J. Photo&em.
Photobiol.
B: BioL, 8 (1991) 371-383
371
Synergism between electricity and ionizing radiation M. A. M. Capella”, M. E. F. Fonsecab and S. Menezes”’ ’ Ynstituto de Bi&sica Carlos Chagas Filho, Centro de G&&as da SaGd.e, UFR.J, 21944 - Rio de Janeiro, RJ (Zkizil) bSetor de Microscopia ElectrGnica, Insttitio d.e Microbiologia, Centro o!e CWias da SaGd.e, UFRJ, 21941 - Rio de Janeiro, RJ (Brazil) (Received February 13, 1990; accepted June 25, 1990)
Keywords. Radiotherapy, X-rays, p particles, ultraviolet (UV), DNA breaks, electricity.
Abstract Weak direct electric currents which produce little (or no) lethal damage to Escherkhia coli bacteria are shown to act synergistically with ionizing radiation, both electromagnetic radiation (X-ray) and charged particles (p radiation). This synergism greatly enhances the lethal effect of ionizing radiation on bacteria. This is possibly due to increased singlestrand breaks in DNA, as detected by the alkaline sucrose gradient method. It is also shown that in cells with thymidine-3H incorporated into their DNA and treated with electricity, the radioactivity is released from the acid-insoluble fraction to the acid-soluble fraction, so that the ratio of radioactivity in the soluble fraction to that in the insoluble fraction increases from 0.47 in the non-treated control cells to 3.46 in the cells treated with an electric current of 1 .O mA (3.0 V) for 30 min, which indicates extensive degradation of cellular DNA. No synergism is detected between electricity and 254 nm UV radiation nor between electricity and X-rays, when these two agents are used sequentially in any order. Electricity alone produces lesions in cell membranes, as shown by electron microscopy.
1. Introduction Despite the progress in radiotherapy, a signi6cant number of malignant tumours cannot be controlled by ionizing radiation, due chiefly to the limits of tolerance of normal tissue to high doses of radiation. To overcome the limitation of radiation therapy in the treatment of some tumours, studies have been conducted on the utilization of the combined action of radiation and other agents, based on the concepts of additivity, synergism, antagonism, sensitization and protection. These agents may be physical, such as other types of ionizing radiation [ 1, 21, UV light [ 3-51, temperature [ 6-31, magnetic +Author to whom correspondence should be addressed.
loll-1344/91/$3.50
Q Elsevier Sequoia/Printed in The Netherlands
372
fields [9, lo] or ultrasound [ 111, or chemical, such as inorganic [ 12, 131 and organic [ 14, 15) compounds, and chemotherapeutic drugs [16-181. In recent studies, we have demonstrated that in terms of survival there is a strong synergism between the photodynamic action of methylene blue and weak direct electric currents (d.c.) in E. colTi [19]. In an attempt to explain this synergism, we presented the hypothesis that the action of the electric current on the charged particles (ions and free radicals) produced in the cells by the photodynamic action could account for the interactions described. If this assumption is correct, a similar interaction is expected between electricity and ionizing radiation, since the latter can be quite efficient in producing charged particles in cells. This paper is a report of the results of preliminary experiments in which E. coli cells were treated with electricity concomitantly with X-rays or p particles emitted by 3H, 14C or 35S.
2. Materials
and methods
2.I. Chemicals
Thymidine-methyL3H (2 Ci nunol-‘), thymidine-2-14C (50 mCi nunol-‘) and uraciL3H (20-30 Ci nunol-‘) were obtained from New England Nuclear. Methionine-35S (38 mCi ml-‘) was prepared in our institute. 2.2. Cells The strains of E. coli used and their nutritional requirements are listed
in Table 1. 2.3. Growth media
Stock bacteria were maintained as individual colonies in Petri dishes containing a BT medium solidified by agar and stored at 4-6 “C. For the experiments, one of these colonies was inoculated either into liquid BT or TABLE 1 Strains of E. coli and their nutritional requirements E. coli strains
K12 K12 K12 K12 K12 K12
-1157 AR2463 AR1886 -2480 JG113 (W3110) JG112 (P3478)
Characteristics Repair
Nutritional requirements
Wild-type recA13 uv-?-Al6 uwA16, recA13 Wild-type pozA1
Thr, leu, thi, his, arg, pro As AB1167 As AB1167 As AR1157 nur, niac As w3110
Thr, threonine; leu, leucine; thi, thiamine; his, hi&dine; arg, arginine; pro, proline; thy, thymidine; niac, niacin.
