Br. J. Cancer (1978) 37, Suppl. III, 103
RADIOSENSITIZATION OF SERRATIA MARCESCENS BY NITROPYRIDINIUM COMPOUNDS R. F. ANDERSON,* K. B. PATEL* AND C. E. SMITHENt From the *Cancer Research Campaign, Gray Laboratory, Mount Vernon Ho8pital, Northwood, Middlesex HA6 2RN, and fRoche Product8 Ltd, Welwyn Garden City, Hertford8hire AL7 3A Y, U.K.
Summary.-The two nitropyridinium compounds tested sensitize hypoxic Serratia marcescens to irradiation up to the oxygen enhancement level by two components which can be separated as a function of compound concentration. Sensitization above the initial plateau level is in order of their determined one-electron reduction potentials, Ro 03-5580 (E71= -335 mV) being more efficient than Ro 03-5637 (E71= -358 mV). Additivity in sensitization up to a maximum enhancement level of 2-1 ± 0 1 is found on combining these hydrophilic compounds, at concentrations to give sensitization at the plateau level, with the hydrophobic sensitizer paranitroacetophenone (PNAP). It is concluded that the nitropyridinium compounds and PNAP sensitize the same site.
EXPERIMENTS with Bacillus megaterium spores (Tallentire, Jones and Jacobs, 1972), double pulse experiments with Escherichia coli (Epp et at., 1973) and rapid-mix experiments with mammalian cells (Shenoy et al., 1975) have shown that sensitization afforded by oxygen occurs by at least two components. Bipyridinium compounds have been used as probes to investigate these so-called oxygen effect components (Anderson and Patel, 1977). The two components are separated on the basis that the bivalent cations, where there is no charge delocalization, do not penetrate the bacterium cell wall and sensitization occurs at an external or membrane site. The sensitization efficiency of the bipyridinium compounds increases with increasing one-electron reduction potential. On the formation of the radical cation, delocalization of charge occurs which enables these compounds to penetrate into gram-negative bacteria (Jones and Garland, 1977). Dihydroimidazo 1,2-a pyridinium compounds are another class of pyridinium compounds which carry a delocalized positive charge and this study of their nitro derivatives was undertaken to further investigate sensitization at an internal cellular site.
MATERIALS AND METHODS
Cultures of Serratia marcescens were grown to stationary phase in Davies' Minimal Medium supplemented with 0-1% glucose. The suspension was diluted to a concentration of 2 x 107 cells/ml in phosphate buffer (15 mmol/dm3, pH 7.2) and the cells starved for 30 min before addition of the compounds. Irradiations were carried out on 5-ml samples using a 60Co y-ray source at a dose rate of about 1.5 krad/min. Irradiations in anoxia were performed by bubbling 02-free nitrogen ( < 5 p.p.m.) for 10 min before and during irradiations. Estimation of viable cell counts were made after suitable dilution and plating in triplicate on nutrient agar and incubation. Plots of log surviving fraction of cells against accumulated dose gave an extrapolation number of one. Enhancement ratios are calculated from the ratio of the slope of the survival curve obtained for nitrogen alone to the slope of the survival curve with the sensitizer present. The nitropyridinium compounds were kindly supplied by Roche Products Ltd. The compounds were synthesized in the following manner: 5-nitro-2-phenylaminopyridine, prepared according to the literature (Mangini, 1937), was condensed with refluxing 1, 2-dibromo-ethane for 24 h and the cooled reaction mixture partitioned between ether and water. The aqueous solution was washed twice with
104
R. F. ANDERSON, K. B. PATEL AND C. B. SMITHEN
fresh ether, treated with saturated aqueous sodium perchlorate and stored overnight at 5°C. The solid product was separated and recrystallized from water to yield I-phenyl2,3-dihydro-6-nitroimidazo [1,2-a] pyridinium perchlorate (Ro 03-5580) as yellow needles, m.p. 192-193°C decomp. In a similar way, 2-ethylamino-5-nitropyridine, m.p. 119-121°C from benzene or ether, was converted into I-ethyl-2,3-dihydro-6-nitroimidazo [1,2-a] pyridinium perchlorate (Ro 03-5637/001), recrystallized from water as yellow needles, m.p. 160-161°C. These new compounds were fully characterized by means of IR, NMR and UV spectroscopy and elemental analysis. Paranitroacetophenone (PNAP) was obtained from B.D.H.
