An absorption spectrum for DNA is included (Jagger, 1967).
of the capacity survival curves begins to increase in value below 260nm. This seems to indicate that another molecule (or combination of molecules) is inDlSCUSSlON volved in determining the cell's capacity response at It is evident from Fig. 8 that the UV action spec- wavelengths less than 260 nm. This effect may modertrum for inactivation of host capacity closely follows ate that caused by the nucleic acid response. The the absorption spectrum for nucleic acid, for wave- action spectrum continues to decrease in value even lengths longer than the peak value (260nm). How- at the lowest wavelengths tested (235 nm). Since proever, for wavelengths less than 260 nm the capacity teins begin to absorb heavily at wavelengths below action spectrum drops more sharply than the nucleic 250nm, the continued decrease in value even at acid absorption spectrum. This phenomenon has been 235 nm gives additional reason to exclud'e direct proobserved in other systems (Coohill and Deering, 1969; tein involvement. However it may be reasonable to Giese, 1964). In addition, the extrapolation number assume that some molecule (or combination of molecules) is shielding the target DNA from UV radiation at these lower wavelengths. This indirect effect could account for the rapid loss of response below a -1.0+ 260nm. The actual cause of this discrepancy awaits further experimentation. Whether the target molecule for this effect is DNA or RNA cannot be determined by action spectroscopy alone. However, several authors have reported corollary data which indicate DNA as the target material c7 z for mammalian cell capacity. Lytle and Benane (1975) A = 3 1 3 nm z in their study of herpes virus infection of mammalian 0 =334nm cells showed that loss of capacity due to UV irradia4 6 5nm a tion can be photoreactivated in potoroo cells but not in CV-1 cells. The latter is consistent with the results of others showing the lack of an in oiuo expression of photoreactivation in cells from placental mammals (Cook, 1970). Photoreactivation is much more evident in DNA than in RNA, and photoreactivability is often considered a justification for choosing between the two molecules (Jagger, 1967). 50 I00 2000 3000 4000 Lytle et al. (1976) using a Xeroderma pigmentosum cell-herpes simplex virus system showed that the cell's FLUENCE (JH] ability to host virus decreases more rapidly in those cells lacking certain DNA repair enzymes. This again Figure I .
Wavelength dependence of cell capacity for virus growth points to DNA as the target molecule for capacity. Coppey and Nocentini (1976) working with the same CV-1 cell-herpes virus system used in the present study showed that cell capacity for “HSV production seems to be bound to the excision repair process working on UV-induced lesions in eukaryotic cells.” In addition they showed that caffeine, a known inhibitor of repair of UV-irradiated DNA, inhibits the recovery of the UV-mediated decrease in mammalian cell capacity. A smaller but similar effect of caffeine on capacity in the same cell-virus system was reported by Hellman et a/. (1976). The target molecule for capacity in prokaryotes does not appear to be DNA. Day and Muel (1974) in their study of capacity in the E . coli cell-T, bacteriophage system argued against nucleic acid as the target molecule. They cite evidence from their action spectrum in the region of 240-297 nm as “characteristic of (some) protein absorption.” It is difficult to compare our action spectrum with theirs since their means of obtaining monochromatic light (chemical filters) and the wavelengths used in their study were different from ours. The only major discrepancy
39 1
between the spectra when similar data points are compared is that they find their values increase over the region 254-240nm while ours decrease over the same region. In addition, unlike the results of Lytle et a/. (1976) in mammalian cells, Harm (1965) has found that in bacteria both E . coli B and its excisionless derivative E . coli B,- lose capacity with similar fluence response kinetics. Fluke (1966) has reported that capacity of E. coli for T1 phage cannot be photoreactivated, a result that differs from that of Lytle and Benane (1975) in mammalian cells. Hence it would appear that the target molecules for cell capacity are different in mammalian and bacterial cells. In summary, the target molecule for UV effects on mammalian cell capacity for herpes simplex virus plaque formation appears to be DNA. would like to thank Leslie James, Ed Ryan and Timothy Eichenbrenner for their help in performing these experiments. We would also like to express our appreciation to L. E. Bockstahler and C. D. Lytle 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. Acknowledgements-We
