INT. J . HYPERTHERMIA,

1992,

VOL.

8,

NO.

5 , 689-699

Lethal interaction between heat and methylene blue in Escherichia coli S . MENEZESt and P. TEIXEIRA Instituto de Biofisica Carlos Chagas Filho. Centro de Ciencias da Saude. Universidade Federal do Rio de Janeiro. 21.949, Rio de Janeiro, RJ-Brazil

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(Received 1 I December 1990; revised 28 October I991 and I0 March I992; accepted 17 March 1992) Hyperthermia treatment is shown to act synergistically with methylene blue (MB), from the end point of lethality in Gram-negative Escherichia coli bacteria. That this lethality is correlated to the damage produced in DNA by the dye is deduced from the fact that bacteria differing in capacity for repair are almost equally sensitive to heat, but differ considerably in sensitivity to concomitant heat and dye treatment. It is demonstrated that the damage is repairable by the excision-repair system. The role of temperature seems to be that of facilitating the incorporation of the dye, which enables the latter to intercalate into the DNA. Ability of the outer membrane of E. coli ABI 157 bacteria to act as a barrier to the penetration of MB remains almost intact up to 46"C, but above this temperature it seems to disrupt abruptly (but reversibly), leading to inactivation of the cells by the dye. Since hyperthermia is in current use for the treatment of cancer, it is suggested that if this synergism also exists in mammalian cells, MB could eventually be used independently of its photodynamic action as an adjuvant in cancer therapy.

Key words: Hyperthermia. inethylene blue, synergism, cell lethality

1. Introduction The observation that hyperthermia could be an effective agent in causing tumour regression and could, therefore, have the potential for use in the treatment of cancer was made many years ago (Overgaard and Overgaard 1972. Suit and Shwayder 1974). It has been demonstrated that heat is a radiosensitizer (Stewart and Gibbs 1984, Yerushalmi 1975, Stewart and Denekamp 1978, Sapareto et al. 1978), chemosensitizer (Hahn et al. 1975, 1977, Hahn 1979, Marmor 1979) and an interactant in the photodynamic therapy of cancer (Waldow and Dougherty 1984, Mang and Dougherty 1985). These properties have led to the use of hyperthermia as an adjuvant in other cancer therapies. Methylene blue (MB) is a thiazin dye with stgrong photodynamic properties. In contact with cells, both pro- and eucaryotic, it sensitizes them to the action of visible light, chiefly in the red band of the electromagnetic spectrum. MB, like other dyes, has been shown to accumulate and be retained in some malignant tissues more efficiently than in normal ones (Fukui e? a/. 1977, Lavelle 1980, Fukui et af. 1983, Konig et a f . 1987), which is the rationale for the use of this dye in photodynamic therapy. This work is a report on experimental results that indicate the existence of marked lethal interaction between MB and heat in E. coli.

2. Materials and methods

2.1 . Bacterial strains Strains used in this work and their characteristics are listed in Table 1. ?To whom correspondence should be addressed. 0265-6736/92 $3.00 01992 Taylor & Francis Ltd.

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Table 1. Escherichia coli strains: repair characteristics and nutritional requirements. Characteristics Nutritional requirements" E. coli strains K12 AB1157 Wild type Thr, Leu, Thi, His, Arg, Pro As for ABI 157 uvrA6 K12 AB1886 As for AB1157 K 12 A82463 recA I3 uvrA6 recA I3 As for AB1157 K I2 A82480 K12 JG113 (W3110) wild type Thy, Niac K12 JGI 12 (P3478) polA 1 Thy, Niac wild type His, Leu, Thr, Thi, Pyr K12 PQ35 B/r wild type Thy. Trp ~~

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B/R hcr

uvrA

~~

As for B/r

"Thr. threonine; Leu. leucine; Thi, thiamine: His, histidine; Arg. arginine; Pro. proline; Thy, thymine: Niac. niacin; Pyr. pyrimidine: Trp. tryptophan.

2.2. Growth and survival experiments Stock cells were maintained as individual colonies in agar solidified BT medium (Marcovich 1956) at 4°C. Cells from one of these colonies were grown overnight in BT medium in a shaking incubator at 37°C. The culture was then diluted 40-fold (0.25 to 10 ml) and the cells left to grow under the same conditions until they reached the midexponential growing phase (1-2 x 10' cells/ml). Cells were then collected by centrifugation, washed and resuspended in phosphate buffer with or without methylene blue (Merck, Darmstadt, Germany) in the desired concentration, and incubated in constanttemperature water baths, monitored by a precision thermometer. Following this, 0 . 1 ml of the culture was removed, diluted in phosphate buffer and aliquots of 0 . 1 ml were spread in duplicate Petri dishes containing BT solidified medium. Dishes were incubated overnight at 37°C to form colonies, which were then counted and the surviving fraction calculated with the survival of non-treated control cells as reference. After the cells were put in contact with the dye, they were maintained in subdued yellow light or in darkness, to avoid photodynamic effects. 2.3. DNA degradation experiments Radioactively-labelled bacteria were prepared by growing the cell cultures in M9 medium (Anderson 1946) containing 0.4% glucose, supplemented by 10 pCi/ml [3Hmethyllthymidine from New England Nuclear (specific activity, 2 Ci/mmol), 2 a 5 mg/ml casaminoacids, 1 pg/ml thiamine and 200 pg/ml 2-deoxyadenosine. After allowing 2 h 15 min for growth in this medium, the cells were centrifuged and washed three times, resuspended in phosphate buffer and incubated with methylene blue at the temperature indicated, in darkness. Cells were then centrifuged and washed again, resuspended in supplemented M9 medium and incubated at 37°C. At suitable intervals, samples of 100 pI were taken and placed on paper rectangles (Whatman 17) measuring 1 8 X 1 - 5 cm. The paper rectangles were then washed twice in cold trichloroacetic acid (TCA, lo%), once in 95 % ethanol and once in acetone. After drying, their radioactive content was measured in a Beckman liquid scintillation counter. Degradation of DNA was inferred from the disappearance of radioactivity from the TCA insoluble fraction.