02
O
02N
,CH CH
Li~~~~
Ro 03-5580
Ro 03-5637 RESULTS
0
a)Ec -C 0)
FIG. 1.-Dependence of enhancement ratio for sensitization of hypoxic S. marce8cen8 on nitropyridinium compound concentration.
When anoxic suspensions of Serratia marcescens were irradiated in the presence of the nitropyridinium compounds sensitization was observed. The survival curves were straight and the enhancement ratios obtained at various concentrations of the compounds are presented in Fig. 1. The plot for both compounds is biphasic, an
Pulse radiolysis was used to determine the one-electron reduction potentials of the nitropyridinium compounds using benzylviologen (E71 --354 mV) as the reference standard. These values are presented in the table together with octan1-ol: water partition data.
10-1 c 0
TABLE .5
log P Compound E71/mV Ro 03-5580 -335 -2-33 Ro 03--5637 -356 -2 -64 PNAP -355* +1 49t * Meisel and Neta, 1975; t Leo, Hansch and Elkins, 1971.
Sensitization by nitropyridinium compounds Anoxic toxicity limited the maximum concentration of Ro 03-5580 used to study
enhancement ratio as a function of sensitizer concentration whereas Ro-03-5637 is non-toxic up to its solubility limit. No sensitization of aerobic cells was observed in the presence of either compound.
i2'
.
104
8 10 12 14 16 Dose / krad FIG. 2.-The effect of combining PNAP with the nitropyridiniumn compounds on the sensitization of hypoxic S. marce8cen,. 0 = 02 bubbled, O = N2 bubbled, V = PNAP (1.5 mmol/dm3), O = Ro 035580 (250 ,tmol/dm3), A = Ro 03-5637 (750 ,umol/dm3), C = V + D, A = 2
V + A.
4
6
SENSITIZATION BY NITROPYRIDINIUM COMPOUNDS
initial plateau at 1-5 ± 0-1 rising at higher concentrations to the full oxygen enhancement level. Ro 03-5580 effects sensitization above the plateau level at lower concentrations than for Ro 03-5637. This compound also exhibits sensitization above the oxygen enhancement level. Combination of sensitizers Sensitization of mammalian cells by PNAP has been shown to reach a plateau at part of the oxygen sensitization level (Adams et al., 1971). This sensitization plateau has been associated with one of the components of the oxygen effect (Adams et al., 1975). The sensitizing effect of combining sufficient of the nitropyridinium compounds to give plateau sensitization with PNAP (1.5 mmol/dm3) was studied (Fig. 2). The plateau in sensitization effected by PNAP (2.1 + 0-1) is reached at this concentration and in both experiments the addition of the nitropyridinium compounds did not increase the level of sensitization. In another experiment a lower concentration of PNAP (100 ,tmol/dm3) sufficient to give an enhancement ratio of 1-4 was added to Ro 03-5580 (250 ,tmol/dm3). In this case combining the sensitizers produced an additive effect, giving an enhancement ratio of 2-0 ± 041. DISCUSSION
The positive charge carried by the nitropyridinium compounds and their high partitioning into the aqueous phase probably results in the formation of a concentration gradient at the cell surface. The plateau at an enhancement ratio of 1*5 ± 0*1 is lower than that found with the bipyridinium compounds where a plateau is seen at 2. 1 ± 0.1 (Anderson and Patel, 1977). Differences in chemical structure between the two classes of compounds may determine the maximum concentration each can achieve at a particular site. Unlike the dicationic form of the bipyridinium compounds, sensitization above
105