M9 supplemented with casaminoacids (2.5 mg ml-‘), glucose (2 mg ml-‘) and thiamin (10 pg ml- ‘). These cultures were grown overnight at a temperature of 37 “C with shaking. The next day, the cultures were diluted 1:40 into a fresh medium and incubated to reach approximately 1 X 10’ cells ml- ‘. In the experiments using thymidine-3H or thymidine-14C, these bases were introduced into the growth medium with activities of 10 PCi ml-’ and 5 PCi ml-’ respectively. In these cases, deoxyadenosine (250 pg ml- ‘) was added to improve thymidine incorporation into the DNA. Uracil-3H and methionine-35S were added to the medium with activities of 10 &i ml-’ and 2 &i ml- ’ respectively. The labelling time was 3 h for all four compounds. 2.4. Irradiation with X-rays To irradiate the cells with X-rays, a Machlet Laboratories AEG 50 tube operating at 20.4 kV and 15 mA was used. Dosimetry was carried out by the F’ricke chemical dosimeter method [ 201. The dose rate applied to the cells was 0.15 Gy s-l. In the figures, X-ray doses are designated as “minutes of irradiation” to account for the time of application of electricity (stated in the figure captions). To determine the X-ray dose at each point of the curve, the time of exposure (s) is multiplied by 0.15. 2.5. Treatment with electricity Electrical treatment of the suspensions of bacteria was performed in a methylmethacrylate cell (3 cm X 1 cm X 1 cm) fitted with platinum electrodes placed face to face to ensure a homogeneous distribution of the electric field. The d.c. source was a 6 V Ray-o-Vat battery, series coupled to a multimeter (SH-105, Shimizu Industry Co.) and a potentiometer to adjust the trans-sample current. 2.6. Single-strand breaks Single-strand breaks of DNA were detected by the alkaline sucrose gradient method described by McGrath and Williams [21] but with minor modifications. To quantify the loss of radioactivity from the acid-insoluble fraction as it passed to the acid-soluble fraction, AR1 157 bacteria were grown in M9 media supplemented as described above, plus thymldlne-3H (10 PCi ml-‘). The cells were then treated with an electric current (d.c., 1.0 m4, 3 V); aliquots (50 ~1) were taken every 5 min and added to 150 ~1 of a lysing solution. After 30 min of incubation at ambient temperature, 500 ,ul of cold TCA (10%) was added and the solution was incubated on ice for an additional 10 min. The solution was then filtered through a Millipore borosilicate microfibre glass filter membrane (AR 15 type) and the acid-soluble fraction was collected in separate vials. An aliquot of this fraction (200 ~1) was added to vials containing 10 ml of a mixture of scintillation solution and Triton X-100 (2: 1) to measure radioactivity. The membranes were placed in vials containing 3 ml of the scintillation solution to determine the amount of radioactivity in the acid-insoluble fraction.
374
A Beckman LS-250 liquid scintihation counter was used to determine the radioactivity counts. 2.7. Irradiation with 254 nm UV light Exponentially growing E. coli AI31157 bacteria were irradiated with 254 nm UV Iight from a GE 30 W germicidal lamp (G30TS). UV dosimetry was performed using a Latarjet meter. A dose rate of 12.5 J me2 min-’ was used to permit concomitant treatment with UV and electricity, since the latter required times of 2-8 min. 2.8. Electron microscopy 2.8.1. Negative staining Stationary phase cells were negatively stained with a 4% aqueous solution of phosphotungstic acid (PTA) and uranyl acetate in 300 mesh copper grids previously covered with Formvar and carbon. 2.8.2. Ultrathin sections The ceI.Is (1 X 10’ ml- ‘) were centrifuged and resuspended in a solution of 1.25% ghrtaraldehyde plus 0.05 M CaC12in sodium cacodylate buffer and incubated for 1 h for pre-fixation. At the end of this period, the cells were fixed with 2.5% gIutaraIdehyde for 2 h, and then post-fixed in 1% osmium tetroxide for 2 h. After fixation, the ceils were embedded in 1.5% agar, dehydrated and embedded in PoIilyte 8001 resin. Ultrathin sections were prepared with a diamond knife in an LKB uhramicrotome, collected in 300 mesh copper grids, counter-stained with many1 acetate and lead citrate, and examined under a Philips EM-301 electron microscope.
3. Results and discussion 3.1. Bacterial survival ccfter combined electricity and X-ray treatment To verify the existence of an interaction between electricity and X-rays, similar to that found between electricity and methylene blue photodynamic action [ 191, bacteria were irradiated with X-rays and treated simuItaneously with weak direct electric currents. Figure 1 shows the synergism between X-rays and electricity when applied concomitantly to E. coli K12 AB1157 (wild-type), AI31886 (uvrAl6), AI32463 (recA13) and Al32480 (uvrAl6, recA13). It can be seen that the synergism is very marked in strains AB1157 and AB1186, which contain the recA gene product, but weak in the other two strains, which lack this repair system. Figure 2 shows that the synergism is also very strong in isogenic strains W3110 (wild-type) and P3478 (PO&U). These results are difhcult to interpret, since they indicate that the, presence of the recA gene product has an important role in the synergism, determining its extent in AI31157 and AI31886 strains (Fig. l), whereas the absence of
375
Fig. 1. Synergism between X-rays and electricity: l , electricity; v, X-ray; W, electricity and X-ray simultaneously. Cells were grown in BT medium, centrifuged, washed and resuspended in phosphate-buffered saline (PBS). Thii suspension (1.5 ml) was X irradiated and treated with a direct electric current (1.0 mA, 3 v) in a methylmethacrylate cell fitted with platinum electrodes. The X-ray dose rate was 0.15 Gy s-‘.
the polA1 gene product determines a higher degree of synergism in strain P3478 than in strain W3 110 (wild-type). Since all six strains are almost equally sensitive to electricity under the conditions used in these experiments and since single-strand breaks of DNA represent one of the predominant damaging effects of X-rays, we may expect that the synergism is mediated by the increase in single-strand breaks induced in DNA by X-rays in the presence of electricity, and that, in the absence of the recombination repair system or polymerase 1, the synergism will be greater than that in the wild-type strain. Figure 2 shows that the presence or absence of the polA1 gene product fits this rationale, but the same is not true of the recA gene. We can offer no explanation for the weak synergism between electricity and X-rays in the strains deficient in the recA gene product. When electricity is applied immediately after X-rays (or vice versa) no synergism is detected.
376
TIME
(MINI
Fig. 2. Synergism between X-rays and electricity. Growth conditions, treatment and symbols are the same as in Fig. 1.