REFERENCES
Anderson, T. F. (1948) J. Bacteriol. 56, 403410. Benzer, S . and F. Jacob (1953) Ann. Inst. Pasteur 84, 18G204. Bockstahler, L. E. and C. D. Lytle (1970) Biochem. Biophys. Res. Commun. 41, 184-189. Bockstahler, L. E., C . D. Lytle, J. E. Stafford and K. F. Haynes (1976) Mutat. Res. 35, 189-198. Coohill, T. P. and R. A. Deering (1969) Radiat. Res. 39, 374-385. Cook, J. S . (1970) In Photophysiology (Edited by A. C. Giese), Vol. V, pp. 191-233. Academic Press, New York. Coppey, J. (1972) J . Gen. Virol. 14, 9-14. Coppey, J. and S . Nocentini (1976) J. Gen. Virol. 32, 1-15. Day, R. S., 111 and B. Muel (1974) Photochem. Photobiol. 20, 95-102. Fluke, D. J. (1966) Abstracts, 20th Meeting of Biophys. Soc., Boston, p. 70. Ciese, A. C. (1964) In Photophysiology (Edited by A. C. Giese), Vol. TI, pp. 2-16. Academic Press, New York. Harm, W. (1965) Photochem. Photobiol. 4, 575-585. Hellman, K. B., C. D. Lytle and L. E. Bockstahler (1976) Mutat. Res. 37, 249-256. Jagger, J. (1967) Introducfion to Research in Ultraviolet Photobiology. Prentice-Hall, Englewood Cliffs, NJ. Lytle, C. D. and S . G. Benane (1975) Int. J. Radiat. Biol. 27, 487-491. Lytle, C. D., R. S . Day 111, K. B. Hellman and L. E. Bockstahler (1976) Mutat. Rex 36, 257-264. Powell, W. F. (1959) Virology 9, 1-19.
Photobroloyy, 1911, Vol. 26, pp. 387-391.
Pergamon Press. Printed in Great Britain
THE WAVELENGTH DEPENDENCE OF ULTRAVIOLET INACTIVATION OF HOST CAPACITY IN A MAMMALIAN CELL-VIRUS SYSTEM THOMAS P.COOHILL, SHARON P. MOOREand STEPHANIE DRAKE Departments of Biology and Physics, Western Kentucky University, Bowling Green, ICY 42101, U.S.A. (Received 1 April 1977; accepted 3 June 1977)
Abstract-The ability of UV-irradiated African green monkey kidney cells (CV-1) to support the growth of unirradiated herpes simplex virus type 1 as measured by plaque forming ability has been investigated. The lowering of plaque formation by the virus when the host cell was irradiated was examined at thirteen different wavelengths. An action spectrum for this cellular parameter (capacity) was obtained in the wavelength region of 235-302nm. This action spectrum points to nucleic acid as the critical target molecule for this effect.
INTRODUCTION
units penicillin, 0.08 g streptomycin (International Scientific Industries, Cary, IL), 100,000 units mycostatin (E. R. Capacity is defined as the ability of a cell to support Squibb, Princeton, NJ) and buffered to pH 7.5 with 0.3 g the growth of a particular virus (Benzer and Jacob, sodium bicarbonate. Cells were incubated in closed Basks 1953). This cellular function is sensitive to ultraviolet or in petri dishes in gassed (approximately 5% COz) at 37°C. (UV) radiation, irradiation of the cell usually decreas- chambers Virus assay: Plaque forming ability kfaj. A macro-plaque ing its capacity to support viral growth (Anderson, strain of HSV-1 (Nerpesuirus hominus) was given to us by 1948; Bockstahler and Lytle, 1970; Coppey, 1972; C. D. Lytle of the Bureau of Radiological Health. These Powell, 1959). In some cases an initial increase in viruses were inoculated into confluent monolayers of capacity is seen at low fluences of UV radiation, but CV-1 cells, harvested, and kept in vials at -40°C until use. For virus assay, freshly confluent monolayers of cells this is followed by a rapid decrease as the Ruence were inoculated with an appropriate viral titre in a mainis raised (Bockstahler et al., 1976; L. E. Bockstahler, tenance medium consisting of 1X MEM supplemented with 0.26 g/t L-glutamine and 2% fetal calf serum and kept personal communication). In a bacterial cell-virus system, Day and Muel at 37°C for 90min. The inoculum was then removed and (1974) argued against nucleic acid as the target mol- 4 mY of growth medium containing 0.2572 immune serum globulin (Armour Pharmaceutical, Kankakee, 1L) was ecule for UV radiation effects on capacity. They cite added to prevent