the plateau level is seen for the nitropyridinium compounds. This result supports the contention that the nitropyridinium compounds do penetrate into the cell. The more electron-affinic compound, Ro 03-5580, is the more efficient compound at effecting sensitization above the plateau level. This correlation of one-electron reduction potential with sensitization efficiency has been reported for both mammalian cells (Adams et al., 1976) and bacteria (Anderson and Patel, 1977). Sensitization levels effected by PNAP and by the nitropyridinium compounds are additive up to an enhancement level of 241 ± 0.1. Above this level no additivity in sensitization is seen, showing that PNAP and the nitropyridinium compounds sensitize the same site. The maximum sensitization possible at this site corresponds to approximately 40% of the overall oxygen enhancement level. We would like to thank Mr D. Brown for technical assistance in the preparation of the compounds. This work is financially supported by the Cancer Research Campaign. REFERENCES ADAMS, G. E., ASQUITH, J. C., DEWEY, D. L., FOSTER, J. L., MICHAEL, B. D. & WILLSON, R. L. (1971) Electron-affinic Sensitization II. ParaNitroacetophenone: A Radiosensitizer for Anoxic Bacterial and Mammalian Cells. Int. J. Radiat. Biol., 19, 575. ADAMS, G. E., MICHAEL, B. D., ASQUITH, J. C., SHENOY, M. A., WATTS, M. E. & WHILLANS, D. W. (1975) Biomedical, Chemical and Physical Perspectives, In Radiation Research. Ed., NYGGARD, 0. F., ADLER, H. I. & SINCLAIR, W. K., New York: Academic Press, Inc. p. 478. ADAMS, G. E., FLOCKHART, I. R., SMITHEN, C. E., STRATFORD, I. J., WARDMAN, P. & WATTS, M. E. (1976) Electron-affinic Sensitization VII. A Correlation between Structures, One-electron Reduction Potentials and Efficiencies of Nitroimidazoles as Hypoxic Cell Radiosensitizers. Radiat. Res., 67, 9. ANDERSON, R. F. & PATEL, K. B. (1977) Radiosensitization of Serratia mtarcescens by Bipyridiniuim Compounds. Int. J. Radiat. Biol., 32, 471. EP, E. R., WEISS, H., KEssARIs, N. D., SANTOMASSO, A., HESLIN, J. & LING, C. C. (1973) Oxygen Diffusion Times in Bacterial Cells Irradiated with High-intensity Pulsed Electrons: New Upper Limit to the Lifetime of Oxygen-sensitive Species Suspected to be Induced at Critical Sites in Bacterial Cells. Radiat. Res., 54, 171.
106
R. F. ANDERSON, K. B. PATEL AND C. B. SMITHEN
JONES, R. W. & GARLAND, P. B. (1977) Sites and Specificity of the Reaction of Bipyridinium Compounds with Anaerobic Respiratory Enzymes of E.scherichia coli. Biochem. J., 164, 199. LEO, A., HANSCH, C. & ELKINs, D. (1971) Partition Coefficients and their Uses. Chenmical Reviews, 71, 525. MANGINI, A. (1937)Nitro Derivatives, XIV. Substituted Nitropyridylphenylamines. Ricerca Sci., 8, 427. MEISEL, D. & NETA, P. (1975) One-electron Redox Potentials of Nitro Compounds and Radiosen-
sitizers. Correlation with Spin Densities of their Radical Anions. J. Amer. Chem. Soc., 97, 5198. SHENOY, M. A., AsQUITH, J. C., ADAMS, G. E., MICHAEL, B. D. & WATTs, M. E. (1975) Timeresolved Oxygen Effects in Irradiated Bacteria and Mammalian Cells: A Rapid-mix Study. Radiat. Res., 62, 498. TALLENTIRE, A., JoN-E, A. B. & JACOBS, G. P. (1972) The Radiosensitizing Actions of Ketonic Agents and Oxygen in Bacterial Spores Suspended in Aqueous and Nonaqueous Milieu. Molecular Basis of Radiation Biology. 24th Farkas Memorial Symposium, Jerusalem. I8r. J. Chem., 10, 1185.