3.2. Bacterial survival Q&T combined electricity and p particle treatment Figure 3 shows the interaction between electricity and /3 particles emitted by radioisotopes incorporated into the DNA (3H, 14C), RNA CH) or proteins (35S) in strain Al31 157. The cells treated only with electricity for 8 ruin have a lower survival rate (Fig. 3) than similar cells submitted to the treatment shown in Fig. 1. This is because the cells used in the experiments represented in Fig. 1 were grown in complete BT medium, whereas those in Fig. 3 were grown in an M9 mineral medium. The difference in sensitivity to electricity according to the growth medium used (data not shown) is consistently observed. However, without a mineral medium the incorporation of radioactively labelled bases into the DNA is not possible. Under our experimental conditions, no synergism is observed between electricity and p particles emitted from 35S incorporated into proteins. For uraciL3H incorporated into RNA, an enhancement of the lethality induced by electricity alone can be seen, indicating a weak synergism. However, the synergism between electricity and 6 radiation emitted from radionuclides incorporated into the DNA is very marked. It is interesting to note that the synergism is greater with 3H (energy of the p particle, 0.018 MeV) than with 14C(energy of the p particle, 0.156 MeV). This may reflect the fact that the less energetic beta particle emitted from 3H incorporated in the DNA cannot travel far from this molecule as it is rapidly absorbed, and thus produces ionization and charged particles
377
2
4
TIME
6
8
(MIN.)
Fig. 3. Synergism between p radiation and electricity: 0, electricity alone; A, electricity and p radiation from methionine-“5S; V, electricity and p radiation from uraciL3H; 0, electricity and p radiation from thymklme-I%; n , electricity and B radiation from thymidiie-3H. AEU157 was grown in M9 mineral medium, supplemented as described in the text, plus methionine35S, uracil-“H, thymidheJH or thymidine-14C. Electrical treatment was performed as described in Fig. 1 and the text.
in the vicinity of the DNA; in contrast the /? particle emitted by 14C (ten times more energetic), can certainly penetrate more deeply into cellular components, thus producing charged particles in sites far from the DNA. Coincidently, the synergism between electricity and 14C is about ten times less than the synergism between electricity and ‘H. It is worth noting that in these experiments, the numbers of disintegrations per minute (d.p.m.) calculated for 3H and 14C are almost the same (17 292 d.p.m. per 10’ cells for 3H and 15 269 d.p.m. per lo7 cells for 14C). Hence, the difference in disintegrations per minute cannot account for the difference in synergism. In an effort to find a molecular event that can account for the enhanced lethality induced by electricity, E. coli AH1 157 were grown in M9 medium containing thymidine-3H, treated or not with electricity and assayed for
378
6 moW3
9
12
18
16
21
FRACTION
24
27
30
TOP
Fig. 4. Sedimentation profile of the DNA of AB1157 bacteria in an alkaline sucrose gradient. The cells were grown as described in Fig. 3, with thymidine-3H, and treated (or not) with electricity to reach 0.04% survival: 0, control; 0, electricity and p radiation. Upper right: total counts detected in the acid-insoluble fraction of control cells (C) and electricity-treated cells (T).
survival and single-strand breaks. Figure 4 shows the sedimentation profiles of the control cells (100% survival) and the cells treated with electricity (0.04% survival). It is clear from this figure that the radioactive decay of 3H incorporated in the DNA structure, submitted to an electric current, induces single-strand breaks in the DNA. However, an alternative methodology different from the alkaline sucrose gradient technique is required to determine if electricity alone induces breaks in DNA. The latter seems unlikely if we compare the survival of cells with and without 3H in their DNA submitted to the same electrical treatment. Moreover, when cells containing radiolabelled DNA are treated with electricity, a considerable amount of radioactivity is released from the acid-insoluble fraction to the acid-soluble fraction, which indicates DNA degradation. In the experiment shown in Fig. 4, it is seen that the total amount of radioactivity incorporated in the cells (29 000 counts per minute (c.p.m.)) and retained in the acid-insoluble fraction falls to 15 300 c.p.m. after the electrical treatment (Fig. 4, upper right). As this observation was found repeatedly, an experiment was performed to verify the transfer of radioactivity from the acid-insoluble to the acid-soluble fraction after the electrical treatment of cells containing thymidine-3H in their DNA. Table 2 shows that the quantity released is proportional to the length of time the electricity is applied. The high number of counts seen in the acid-soluble fraction at time zero certainly reflects a large pool of non-incorporated thymidine-3H. Finally, it should be noted that the synergistic effect between electricity and 0 particles is proportional to the radioactivity incorporated
379 TABLE 2 Quantity of radioactivity released from the acid-insoluble fraction to the acid-soluble fraction as a function of time of electrical treatment Time of electrical treatment (mW 0
5 10 15 20 25 30
Acid-soluble (S) fraction (c.p.m.)
Acid-insoluble (I) fraction (c.p.m.)