non-cellular viral transfer. The cells were evidence from their study of this effect at several incubated for 2 days and then stained by adding to the wavelengths in the UV region and from the reports growth medium one drop of a 3% aqueous solution of of others involving genetic and photobiological data crystal violet in 20% ETOH and 0.8% ammonium oxalate (Carolina Biological Co., Burlington, NC). The resulting that support the exclusion of DNA as the critical mol- solution was poured off after 10niin and plaques were ecule for bacterial capacity (Harm, 1965; Fluke, 1966). counted. Virus dilutions were adjusted to give approxiHowever, Lytle et al. (1976), Lytle and Benane (1975), mately 80 plaques on unirradiated monolayers. Monochromatic exposures. To obtain sufficient exposure and Coppey and Nocentini (1976) have recently rates, a 2.5 kW high pressure mercury-xenon lamp was reported data that support DNA as the target mol- used (929B Hanovia Lamp, Newark, N.J.). This source was ecule for mammalian cell capacity of herpes virus rep- air cooled to remove ozone, and infrared radiation was lication. Accordingly, we conducted experiments in- partially removed from the beam by means of a circulating volving the UV wavelength dependence of mam- water filter. Spectral separation was achieved by means malian cell (CV-1) capacity to host the virus herpes of two 25 cm grating monochromators coupled in tandem (GM 250, Schoeffel Inst., Westwood, NJ) as shown in Fig. simplex type 1 (HSV 1). Here we present results in 1. Half band widths were measured at several wavelengths the UV region (235-365 nm). and varied from 1.5 to 6.0nm depending on the slit width ( 1 - 4 mm). The spectral purity of the monochromator output was further checked by placing a series of cut-off filters into the exit beam and checking for the presence of conMATERIALS AND METHODS taminating wavelengths. For our studies only wavelengths Cell cultures. A permanent line of African green monkey corresponding to individual lines of the mercury spectrum kidney cells (CV-1) was obtained from L. E. Bockstahler were used, and thebe were found to be spectrally pure of the Bureau of Radiological Health, Rockville, MD. within the limits of our testing. Exposure rates were These cells were maintained in a growth medium consist- measured at different times during an experiment by placing of 1X Minimum Essential Medium (Eagle’s) with the ing a calibrated UV-sensitive photodiode [Cal-UV, United following components added per liter: 10% fetal calf serum Detector Technology (UDT), Inc., Santa Monica, CAI in Reheis Chemical Co., Kankakee, IL), 0.26 g ~-glutamine, the sample position and measuring the photocurrent pro16 m/ of 50X amino acids, 8 md of lOOX vitamins, 80,000 duced by the radiation beam with a Keithley 610B elec387 P A P 2614-0
388
THOMASP. COOHILL, SHARON P. MOOREand STEPHANIE DRAKE
.
RADIATION SOURCE
.
DOUBLE MONOCHROMATOR
OU PUQTODIODE
Figure 1. Source and dispersion system for the production of monochromatic radiation beams (see Materials and Methods). trometer (Keithley Inst., Cleveland, OH). The conversion factor for converting photocurrent output to energy fluence rate at each wavelength was obtained by combining a relative spectral response curve with the absolute calibrations at 250, 320 and 360nm supplied by UDT. These values were reliable within an accuracy of 2% (UDT, personal communication). Cells were irradiated in horizontally oriented open petri dishes. The UV beam was deflected downward onto the dishes by means of a 45-degree frontsurfaced mirror. Dishes were rotated at 0.5 rps to compensate for possible inhomogeneities in the radiation beam. Exposure times varied from 1 to 120s. Exposure rates .varied from 0.05 to 21 W/m2 (Table 1). Reciprocity of time and intensity was tested over a factor of at least three at each wavelength and no dependence of capacity on intensity was observed. Before exposure to radiation the growth medium was removed from confluent monolayers in plastic petri dishes. These monolayers were then rinsed twice in Dulbecco's phosphate buffered saline (PBS) to remove UV-absorbing components in the medium. The cell layers were kept moist during irradiation by the presence of 2m/ PBS. Immediately after irradiation the PBS was removed, and the cells were inoculated with unirradiated virus in maintenance medium.