RADIOSENSITIZATION OF SERRATIA MARCESCENS BY NITROPYRIDINIUM COMPOUNDS R. F. ANDERSON,* K. B. PATEL* AND C. E. SMITHENt From the *Cancer Research Campaign, Gray Laboratory, Mount Vernon Ho8pital, Northwood, Middlesex HA6 2RN, and fRoche Product8 Ltd, Welwyn Garden City, Hertford8hire AL7 3A Y, U.K.
Summary.-The two nitropyridinium compounds tested sensitize hypoxic Serratia marcescens to irradiation up to the oxygen enhancement level by two components which can be separated as a function of compound concentration. Sensitization above the initial plateau level is in order of their determined one-electron reduction potentials, Ro 03-5580 (E71= -335 mV) being more efficient than Ro 03-5637 (E71= -358 mV). Additivity in sensitization up to a maximum enhancement level of 2-1 ± 0 1 is found on combining these hydrophilic compounds, at concentrations to give sensitization at the plateau level, with the hydrophobic sensitizer paranitroacetophenone (PNAP). It is concluded that the nitropyridinium compounds and PNAP sensitize the same site.
EXPERIMENTS with Bacillus megaterium spores (Tallentire, Jones and Jacobs, 1972), double pulse experiments with Escherichia coli (Epp et at., 1973) and rapid-mix experiments with mammalian cells (Shenoy et al., 1975) have shown that sensitization afforded by oxygen occurs by at least two components. Bipyridinium compounds have been used as probes to investigate these so-called oxygen effect components (Anderson and Patel, 1977). The two components are separated on the basis that the bivalent cations, where there is no charge delocalization, do not penetrate the bacterium cell wall and sensitization occurs at an external or membrane site. The sensitization efficiency of the bipyridinium compounds increases with increasing one-electron reduction potential. On the formation of the radical cation, delocalization of charge occurs which enables these compounds to penetrate into gram-negative bacteria (Jones and Garland, 1977). Dihydroimidazo 1,2-a pyridinium compounds are another class of pyridinium compounds which carry a delocalized positive charge and this study of their nitro derivatives was undertaken to further investigate sensitization at an internal cellular site.
MATERIALS AND METHODS
Cultures of Serratia marcescens were grown to stationary phase in Davies' Minimal Medium supplemented with 0-1% glucose. The suspension was diluted to a concentration of 2 x 107 cells/ml in phosphate buffer (15 mmol/dm3, pH 7.2) and the cells starved for 30 min before addition of the compounds. Irradiations were carried out on 5-ml samples using a 60Co y-ray source at a dose rate of about 1.5 krad/min. Irradiations in anoxia were performed by bubbling 02-free nitrogen ( < 5 p.p.m.) for 10 min before and during irradiations. Estimation of viable cell counts were made after suitable dilution and plating in triplicate on nutrient agar and incubation. Plots of log surviving fraction of cells against accumulated dose gave an extrapolation number of one. Enhancement ratios are calculated from the ratio of the slope of the survival curve obtained for nitrogen alone to the slope of the survival curve with the sensitizer present. The nitropyridinium compounds were kindly supplied by Roche Products Ltd. The compounds were synthesized in the following manner: 5-nitro-2-phenylaminopyridine, prepared according to the literature (Mangini, 1937), was condensed with refluxing 1, 2-dibromo-ethane for 24 h and the cooled reaction mixture partitioned between ether and water. The aqueous solution was washed twice with
104
R. F. ANDERSON, K. B. PATEL AND C. B. SMITHEN
fresh ether, treated with saturated aqueous sodium perchlorate and stored overnight at 5°C. The solid product was separated and recrystallized from water to yield I-phenyl2,3-dihydro-6-nitroimidazo [1,2-a] pyridinium perchlorate (Ro 03-5580) as yellow needles, m.p. 192-193°C decomp. In a similar way, 2-ethylamino-5-nitropyridine, m.p. 119-121°C from benzene or ether, was converted into I-ethyl-2,3-dihydro-6-nitroimidazo [1,2-a] pyridinium perchlorate (Ro 03-5637/001), recrystallized from water as yellow needles, m.p. 160-161°C. These new compounds were fully characterized by means of IR, NMR and UV spectroscopy and elemental analysis. Paranitroacetophenone (PNAP) was obtained from B.D.H.