s+I
SII
7812 9562 10969 13706 15498 16695 20258
16796 12828 10648 9984 8664 6332 5850
24608 22390 21617 23690 24162 23027 26108
0.47 0.75 1.03 1.37 1.79 2.64 3.46
in the DNA, i.e. for any given length of time in which the electricity is applied, the death of radiolabelled bacteria increases proportionally to the increasing incorporation of 3H (data not shown). 3.3. Interaction between electricity and 254 nm UV light To explain the synergism between electricity and ionizing radiation, we have supposed that if the cell is traversed by an electric current during the formation of charged particles by a certain treatment, the current will protect of these charged particles by preventing their against the “quenching” recombination with one another. These particles, by being attracted to opposite directions, will function as “projectiles” triggered from the interior of the cells, thus enhancing the possibility of collisions with other molecules in the cells and the production of lesions. If this rationale is correct, agents which inactivate cells without producing charged particles will not be synergistic with electricity. To verify this hypothesis, E. coli cells were treated with UVC (254 run) light alone (which inactivates the cells by mechanisms that do not include the formation of charged particles) or with the simultaneous application of electricity. Figure 5 shows that there is no synergism between electricity and UV radiation. The slight difference in the survival curves for UV alone and UV plus electricity is certainly due to the additive effect of the two treatments. This result confirms the hypothesis that the synergism is mediated by the action of the electric current on the charged particles formed in the cells by ionizing radiation. 3.4. Membrane damage induced by electric current In Figs. 1, 2 and 3 it can be seen that the synergism starts to manifest itself after 4 min of application of electricity, increasing rapidly after 6 mm. This seems to reflect the dielectric nature of the membrane, since, under our experimental conditions, a period of about 4 min is probably required for the applied electric current to overcome this barrier, passing through
Fig. 5. Interaction between electricity and WC light: A, electricity; 0,254 nm Uv; 0, electricity plus W. Exponentially growing E. coli -1157 were irradiated with 254 nm W light (12.5 J mm2 mix-‘) alone or treated simultaneously with electricity (1.0 mA, 3.0 V).
the cell and acting on the charged particles produced by X-rays or p radiation. If this is true, we may expect that some type of damage will be inflicted on the cell membrane by the electric current. To test this hypothesis, E. coli cultures were treated with electricity (10% survival) and examined under an electron microscope by negative staining and thin section technifiues. By comparing negatively-stained control cells (Fig. 6(A)) with cells treated with electricity (Fig. 6(B)), it can be seen that there is a strong penetration of the stain into the latter. In addition, by comparing thin-sectioned control cells (Fig. 6(C)) with treated cells (Fig. 6(D)), zones of disrupted membranes in the treated cells can be seen, as well as blebs, similar to those produced in E. coli membranes by heat treatment (55 “C) [22]. It is important to note that no signilicant rise in temperature is observed in cell suspensions even after 20 min of application of electricity. This result provides direct evidence of modifications induced in the cellular membrane by electricity, indicating that it is very likely that these alterations are essential for the occurrence of the synergisms reported here. The results presented in this work were obtained using bacteria; however, preliminary results with a mammalian cell line (P815, a mouse mastocytoma, ATCC TiB B64) indicate the existence of a strong synergism between Xrays and electricity in these cells. If the interactions between direct electric
381
Fig. 6. Electron micrographs of E. coli AB1157 treated or not with electricity (1.0 mA, 3 V for 10 min). Negatively-stained cells: (A) control (original magnification X 11 000); (B) cells treated with electricity (original magnification X 11 000). Thin-sectioned cells: (C) control (original magnification X25 000); (D) cells treated with electricity (original magnification x45 000). B, blebs; R, ruptured regions in the membrane.
currents and ionizing radiation can be shown to exist in mammalian cells, either in culture or in viwo,they may be able to play an important role in the radiotherapy of cancer, particularly since it would not be difficult to combine radiations with common electrotherapy procedures.
382
Acknowledgments
We are thankful to the following Brazilian agencies: Conselho National de Pesquisas; Funda@o de Amparo & Pesquisa do Estado do Rio de Janeiro; Comiss~o National de Energia Nuclear; and Conselho de En&no para Graduados da Universidade Federal do Rio de Janeiro.
References 1 F. Q. H. Ngo, A. Han and M. M. Elkind, On the repair of sub-lethal damage in V-79 Chinese hamster cells resulting from irradiation with fast neutrons or fast neutrons combined with X-rays, Int. J. Radiat. &i~l., 32 (1977) 507-511. 2 R. P. Bird, H. H. Rossi and N. Rohrlg, Cell inactivation by a mixture of high and low-LET radiations, Radiat. Res., 83 (1980) 469-470. 3 R. H. Raynes, Role of DNA repair mechanisms ln microbial inactivation and recovery phenomena, Photochem. Photobiol., 3 (1964) 429-450. 4 Y. Yan and S. Kondo, Synergistic effects of 32Pdecay and ultraviolet irradiation on inactivation of Salmonella, Radiat. Res., 22 (1964) 440-456. 5 A. Han and M. M. Elkind, Additive action of ionizing and non ionizing radiation throughout the Chinese hamster cell-cycle, ht. J. Radiut. Biol., 31 (1977) 275-282. 6 S. A. Sapareto, L. E. Hopwood and W. C. Dewey, Combined effects of X-irradiation and hyperthermia on CHO cells for various temperatures and orders of application, Radiat. Res., 73 (1978) 221-233. 7 M. L. Freeman, C. P. Raaphorst and W. C. Dewey, The relationship of heat IdIIing and thermal radiosensitization to the duration of heating at 42 “C, Radiut. Res., 78 (1979) 172-175. 8 G. P. Raaphorst, S. L. Romano, J. B. Mitchell, J. S. Bedford and W. C. Dewey, Intrinsic differences in heat and/or X-ray sensitivity of seven mammalian cell lines cultured and treated under identical conditions, Cancer Res., 39 (1979) 396-401. 9 R. Nath, R. J. Schulz and P. Bongiornl, Response of mammalian cells irradiated with 30 MeV X-rays in the presence of a uniform 20 kilogauss magnetic field, Int. J. Radiut. Biol., 38 (1980)
285-292.
