Br. J. Cancer (1978) 37, Suppl. III, 111
SENSITIZATION OF ULTRAVIOLET RADIATION DAMAGE IN BACTERIA AND MAMMALIAN CELLS G. J. FISHER,* M. E. WATTS, K. B. PATEL AND G. E. ADAMSt From the Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, England
Summary.-Bacteria (Serratia marcescens) and mammalian cells (Chinese hamster V79-379A) were irradiated in monolayers with ultraviolet light at 254 nm or 365 nm in the presence or absence of radiosensitizing drugs. At 254 nm, killing is very efficient (D37 1 J m-2 exposure, or 6 x 104 photons absorbed by DNA per bacterium), and sensitizers have no effect. At 365 nm, cells are not killed in buffer, but are inactivated in the presence of nifurpipone or misonidazole. Lethal exposures ("5 x 103 J m-2 at 10 mM misonidazole) correspond to about 107 photons absorbed by sensitizer molecules per bacterium. Toxicity of stable photoproducts of the drugs is not involved, nor is oxygen required. Hence the transient species formed by photo-excitation of radiosensitizer molecules are capable of killing cells in the absence of other types of radiation damage. METHODS
APART from several biochemical effects of electron-affinic radiosensitizers (depletion of thiols, differential toxicity to hypoxic cells, changes in metabolic rate, and inhibition of repair), a number of general radiation chemical mechanisms have been proposed to account for radiosensitization by such drugs. These include: (1) the formation of stable cytotoxic products; (2) sequestration or trapping of electrons, to prevent normal charge recombination; (3) radical fixation, through addition or charge transfer reactions of sensitizer molecules with radiationinduced radicals on target biomolecules; and (4) attack on biomolecules by transient sensitizer radicals, to give additional damage. Since ionizing radiation deposits energy nonspecifically and forms a large variety of initial species throughout the cell, these processes may all occur. Ultraviolet light (UV) is much more specific in its action, and the present results suggest that the fourth class of reaction can lead to cell death.
UV irradiations were carried out at two different wavelengths, using a 6-watt low pressure mercury lamp emittingf at 254 nm and a 100-watt medium pressure mercury lamp emitting largely at 365 nm. Glass filters were used to improve spectral purity. Exposure rates were controlled by blocking off all but a few cm of each lamp and by adjusting the lamp heights. Potassium ferrioxalate actinometry at 365 nm (Hatchard and Parker, 1956) was used to obtain an exposure rate of about 3 x 103 J m-2m in-1 at a distance of 5 cm. Since the filter transmitted some light of longer wavelengths, the effective rate and total exposure were somewhat lower than the calculated values. At 254 nm, malachite green leukocyanide (Fisher, LeBlanc and Johns, 1967) was used to measure exposure rates of about 2-0 J m-2 min-1 at 30 cm from the lamp. Rapid solarization or darkening of the filter led to decreasing transmission during the course of this work; hence frequent intensity measurements were necessary. Because of the high spectral purity at 254 nm and the sensitivity of the actinometer, exposures are accurate to better than 5%. * Present address: Departement de M6decine Nucl6aire et de Radiobiologie, Centre Hospitalier Universi-
taire, Sherbrooke, Qu6bec, Canada J1H 5N4. t Present address: Physics Division, Institute of Cancer Research, Clifton Ave., Sutton, Surrey SM2 5PX, England.