02
O
02N
,CH CH
Li~~~~
Ro 03-5580
Ro 03-5637 RESULTS
0
a)Ec -C 0)
FIG. 1.-Dependence of enhancement ratio for sensitization of hypoxic S. marce8cen8 on nitropyridinium compound concentration.
When anoxic suspensions of Serratia marcescens were irradiated in the presence of the nitropyridinium compounds sensitization was observed. The survival curves were straight and the enhancement ratios obtained at various concentrations of the compounds are presented in Fig. 1. The plot for both compounds is biphasic, an
Pulse radiolysis was used to determine the one-electron reduction potentials of the nitropyridinium compounds using benzylviologen (E71 --354 mV) as the reference standard. These values are presented in the table together with octan1-ol: water partition data.
10-1 c 0
TABLE .5
log P Compound E71/mV Ro 03-5580 -335 -2-33 Ro 03--5637 -356 -2 -64 PNAP -355* +1 49t * Meisel and Neta, 1975; t Leo, Hansch and Elkins, 1971.
Sensitization by nitropyridinium compounds Anoxic toxicity limited the maximum concentration of Ro 03-5580 used to study
enhancement ratio as a function of sensitizer concentration whereas Ro-03-5637 is non-toxic up to its solubility limit. No sensitization of aerobic cells was observed in the presence of either compound.
i2'
.
104
8 10 12 14 16 Dose / krad FIG. 2.-The effect of combining PNAP with the nitropyridiniumn compounds on the sensitization of hypoxic S. marce8cen,. 0 = 02 bubbled, O = N2 bubbled, V = PNAP (1.5 mmol/dm3), O = Ro 035580 (250 ,tmol/dm3), A = Ro 03-5637 (750 ,umol/dm3), C = V + D, A = 2
V + A.
4
6
SENSITIZATION BY NITROPYRIDINIUM COMPOUNDS
initial plateau at 1-5 ± 0-1 rising at higher concentrations to the full oxygen enhancement level. Ro 03-5580 effects sensitization above the plateau level at lower concentrations than for Ro 03-5637. This compound also exhibits sensitization above the oxygen enhancement level. Combination of sensitizers Sensitization of mammalian cells by PNAP has been shown to reach a plateau at part of the oxygen sensitization level (Adams et al., 1971). This sensitization plateau has been associated with one of the components of the oxygen effect (Adams et al., 1975). The sensitizing effect of combining sufficient of the nitropyridinium compounds to give plateau sensitization with PNAP (1.5 mmol/dm3) was studied (Fig. 2). The plateau in sensitization effected by PNAP (2.1 + 0-1) is reached at this concentration and in both experiments the addition of the nitropyridinium compounds did not increase the level of sensitization. In another experiment a lower concentration of PNAP (100 ,tmol/dm3) sufficient to give an enhancement ratio of 1-4 was added to Ro 03-5580 (250 ,tmol/dm3). In this case combining the sensitizers produced an additive effect, giving an enhancement ratio of 2-0 ± 041. DISCUSSION
The positive charge carried by the nitropyridinium compounds and their high partitioning into the aqueous phase probably results in the formation of a concentration gradient at the cell surface. The plateau at an enhancement ratio of 1*5 ± 0*1 is lower than that found with the bipyridinium compounds where a plateau is seen at 2. 1 ± 0.1 (Anderson and Patel, 1977). Differences in chemical structure between the two classes of compounds may determine the maximum concentration each can achieve at a particular site. Unlike the dicationic form of the bipyridinium compounds, sensitization above
105
