INT. J . RADIAT . BIOL .,
1978,
VOL .
34,
NO .
6, 587-588
Does singlet oxygen contribute to the oxygen effect? JVRGEN KIEFER Strahlenzentrum der Justus-Liebig-Universitat Giessen, 6300 Giessen, Leihgesterner Weg 217, Germany
Int J Radiat Biol Downloaded from informahealthcare.com by Mcgill University on 11/14/14 For personal use only.
(Received 15 May 1978 ; accepted 31 May 1978)
The molecular mechanism of the oxygen effects still remains obscure and reaction pathways and intermediates are not known . It cannot be excluded a priori that singlet oxygen may play a role since it is known from experiments on photodynamic action that it is able to kill cells (Ito 1977) . It may also be produced by ionizing radiation via reactions between oxygen and radiationgenerated free radicals (Koppenol and Butler 1977) . It appears, therefore, feasible to assume that it contributes to the radiobiological oxygen effect . This hypothesis was tested with diploid yeast . It has been shown that azide quenches singlet oxygen in this system (Kobayashi and Ito 1976) . If this reactive intermediate were to play a role in the oxygen effect one would expect a decreased oxygen enhancement ratio when azide is present during the exposure . Another way of checking the involvement of singlet oxygen is to irradiate with D 20 as suspending medium since the life-time of singlet oxygen is increased under these circumstances (Kobayashi and Ito 1976) . Diploid yeast strain 211 was used for the experiments . Culture conditions were as described by Kiefer (1971). Stationary phase cells were washed off agar plates and irradiated either in air or in nitrogen in stirred aqueous suspensions . For the D 2 0 experiments this was used as a suspending medium . Sodium azide was added to the aqueous samples at a concentration of 0 . 05 moll-' . This amount suffices to quench singlet oxygen as evidenced by its effect on photodynamic inactivation in our system (Adam, Baker and Kiefer, to be published) . All samples were diluted with water by at least tenfold immediately after irradiation . Control viability was not impaired by either of these treatments .
I
;. DOSE/krad 94 141
188
235
1
U
n
\ 01
Z 0 .01 CY U,
OD01
Figure 1 . Survival of diploid yeast Saccharomyces cerevisiae after exposure to 85 kVp X-rays in air and in nitrogen with and without sodium azide . 0 = air, no azide ; • = nitrogen, no azide ; + = air, with azide, x = nitrogen, with azide . Pooled results of three experiments . Error bars derived from interexperimental variation .
588
Correspondence Figure 1 shows the effect of azide . There is no change in survival, either
after exposure in air or nitrogen . The effect of D2 0 as a suspending medium is depicted in figure 2 . Oxic sensitivity is not changed but there is significantly less survival after irradiation in nitrogen . The results described appear to rule out the possibility that singlet oxygen contributes to the oxygen effect since no change in the ' air curves ' could be detected . The increased sensitivity with D2 0 and nitrogen is presumably due to the increased life-time of OHradicals (Fielden and Hart 1968) .
A similar effect has been found in Bacillus
Int J Radiat Biol Downloaded from informahealthcare.com by Mcgill University on 11/14/14 For personal use only.
megatherium spores (Powers 1964) .
Figure 2 . Survival of X-irradiated yeast cells with H20 or D20 as suspending medium . 0 = air, H20 ; 0 = Ns, H20 ; + = air, D 2 0 ; x = N1, D 1 0 . Data from one typical experiment .
ACKNOWLEDGMENTS
This study was supported by a grant from the German Bundesministerium fur Forschung and Technologie . The author wishes to thank Klaus Escher for his technical assistance .
REFERENCES ADAM BAKER, Y . M ., and KIEFER, I . J ., 1978 (to be published) . FIELDEN, E . M ., and HART, E . J ., 1968, Radiat . Res., 33, 426 . ITO, T ., 1977, Photochem . Photobiol ., 25, 47 . KIEFER, J ., 1971, Int . J. Radiat . Biol., 20, 325 . KOBAYASHI, K ., and ITO, T ., 1976, Photochem . Photobiol ., 23, 21 . KOPPENOL, W . H ., and BUTLER, J ., 1977, FEBS Lett., 83, 1 . POWERS, E . L ., 1964, Cellular Radiation Biology (Baltimore : Williams & Wilkins),
286-304 .
pp .