112
G. J. FISHER, M. E. WATTS, K. B. PATEL AND G. E. ADAMS
Bacteria and cells were irradiated in monolayers to avoid problems of beam scatter (by cells) and attenuation (by medium and dissolved sensitizers: 10 mM misonidazole can absorb over 90% of the light in 1 mm). Therefore, no corrections were required for exposures. Irradiations were carried out at room temperature. Serratia marcescens were spread on Millipore filters (0-22 ,um pore size) on Ion-agar. The filters were transferred to 15 A drops of buffer or of sensitizer solution in the quartztopped irradiation jig, and equilibrated under air or nitrogen (
SENSITIZATION OF ULTRAVIOLET RADIATION DAMAGE IN BACTERIA AND MAMMALIAN CELLS G. J. FISHER,* M. E. WATTS, K. B. PATEL AND G. E. ADAMSt From the Cancer Research Campaign, Gray Laboratory, Mount Vernon Hospital, Northwood, Middlesex HA6 2RN, England
Summary.-Bacteria (Serratia marcescens) and mammalian cells (Chinese hamster V79-379A) were irradiated in monolayers with ultraviolet light at 254 nm or 365 nm in the presence or absence of radiosensitizing drugs. At 254 nm, killing is very efficient (D37 1 J m-2 exposure, or 6 x 104 photons absorbed by DNA per bacterium), and sensitizers have no effect. At 365 nm, cells are not killed in buffer, but are inactivated in the presence of nifurpipone or misonidazole. Lethal exposures ("5 x 103 J m-2 at 10 mM misonidazole) correspond to about 107 photons absorbed by sensitizer molecules per bacterium. Toxicity of stable photoproducts of the drugs is not involved, nor is oxygen required. Hence the transient species formed by photo-excitation of radiosensitizer molecules are capable of killing cells in the absence of other types of radiation damage. METHODS
APART from several biochemical effects of electron-affinic radiosensitizers (depletion of thiols, differential toxicity to hypoxic cells, changes in metabolic rate, and inhibition of repair), a number of general radiation chemical mechanisms have been proposed to account for radiosensitization by such drugs. These include: (1) the formation of stable cytotoxic products; (2) sequestration or trapping of electrons, to prevent normal charge recombination; (3) radical fixation, through addition or charge transfer reactions of sensitizer molecules with radiationinduced radicals on target biomolecules; and (4) attack on biomolecules by transient sensitizer radicals, to give additional damage. Since ionizing radiation deposits energy nonspecifically and forms a large variety of initial species throughout the cell, these processes may all occur. Ultraviolet light (UV) is much more specific in its action, and the present results suggest that the fourth class of reaction can lead to cell death.
UV irradiations were carried out at two different wavelengths, using a 6-watt low pressure mercury lamp emittingf at 254 nm and a 100-watt medium pressure mercury lamp emitting largely at 365 nm. Glass filters were used to improve spectral purity. Exposure rates were controlled by blocking off all but a few cm of each lamp and by adjusting the lamp heights. Potassium ferrioxalate actinometry at 365 nm (Hatchard and Parker, 1956) was used to obtain an exposure rate of about 3 x 103 J m-2m in-1 at a distance of 5 cm. Since the filter transmitted some light of longer wavelengths, the effective rate and total exposure were somewhat lower than the calculated values. At 254 nm, malachite green leukocyanide (Fisher, LeBlanc and Johns, 1967) was used to measure exposure rates of about 2-0 J m-2 min-1 at 30 cm from the lamp. Rapid solarization or darkening of the filter led to decreasing transmission during the course of this work; hence frequent intensity measurements were necessary. Because of the high spectral purity at 254 nm and the sensitivity of the actinometer, exposures are accurate to better than 5%. * Present address: Departement de M6decine Nucl6aire et de Radiobiologie, Centre Hospitalier Universi-
taire, Sherbrooke, Qu6bec, Canada J1H 5N4. t Present address: Physics Division, Institute of Cancer Research, Clifton Ave., Sutton, Surrey SM2 5PX, England.
112
G. J. FISHER, M. E. WATTS, K. B. PATEL AND G. E. ADAMS
Bacteria and cells were irradiated in monolayers to avoid problems of beam scatter (by cells) and attenuation (by medium and dissolved sensitizers: 10 mM misonidazole can absorb over 90% of the light in 1 mm). Therefore, no corrections were required for exposures. Irradiations were carried out at room temperature. Serratia marcescens were spread on Millipore filters (0-22 ,um pore size) on Ion-agar. The filters were transferred to 15 A drops of buffer or of sensitizer solution in the quartztopped irradiation jig, and equilibrated under air or nitrogen (