the plateau level is seen for the nitropyridinium compounds. This result supports the contention that the nitropyridinium compounds do penetrate into the cell. The more electron-affinic compound, Ro 03-5580, is the more efficient compound at effecting sensitization above the plateau level. This correlation of one-electron reduction potential with sensitization efficiency has been reported for both mammalian cells (Adams et al., 1976) and bacteria (Anderson and Patel, 1977). Sensitization levels effected by PNAP and by the nitropyridinium compounds are additive up to an enhancement level of 241 ± 0.1. Above this level no additivity in sensitization is seen, showing that PNAP and the nitropyridinium compounds sensitize the same site. The maximum sensitization possible at this site corresponds to approximately 40% of the overall oxygen enhancement level. We would like to thank Mr D. Brown for technical assistance in the preparation of the compounds. This work is financially supported by the Cancer Research Campaign. REFERENCES ADAMS, G. E., ASQUITH, J. C., DEWEY, D. L., FOSTER, J. L., MICHAEL, B. D. & WILLSON, R. L. (1971) Electron-affinic Sensitization II. ParaNitroacetophenone: A Radiosensitizer for Anoxic Bacterial and Mammalian Cells. Int. J. Radiat. Biol., 19, 575. ADAMS, G. E., MICHAEL, B. D., ASQUITH, J. C., SHENOY, M. A., WATTS, M. E. & WHILLANS, D. W. (1975) Biomedical, Chemical and Physical Perspectives, In Radiation Research. Ed., NYGGARD, 0. F., ADLER, H. I. & SINCLAIR, W. K., New York: Academic Press, Inc. p. 478. ADAMS, G. E., FLOCKHART, I. R., SMITHEN, C. E., STRATFORD, I. J., WARDMAN, P. & WATTS, M. E. (1976) Electron-affinic Sensitization VII. A Correlation between Structures, One-electron Reduction Potentials and Efficiencies of Nitroimidazoles as Hypoxic Cell Radiosensitizers. Radiat. Res., 67, 9. ANDERSON, R. F. & PATEL, K. B. (1977) Radiosensitization of Serratia mtarcescens by Bipyridiniuim Compounds. Int. J. Radiat. Biol., 32, 471. EP, E. R., WEISS, H., KEssARIs, N. D., SANTOMASSO, A., HESLIN, J. & LING, C. C. (1973) Oxygen Diffusion Times in Bacterial Cells Irradiated with High-intensity Pulsed Electrons: New Upper Limit to the Lifetime of Oxygen-sensitive Species Suspected to be Induced at Critical Sites in Bacterial Cells. Radiat. Res., 54, 171.
106
R. F. ANDERSON, K. B. PATEL AND C. B. SMITHEN
JONES, R. W. & GARLAND, P. B. (1977) Sites and Specificity of the Reaction of Bipyridinium Compounds with Anaerobic Respiratory Enzymes of E.scherichia coli. Biochem. J., 164, 199. LEO, A., HANSCH, C. & ELKINs, D. (1971) Partition Coefficients and their Uses. Chenmical Reviews, 71, 525. MANGINI, A. (1937)Nitro Derivatives, XIV. Substituted Nitropyridylphenylamines. Ricerca Sci., 8, 427. MEISEL, D. & NETA, P. (1975) One-electron Redox Potentials of Nitro Compounds and Radiosen-
sitizers. Correlation with Spin Densities of their Radical Anions. J. Amer. Chem. Soc., 97, 5198. SHENOY, M. A., AsQUITH, J. C., ADAMS, G. E., MICHAEL, B. D. & WATTs, M. E. (1975) Timeresolved Oxygen Effects in Irradiated Bacteria and Mammalian Cells: A Rapid-mix Study. Radiat. Res., 62, 498. TALLENTIRE, A., JoN-E, A. B. & JACOBS, G. P. (1972) The Radiosensitizing Actions of Ketonic Agents and Oxygen in Bacterial Spores Suspended in Aqueous and Nonaqueous Milieu. Molecular Basis of Radiation Biology. 24th Farkas Memorial Symposium, Jerusalem. I8r. J. Chem., 10, 1185.