10 D. J. Wordsworth, Comparative long-term effects of liver damage in the rat after (a) localized X-irradiation and (b) localized X-irradiation in the presence of a strong homogeneous magnetic field, Radiut. Res., 57 (1974) 442-450. 11 2. Harkanyi, J. Szollar and Z. Vigvari, A search for an effect of ultrasound alone and ln combination with X-rays on chromosomes in vivo, Br. J. Radial., 51 (1978) 46-49. 12 E. Ben-Hur and E. Rlls, Deuterium oxide enhancement of Chinese hamster cell response to gamma-radiation, Radiat. Res., 81 (1980) 224-235. 13 K. D. Held and E. L. Powers, Sensitization to X-rays of transforming DNA by Ag+, Int. J. Radiat. Biol., 38 (1980) 293-307. 14 J. C. Asquith, M. E. Watts, K. Patel, C. E. Smithen and G. E. Adams, Electron afIlnic sensitization. V. Radiosensitization of hypoxlc bacteria and mammalian cells in vitro by some nitroimidazoles and nitropyrazoles, Radiat. Res., 60 (1974) 108-l 18.
15 H. S. Kaplan, Radiosensitlzation by the halogenated pyrimidine analogues: laboratory and clinical investigations, in H. Moroson and M. Quintiliani (eds.), Radiation Protection and Sensitizution, Taylor and Francis, London, 1970, pp. 32-45. 16 T. L. Philips, Chemical modification of radiation effects, Cancer, 39 (1977) 987-999. 17 M. M. Elklnd, G. F. Whitmore and T. Alescio, Actlnomycin D: suppression of recovery in X-irradiated mammalian cells, Science, 143 (1964) 1454-1456. 18 T. L. Philips, M. D. Wharam and L. W. Margoles, Modification of radiation @iury to normal tissues by chemotherapeutic drugs, Cancer, 35 (1975) 1678-1684.
383
19 M. A. M. Capella and S. Menezes, Synergism between electricity and methylene blue photodynamic action, Int. J. Radiat. Oncol., Bid., Phys., submitted for publication. 20 N. Fricke and S. Morse, The chemical action of roentgen rays on diluted ferrous sulphate solutions as a measure of dose, Am. J. Roetngenol., 19 (1927) 426-430. 21 R. A. McGrath and R. W. Wiiiams, Reconstruction in wivo of irradiated E. coli deoxyribonucleic acid; the rejoining of broken pieces, Nature, 212 (1966) 534-535. 22 N. Katsui, T. Tsuchido, R. Hiramatzu, S. Fqjikawa, M. Takano and I. Shibasaki, Heat induced blebbing and vesiculation of the outer membrane of Escherichia coli, J. Bacterial., I51 (1982) 1523-1531.
Photobiol.
B: BioL, 8 (1991) 371-383
371
Synergism between electricity and ionizing radiation M. A. M. Capella”, M. E. F. Fonsecab and S. Menezes”’ ’ Ynstituto de Bi&sica Carlos Chagas Filho, Centro de G&&as da SaGd.e, UFR.J, 21944 - Rio de Janeiro, RJ (Zkizil) bSetor de Microscopia ElectrGnica, Insttitio d.e Microbiologia, Centro o!e CWias da SaGd.e, UFRJ, 21941 - Rio de Janeiro, RJ (Brazil) (Received February 13, 1990; accepted June 25, 1990)
Keywords. Radiotherapy, X-rays, p particles, ultraviolet (UV), DNA breaks, electricity.
Abstract Weak direct electric currents which produce little (or no) lethal damage to Escherkhia coli bacteria are shown to act synergistically with ionizing radiation, both electromagnetic radiation (X-ray) and charged particles (p radiation). This synergism greatly enhances the lethal effect of ionizing radiation on bacteria. This is possibly due to increased singlestrand breaks in DNA, as detected by the alkaline sucrose gradient method. It is also shown that in cells with thymidine-3H incorporated into their DNA and treated with electricity, the radioactivity is released from the acid-insoluble fraction to the acid-soluble fraction, so that the ratio of radioactivity in the soluble fraction to that in the insoluble fraction increases from 0.47 in the non-treated control cells to 3.46 in the cells treated with an electric current of 1 .O mA (3.0 V) for 30 min, which indicates extensive degradation of cellular DNA. No synergism is detected between electricity and 254 nm UV radiation nor between electricity and X-rays, when these two agents are used sequentially in any order. Electricity alone produces lesions in cell membranes, as shown by electron microscopy.
1. Introduction Despite the progress in radiotherapy, a signi6cant number of malignant tumours cannot be controlled by ionizing radiation, due chiefly to the limits of tolerance of normal tissue to high doses of radiation. To overcome the limitation of radiation therapy in the treatment of some tumours, studies have been conducted on the utilization of the combined action of radiation and other agents, based on the concepts of additivity, synergism, antagonism, sensitization and protection. These agents may be physical, such as other types of ionizing radiation [ 1, 21, UV light [ 3-51, temperature [ 6-31, magnetic +Author to whom correspondence should be addressed.
loll-1344/91/$3.50
Q Elsevier Sequoia/Printed in The Netherlands
372
fields [9, lo] or ultrasound [ 111, or chemical, such as inorganic [ 12, 131 and organic [ 14, 15) compounds, and chemotherapeutic drugs [16-181. In recent studies, we have demonstrated that in terms of survival there is a strong synergism between the photodynamic action of methylene blue and weak direct electric currents (d.c.) in E. colTi [19]. In an attempt to explain this synergism, we presented the hypothesis that the action of the electric current on the charged particles (ions and free radicals) produced in the cells by the photodynamic action could account for the interactions described. If this assumption is correct, a similar interaction is expected between electricity and ionizing radiation, since the latter can be quite efficient in producing charged particles in cells. This paper is a report of the results of preliminary experiments in which E. coli cells were treated with electricity concomitantly with X-rays or p particles emitted by 3H, 14C or 35S.