1978,
VOL .
34,
NO .
6, 587-588
Does singlet oxygen contribute to the oxygen effect? JVRGEN KIEFER Strahlenzentrum der Justus-Liebig-Universitat Giessen, 6300 Giessen, Leihgesterner Weg 217, Germany
Int J Radiat Biol Downloaded from informahealthcare.com by Mcgill University on 11/14/14 For personal use only.
(Received 15 May 1978 ; accepted 31 May 1978)
The molecular mechanism of the oxygen effects still remains obscure and reaction pathways and intermediates are not known . It cannot be excluded a priori that singlet oxygen may play a role since it is known from experiments on photodynamic action that it is able to kill cells (Ito 1977) . It may also be produced by ionizing radiation via reactions between oxygen and radiationgenerated free radicals (Koppenol and Butler 1977) . It appears, therefore, feasible to assume that it contributes to the radiobiological oxygen effect . This hypothesis was tested with diploid yeast . It has been shown that azide quenches singlet oxygen in this system (Kobayashi and Ito 1976) . If this reactive intermediate were to play a role in the oxygen effect one would expect a decreased oxygen enhancement ratio when azide is present during the exposure . Another way of checking the involvement of singlet oxygen is to irradiate with D 20 as suspending medium since the life-time of singlet oxygen is increased under these circumstances (Kobayashi and Ito 1976) . Diploid yeast strain 211 was used for the experiments . Culture conditions were as described by Kiefer (1971). Stationary phase cells were washed off agar plates and irradiated either in air or in nitrogen in stirred aqueous suspensions . For the D 2 0 experiments this was used as a suspending medium . Sodium azide was added to the aqueous samples at a concentration of 0 . 05 moll-' . This amount suffices to quench singlet oxygen as evidenced by its effect on photodynamic inactivation in our system (Adam, Baker and Kiefer, to be published) . All samples were diluted with water by at least tenfold immediately after irradiation . Control viability was not impaired by either of these treatments .
I
;. DOSE/krad 94 141
188
235
1
U
n
\ 01
Z 0 .01 CY U,
OD01
Figure 1 . Survival of diploid yeast Saccharomyces cerevisiae after exposure to 85 kVp X-rays in air and in nitrogen with and without sodium azide . 0 = air, no azide ; • = nitrogen, no azide ; + = air, with azide, x = nitrogen, with azide . Pooled results of three experiments . Error bars derived from interexperimental variation .
588
Correspondence Figure 1 shows the effect of azide . There is no change in survival, either
after exposure in air or nitrogen . The effect of D2 0 as a suspending medium is depicted in figure 2 . Oxic sensitivity is not changed but there is significantly less survival after irradiation in nitrogen . The results described appear to rule out the possibility that singlet oxygen contributes to the oxygen effect since no change in the ' air curves ' could be detected . The increased sensitivity with D2 0 and nitrogen is presumably due to the increased life-time of OHradicals (Fielden and Hart 1968) .
A similar effect has been found in Bacillus
Int J Radiat Biol Downloaded from informahealthcare.com by Mcgill University on 11/14/14 For personal use only.
megatherium spores (Powers 1964) .
Figure 2 . Survival of X-irradiated yeast cells with H20 or D20 as suspending medium . 0 = air, H20 ; 0 = Ns, H20 ; + = air, D 2 0 ; x = N1, D 1 0 . Data from one typical experiment .
ACKNOWLEDGMENTS
This study was supported by a grant from the German Bundesministerium fur Forschung and Technologie . The author wishes to thank Klaus Escher for his technical assistance .
REFERENCES ADAM BAKER, Y . M ., and KIEFER, I . J ., 1978 (to be published) . FIELDEN, E . M ., and HART, E . J ., 1968, Radiat . Res., 33, 426 . ITO, T ., 1977, Photochem . Photobiol ., 25, 47 . KIEFER, J ., 1971, Int . J. Radiat . Biol., 20, 325 . KOBAYASHI, K ., and ITO, T ., 1976, Photochem . Photobiol ., 23, 21 . KOPPENOL, W . H ., and BUTLER, J ., 1977, FEBS Lett., 83, 1 . POWERS, E . L ., 1964, Cellular Radiation Biology (Baltimore : Williams & Wilkins),
286-304 .
pp .