RESULTS
Figures 2-7 show the effect on cell capacity of U V radiation of different wavelengths (235-365 nm). On the ordinate is plotted the ability of these irradiated cells to support viral growth (capacity). The abscissa shows the fluence of radiation of the stated wavelength. Each datum point in these figures represents the average value for points of the same fluence obtained from a minimum of three separate experiments a t each wavelength. During any experiment 10 monolayers were assayed at each fluence, and each monolayer contained approximately 20-100 plaques, therefore, each average value corresponds to 600-3000 plaques. Error bars, corresponding to a measure of the standard error, were added only if the error exceeded 10% of the average value. It is evident from Figs. 2-6 that the shapes of all the exposure response curves are similar and exhibit an initial flat shoulder the extent of which depends on the wavelength being tested. After the initial
Table 1. Fluence rate ranges for monochromatic exposures and cell capacity responses to the F,, level Wavelength (nm)
Fluence rate range* (w m-2)
Capacity (J- m2)
Quantum corrected (J-' m2)
235 238 240 245 248 254 260 265 210 275 280 289 297 302
0.50-2.00 0.60-3.50 1.10-3.90 1.30-5.10 1.25-5.40 0.45-1.45 0.05-0.20 0.30-1.10 0.60-2.00 0.75-2.60 0.90-3.70 1.05-3.50 6.00-19.00 5.W21.00
0.0085 0.0091 0.0103 0.0149 0.0176 0.0236 0.364 0.330 0.313 0.272 0.0216 0.0116 0.0017 0.0005
0.0109 0.0115 0.0130 0.0184 0.02 14 0.0281 0.423 0.376 0.350 0.0299 0.0233 0.0121 0.0017 0.0005
l/tilO
W l O
* Upper fluence rate limited to monochromator output at that wavelength.
Wavelength dependence of cell capacity for virus growth
389
1.0
0.5
0.1
LL 0
I
0
I
I
1
I
20
40
60
80
I
I
100
120
FLUENCE (M) Figure 2. Figures 2-7. The effect of radiation of different wavelengths on the capacity of CV-1 cells to support the growth of HSV 1.
shoulder there is an approximate exponential decrease in capacity to at least that fluence which lowers the cell's capacity to 10% (Flo)of the control.(unirra: diated) value. To minimize slight differences (less than 1004) in daily cellular response to UV radiation each experiment was repeated at least three times. In addition, since age-related changes in cellular response were observed, data were limited to that obtained with cells passaged no more than 18 times. As a control in each experiment a group of monolayers .was exposed to 280nm light of an amount sufficient to give an Flo value. This standard response to 280nm
0
20
40
60
FLUENCE Figure 3.
80
(JA)
100
I
0
I
10
I
20
I
30
FLUENCE Figure 4.
I
40
I
50
(J,)
light exposure confirmed that the cells were respwding in a similar manner during each experiment. Experiments conducted at wavelengths of 313, 334 and 365 nm were not at exposures sufficient to obtain an F,, (Fig. 7). At these wavelengths, exposures of 100 times as much radiation (3000 J/mz) as required for an Flo at 260 nm (28 J/m') were not sufficient to lower capacity by this amount. After a value for an Flo at each wavelength was determined, the action spectrum for capacity was plotted (Fig. 8) as the reciprocal of the F , , value against wavelength (Jagger, 1967). Since it is usually assumed that the photochemical changes occurring in biological systems are due to photon absorption rather than to the total energy of exposure, these F,, values were quantum corrected by the method of Jag-
0
10
20
30
FLUENCE Figure. 5.
40
(JJ)
50
THOMAS P.COOHILL, SHARON P. MOOREand STEPHANIE DRAKE
390
.04-
,03-
2 -
n L/
2
.02-
.Ol-
-
0I
1 0
I
400
660
FLUENCE
I
I
I
I
1200 1600 2000 2400
(m)
Figure 6 . ger (p. 83, 1967) (Table 1). For comparison, an absorption spectrum for DNA (Jagger, 1967) is included in Fig. 8 as a dashed line.