2. Materials
and methods
2.I. Chemicals
Thymidine-methyL3H (2 Ci nunol-‘), thymidine-2-14C (50 mCi nunol-‘) and uraciL3H (20-30 Ci nunol-‘) were obtained from New England Nuclear. Methionine-35S (38 mCi ml-‘) was prepared in our institute. 2.2. Cells The strains of E. coli used and their nutritional requirements are listed
in Table 1. 2.3. Growth media
Stock bacteria were maintained as individual colonies in Petri dishes containing a BT medium solidified by agar and stored at 4-6 “C. For the experiments, one of these colonies was inoculated either into liquid BT or TABLE 1 Strains of E. coli and their nutritional requirements E. coli strains
K12 K12 K12 K12 K12 K12
-1157 AR2463 AR1886 -2480 JG113 (W3110) JG112 (P3478)
Characteristics Repair
Nutritional requirements
Wild-type recA13 uv-?-Al6 uwA16, recA13 Wild-type pozA1
Thr, leu, thi, his, arg, pro As AB1167 As AB1167 As AR1157 nur, niac As w3110
Thr, threonine; leu, leucine; thi, thiamine; his, hi&dine; arg, arginine; pro, proline; thy, thymidine; niac, niacin.
M9 supplemented with casaminoacids (2.5 mg ml-‘), glucose (2 mg ml-‘) and thiamin (10 pg ml- ‘). These cultures were grown overnight at a temperature of 37 “C with shaking. The next day, the cultures were diluted 1:40 into a fresh medium and incubated to reach approximately 1 X 10’ cells ml- ‘. In the experiments using thymidine-3H or thymidine-14C, these bases were introduced into the growth medium with activities of 10 PCi ml-’ and 5 PCi ml-’ respectively. In these cases, deoxyadenosine (250 pg ml- ‘) was added to improve thymidine incorporation into the DNA. Uracil-3H and methionine-35S were added to the medium with activities of 10 &i ml-’ and 2 &i ml- ’ respectively. The labelling time was 3 h for all four compounds. 2.4. Irradiation with X-rays To irradiate the cells with X-rays, a Machlet Laboratories AEG 50 tube operating at 20.4 kV and 15 mA was used. Dosimetry was carried out by the F’ricke chemical dosimeter method [ 201. The dose rate applied to the cells was 0.15 Gy s-l. In the figures, X-ray doses are designated as “minutes of irradiation” to account for the time of application of electricity (stated in the figure captions). To determine the X-ray dose at each point of the curve, the time of exposure (s) is multiplied by 0.15. 2.5. Treatment with electricity Electrical treatment of the suspensions of bacteria was performed in a methylmethacrylate cell (3 cm X 1 cm X 1 cm) fitted with platinum electrodes placed face to face to ensure a homogeneous distribution of the electric field. The d.c. source was a 6 V Ray-o-Vat battery, series coupled to a multimeter (SH-105, Shimizu Industry Co.) and a potentiometer to adjust the trans-sample current. 2.6. Single-strand breaks Single-strand breaks of DNA were detected by the alkaline sucrose gradient method described by McGrath and Williams [21] but with minor modifications. To quantify the loss of radioactivity from the acid-insoluble fraction as it passed to the acid-soluble fraction, AR1 157 bacteria were grown in M9 media supplemented as described above, plus thymldlne-3H (10 PCi ml-‘). The cells were then treated with an electric current (d.c., 1.0 m4, 3 V); aliquots (50 ~1) were taken every 5 min and added to 150 ~1 of a lysing solution. After 30 min of incubation at ambient temperature, 500 ,ul of cold TCA (10%) was added and the solution was incubated on ice for an additional 10 min. The solution was then filtered through a Millipore borosilicate microfibre glass filter membrane (AR 15 type) and the acid-soluble fraction was collected in separate vials. An aliquot of this fraction (200 ~1) was added to vials containing 10 ml of a mixture of scintillation solution and Triton X-100 (2: 1) to measure radioactivity. The membranes were placed in vials containing 3 ml of the scintillation solution to determine the amount of radioactivity in the acid-insoluble fraction.
374
A Beckman LS-250 liquid scintihation counter was used to determine the radioactivity counts. 2.7. Irradiation with 254 nm UV light Exponentially growing E. coli AI31157 bacteria were irradiated with 254 nm UV Iight from a GE 30 W germicidal lamp (G30TS). UV dosimetry was performed using a Latarjet meter. A dose rate of 12.5 J me2 min-’ was used to permit concomitant treatment with UV and electricity, since the latter required times of 2-8 min. 2.8. Electron microscopy 2.8.1. Negative staining Stationary phase cells were negatively stained with a 4% aqueous solution of phosphotungstic acid (PTA) and uranyl acetate in 300 mesh copper grids previously covered with Formvar and carbon. 2.8.2. Ultrathin sections The ceI.Is (1 X 10’ ml- ‘) were centrifuged and resuspended in a solution of 1.25% ghrtaraldehyde plus 0.05 M CaC12in sodium cacodylate buffer and incubated for 1 h for pre-fixation. At the end of this period, the cells were fixed with 2.5% gIutaraIdehyde for 2 h, and then post-fixed in 1% osmium tetroxide for 2 h. After fixation, the ceils were embedded in 1.5% agar, dehydrated and embedded in PoIilyte 8001 resin. Ultrathin sections were prepared with a diamond knife in an LKB uhramicrotome, collected in 300 mesh copper grids, counter-stained with many1 acetate and lead citrate, and examined under a Philips EM-301 electron microscope.
3. Results and discussion 3.1. Bacterial survival ccfter combined electricity and X-ray treatment To verify the existence of an interaction between electricity and X-rays, similar to that found between electricity and methylene blue photodynamic action [ 191, bacteria were irradiated with X-rays and treated simuItaneously with weak direct electric currents. Figure 1 shows the synergism between X-rays and electricity when applied concomitantly to E. coli K12 AB1157 (wild-type), AI31886 (uvrAl6), AI32463 (recA13) and Al32480 (uvrAl6, recA13). It can be seen that the synergism is very marked in strains AB1157 and AB1186, which contain the recA gene product, but weak in the other two strains, which lack this repair system. Figure 2 shows that the synergism is also very strong in isogenic strains W3110 (wild-type) and P3478 (PO&U). These results are difhcult to interpret, since they indicate that the, presence of the recA gene product has an important role in the synergism, determining its extent in AI31157 and AI31886 strains (Fig. l), whereas the absence of
375
Fig. 1. Synergism between X-rays and electricity: l , electricity; v, X-ray; W, electricity and X-ray simultaneously. Cells were grown in BT medium, centrifuged, washed and resuspended in phosphate-buffered saline (PBS). Thii suspension (1.5 ml) was X irradiated and treated with a direct electric current (1.0 mA, 3 v) in a methylmethacrylate cell fitted with platinum electrodes. The X-ray dose rate was 0.15 Gy s-‘.
the polA1 gene product determines a higher degree of synergism in strain P3478 than in strain W3 110 (wild-type). Since all six strains are almost equally sensitive to electricity under the conditions used in these experiments and since single-strand breaks of DNA represent one of the predominant damaging effects of X-rays, we may expect that the synergism is mediated by the increase in single-strand breaks induced in DNA by X-rays in the presence of electricity, and that, in the absence of the recombination repair system or polymerase 1, the synergism will be greater than that in the wild-type strain. Figure 2 shows that the presence or absence of the polA1 gene product fits this rationale, but the same is not true of the recA gene. We can offer no explanation for the weak synergism between electricity and X-rays in the strains deficient in the recA gene product. When electricity is applied immediately after X-rays (or vice versa) no synergism is detected.
376
TIME
(MINI
Fig. 2. Synergism between X-rays and electricity. Growth conditions, treatment and symbols are the same as in Fig. 1.
3.2. Bacterial survival Q&T combined electricity and p particle treatment Figure 3 shows the interaction between electricity and /3 particles emitted by radioisotopes incorporated into the DNA (3H, 14C), RNA CH) or proteins (35S) in strain Al31 157. The cells treated only with electricity for 8 ruin have a lower survival rate (Fig. 3) than similar cells submitted to the treatment shown in Fig. 1. This is because the cells used in the experiments represented in Fig. 1 were grown in complete BT medium, whereas those in Fig. 3 were grown in an M9 mineral medium. The difference in sensitivity to electricity according to the growth medium used (data not shown) is consistently observed. However, without a mineral medium the incorporation of radioactively labelled bases into the DNA is not possible. Under our experimental conditions, no synergism is observed between electricity and p particles emitted from 35S incorporated into proteins. For uraciL3H incorporated into RNA, an enhancement of the lethality induced by electricity alone can be seen, indicating a weak synergism. However, the synergism between electricity and 6 radiation emitted from radionuclides incorporated into the DNA is very marked. It is interesting to note that the synergism is greater with 3H (energy of the p particle, 0.018 MeV) than with 14C(energy of the p particle, 0.156 MeV). This may reflect the fact that the less energetic beta particle emitted from 3H incorporated in the DNA cannot travel far from this molecule as it is rapidly absorbed, and thus produces ionization and charged particles
377
2
4
TIME
6
8
(MIN.)
Fig. 3. Synergism between p radiation and electricity: 0, electricity alone; A, electricity and p radiation from methionine-“5S; V, electricity and p radiation from uraciL3H; 0, electricity and p radiation from thymklme-I%; n , electricity and B radiation from thymidiie-3H. AEU157 was grown in M9 mineral medium, supplemented as described in the text, plus methionine35S, uracil-“H, thymidheJH or thymidine-14C. Electrical treatment was performed as described in Fig. 1 and the text.
in the vicinity of the DNA; in contrast the /? particle emitted by 14C (ten times more energetic), can certainly penetrate more deeply into cellular components, thus producing charged particles in sites far from the DNA. Coincidently, the synergism between electricity and 14C is about ten times less than the synergism between electricity and ‘H. It is worth noting that in these experiments, the numbers of disintegrations per minute (d.p.m.) calculated for 3H and 14C are almost the same (17 292 d.p.m. per 10’ cells for 3H and 15 269 d.p.m. per lo7 cells for 14C). Hence, the difference in disintegrations per minute cannot account for the difference in synergism. In an effort to find a molecular event that can account for the enhanced lethality induced by electricity, E. coli AH1 157 were grown in M9 medium containing thymidine-3H, treated or not with electricity and assayed for
378
6 moW3
9
12
18
16
21
FRACTION
24
27
30
TOP
Fig. 4. Sedimentation profile of the DNA of AB1157 bacteria in an alkaline sucrose gradient. The cells were grown as described in Fig. 3, with thymidine-3H, and treated (or not) with electricity to reach 0.04% survival: 0, control; 0, electricity and p radiation. Upper right: total counts detected in the acid-insoluble fraction of control cells (C) and electricity-treated cells (T).