I
I
I
I
I
I
1
230 240 250 260 270 280 290 300
Figure 8. An action spectrum for cellular capacity plotted as the reciprocal of the F,, value for each wavelength (from Figs. 2-6). An absorption spectrum for DNA is included (Jagger, 1967).
of the capacity survival curves begins to increase in value below 260nm. This seems to indicate that another molecule (or combination of molecules) is inDlSCUSSlON volved in determining the cell's capacity response at It is evident from Fig. 8 that the UV action spec- wavelengths less than 260 nm. This effect may modertrum for inactivation of host capacity closely follows ate that caused by the nucleic acid response. The the absorption spectrum for nucleic acid, for wave- action spectrum continues to decrease in value even lengths longer than the peak value (260nm). How- at the lowest wavelengths tested (235 nm). Since proever, for wavelengths less than 260 nm the capacity teins begin to absorb heavily at wavelengths below action spectrum drops more sharply than the nucleic 250nm, the continued decrease in value even at acid absorption spectrum. This phenomenon has been 235 nm gives additional reason to exclud'e direct proobserved in other systems (Coohill and Deering, 1969; tein involvement. However it may be reasonable to Giese, 1964). In addition, the extrapolation number assume that some molecule (or combination of molecules) is shielding the target DNA from UV radiation at these lower wavelengths. This indirect effect could account for the rapid loss of response below a -1.0+ 260nm. The actual cause of this discrepancy awaits further experimentation. Whether the target molecule for this effect is DNA or RNA cannot be determined by action spectroscopy alone. However, several authors have reported corollary data which indicate DNA as the target material c7 z for mammalian cell capacity. Lytle and Benane (1975) A = 3 1 3 nm z in their study of herpes virus infection of mammalian 0 =334nm cells showed that loss of capacity due to UV irradia4 6 5nm a tion can be photoreactivated in potoroo cells but not in CV-1 cells. The latter is consistent with the results of others showing the lack of an in oiuo expression of photoreactivation in cells from placental mammals (Cook, 1970). Photoreactivation is much more evident in DNA than in RNA, and photoreactivability is often considered a justification for choosing between the two molecules (Jagger, 1967). 50 I00 2000 3000 4000 Lytle et al. (1976) using a Xeroderma pigmentosum cell-herpes simplex virus system showed that the cell's FLUENCE (JH] ability to host virus decreases more rapidly in those cells lacking certain DNA repair enzymes. This again Figure I .
Wavelength dependence of cell capacity for virus growth points to DNA as the target molecule for capacity. Coppey and Nocentini (1976) working with the same CV-1 cell-herpes virus system used in the present study showed that cell capacity for “HSV production seems to be bound to the excision repair process working on UV-induced lesions in eukaryotic cells.” In addition they showed that caffeine, a known inhibitor of repair of UV-irradiated DNA, inhibits the recovery of the UV-mediated decrease in mammalian cell capacity. A smaller but similar effect of caffeine on capacity in the same cell-virus system was reported by Hellman et a/. (1976). The target molecule for capacity in prokaryotes does not appear to be DNA. Day and Muel (1974) in their study of capacity in the E . coli cell-T, bacteriophage system argued against nucleic acid as the target molecule. They cite evidence from their action spectrum in the region of 240-297 nm as “characteristic of (some) protein absorption.” It is difficult to compare our action spectrum with theirs since their means of obtaining monochromatic light (chemical filters) and the wavelengths used in their study were different from ours. The only major discrepancy
39 1
between the spectra when similar data points are compared is that they find their values increase over the region 254-240nm while ours decrease over the same region. In addition, unlike the results of Lytle et a/. (1976) in mammalian cells, Harm (1965) has found that in bacteria both E . coli B and its excisionless derivative E . coli B,- lose capacity with similar fluence response kinetics. Fluke (1966) has reported that capacity of E. coli for T1 phage cannot be photoreactivated, a result that differs from that of Lytle and Benane (1975) in mammalian cells. Hence it would appear that the target molecules for cell capacity are different in mammalian and bacterial cells. In summary, the target molecule for UV effects on mammalian cell capacity for herpes simplex virus plaque formation appears to be DNA. would like to thank Leslie James, Ed Ryan and Timothy Eichenbrenner for their help in performing these experiments. We would also like to express our appreciation to L. E. Bockstahler and C. D. Lytle 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. Acknowledgements-We
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