survival and single-strand breaks. Figure 4 shows the sedimentation profiles of the control cells (100% survival) and the cells treated with electricity (0.04% survival). It is clear from this figure that the radioactive decay of 3H incorporated in the DNA structure, submitted to an electric current, induces single-strand breaks in the DNA. However, an alternative methodology different from the alkaline sucrose gradient technique is required to determine if electricity alone induces breaks in DNA. The latter seems unlikely if we compare the survival of cells with and without 3H in their DNA submitted to the same electrical treatment. Moreover, when cells containing radiolabelled DNA are treated with electricity, a considerable amount of radioactivity is released from the acid-insoluble fraction to the acid-soluble fraction, which indicates DNA degradation. In the experiment shown in Fig. 4, it is seen that the total amount of radioactivity incorporated in the cells (29 000 counts per minute (c.p.m.)) and retained in the acid-insoluble fraction falls to 15 300 c.p.m. after the electrical treatment (Fig. 4, upper right). As this observation was found repeatedly, an experiment was performed to verify the transfer of radioactivity from the acid-insoluble to the acid-soluble fraction after the electrical treatment of cells containing thymidine-3H in their DNA. Table 2 shows that the quantity released is proportional to the length of time the electricity is applied. The high number of counts seen in the acid-soluble fraction at time zero certainly reflects a large pool of non-incorporated thymidine-3H. Finally, it should be noted that the synergistic effect between electricity and 0 particles is proportional to the radioactivity incorporated
379 TABLE 2 Quantity of radioactivity released from the acid-insoluble fraction to the acid-soluble fraction as a function of time of electrical treatment Time of electrical treatment (mW 0
5 10 15 20 25 30
Acid-soluble (S) fraction (c.p.m.)
Acid-insoluble (I) fraction (c.p.m.)
s+I
SII
7812 9562 10969 13706 15498 16695 20258
16796 12828 10648 9984 8664 6332 5850
24608 22390 21617 23690 24162 23027 26108
0.47 0.75 1.03 1.37 1.79 2.64 3.46
in the DNA, i.e. for any given length of time in which the electricity is applied, the death of radiolabelled bacteria increases proportionally to the increasing incorporation of 3H (data not shown). 3.3. Interaction between electricity and 254 nm UV light To explain the synergism between electricity and ionizing radiation, we have supposed that if the cell is traversed by an electric current during the formation of charged particles by a certain treatment, the current will protect of these charged particles by preventing their against the “quenching” recombination with one another. These particles, by being attracted to opposite directions, will function as “projectiles” triggered from the interior of the cells, thus enhancing the possibility of collisions with other molecules in the cells and the production of lesions. If this rationale is correct, agents which inactivate cells without producing charged particles will not be synergistic with electricity. To verify this hypothesis, E. coli cells were treated with UVC (254 run) light alone (which inactivates the cells by mechanisms that do not include the formation of charged particles) or with the simultaneous application of electricity. Figure 5 shows that there is no synergism between electricity and UV radiation. The slight difference in the survival curves for UV alone and UV plus electricity is certainly due to the additive effect of the two treatments. This result confirms the hypothesis that the synergism is mediated by the action of the electric current on the charged particles formed in the cells by ionizing radiation. 3.4. Membrane damage induced by electric current In Figs. 1, 2 and 3 it can be seen that the synergism starts to manifest itself after 4 min of application of electricity, increasing rapidly after 6 mm. This seems to reflect the dielectric nature of the membrane, since, under our experimental conditions, a period of about 4 min is probably required for the applied electric current to overcome this barrier, passing through
Fig. 5. Interaction between electricity and WC light: A, electricity; 0,254 nm Uv; 0, electricity plus W. Exponentially growing E. coli -1157 were irradiated with 254 nm W light (12.5 J mm2 mix-‘) alone or treated simultaneously with electricity (1.0 mA, 3.0 V).
the cell and acting on the charged particles produced by X-rays or p radiation. If this is true, we may expect that some type of damage will be inflicted on the cell membrane by the electric current. To test this hypothesis, E. coli cultures were treated with electricity (10% survival) and examined under an electron microscope by negative staining and thin section technifiues. By comparing negatively-stained control cells (Fig. 6(A)) with cells treated with electricity (Fig. 6(B)), it can be seen that there is a strong penetration of the stain into the latter. In addition, by comparing thin-sectioned control cells (Fig. 6(C)) with treated cells (Fig. 6(D)), zones of disrupted membranes in the treated cells can be seen, as well as blebs, similar to those produced in E. coli membranes by heat treatment (55 “C) [22]. It is important to note that no signilicant rise in temperature is observed in cell suspensions even after 20 min of application of electricity. This result provides direct evidence of modifications induced in the cellular membrane by electricity, indicating that it is very likely that these alterations are essential for the occurrence of the synergisms reported here. The results presented in this work were obtained using bacteria; however, preliminary results with a mammalian cell line (P815, a mouse mastocytoma, ATCC TiB B64) indicate the existence of a strong synergism between Xrays and electricity in these cells. If the interactions between direct electric
381
Fig. 6. Electron micrographs of E. coli AB1157 treated or not with electricity (1.0 mA, 3 V for 10 min). Negatively-stained cells: (A) control (original magnification X 11 000); (B) cells treated with electricity (original magnification X 11 000). Thin-sectioned cells: (C) control (original magnification X25 000); (D) cells treated with electricity (original magnification x45 000). B, blebs; R, ruptured regions in the membrane.
currents and ionizing radiation can be shown to exist in mammalian cells, either in culture or in viwo,they may be able to play an important role in the radiotherapy of cancer, particularly since it would not be difficult to combine radiations with common electrotherapy procedures.
382
Acknowledgments
We are thankful to the following Brazilian agencies: Conselho National de Pesquisas; Funda@o de Amparo & Pesquisa do Estado do Rio de Janeiro; Comiss~o National de Energia Nuclear; and Conselho de En&no para Graduados da Universidade Federal do Rio de Janeiro.
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