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2.4. Mutation essays Mutation induction was measured by scoring the reversion to prototrophy in restrictive medium agar plates (semi-enriched medium, SEM) of tryptophan auxotrophic bacteria after 48 h incubation at 37°C. Frequency of induced mutation was calculated according to the method of Webb (1978).

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Synergism between heat and MB

3. Results 3.1. Bacterial survival afrer combined MB and hyperthermia treatment of E. coli K12 AB11.57 Bacteria K12 ABll57 (wild type for repair) were incubated at temperatures varying from 25°C (room temperature) to 50"C, with or without MB at a concentration of 6 pg/ml (19 pM). Figure 1 shows that temperature alone, up to 46"C, is not lethal to these bacteria within 2 h of incubation, while some degree of lethality occurs at 48 and 50"C, proportional to the increase in temperature and the length of incubation. MB at a concentration of 6 pg/ml was not lethal at 25, 37 and 46°C. However, the same concentration of MB was strongly lethal at 48 and 50°C. It is interesting to note that the survival curves at 48 and 50°C without MB are straight exponential lines, while those with MB show an inflexion at 30 min (50°C)and 90 min (48°C). To examine the effect of the concentration of MB on the lethal interaction of this dye with heat, ABI 157 cells were incubated for 2 h with increasing concentrations of MB (0-30 pg/ml) at temperatures ranging from 25 to 5 0 ° C and the surviving fraction plotted against dye concentration for each temperature. Figure 2 shows that at 25 and 30°C no lethality occurs with any concentration of MB, that at 37 and 46°C lethality starts only after 5 pg/ml and increases proportionally with the increase in MB concentration up to 30 pg/ml, and that at 48, 49 and 50"C, heat alone presents a certain degree of lethality. The interaction with the dye is so marked (also shown in Figure 1) that with 7.5 pg/ml (at 50"C), 10 pg/ml (at 49°C) and 12.5 pg/ml (at 48"C), it was

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Figure 1. Cytotoxic synergism between heat and merhylene blue in Eschen'chia coli AB1157 bacteria as a function of temperature. Exponentially growing cells were incubated at different temperatures for 2 h without (open symbols) or with 6 pg/rnl(19 pm) MB (closed symbols) in phosphate buffer (pH 7-0). Survival was determined every 30 min. 25, 37 and 46°C (0, 0);48°C (A, A);50°C (0,n.

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no longer possible to detect any viable colony-forming units. Figure 2 also shows that above 46°C the slope of the curves rises rapidly, indicating a drastic lethal effect with MB and heat simultaneously. Above SOT, heat alone inactivates the cells, making it difficult to detect interaction between heat and the dye.

3.2. Incorporation of MB into AB1157 bacteria as a hnction of temperature and dye concentration ABll57 bacteria were incubated with 6 pg/ml MB for 2 h at various temperatures. After incubation, the cells were centrifuged and the supernatant examined in a spectrophotometer at 660 nm to determine the remaining amount of dye and, indirectly, the amount of dye incorporated into the cells. Figure 3 shows that the maximum amount of incorporation occurs in the range between 25 and 4O"C, and that from 40 to 46°C there is a surprising decrease in the incorporation of MB, so that the amount of dye bound to the cells at 46°C is half of that at 40°C. Amount of dye bound to the cells was evaluated by considering the linear relationship between optical density, as read in a spectrophotometer(660 nm), and pre-established dye concentration solutions (Figure 3, upper right). At 37°C the incorporation of dye increases proportionally with the increase in dye concentration (data not shown). I

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Figure 2. Effect of dye concentration on the synergism between heat and MB at various temperatures in ABll57 bacteria. Cells were incubated for 2 h with different concentrations of the dye for each temperature and survival determined after incubation. 25 and 30°C (0); 37°C (@); 46°C (A); 48°C (A);49°C (0);50°C (W).

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Figure 3. Incorporation of MB in ABI 157 bacteria as a function of the temperature of incubation. Cells were incubated with 6 pg/ml MB for 2 h at 25, 30, 37, 40, 43, 46, 48 and 50°C. centrifuged, and the amount of dye remaining in the supernatant determined by means of a spectrophotometer (660 nm), with a standard curve (upper right) used for comparison.

3.3, Interaction between heat and MB as a function of cell membranes Gram-negative bacteria such as E. coli are resistant to a number of chemicals, probably due to the lipopolysaccharide component of the outer membrane, which removes the molecules that can penetrate this permeability barrier (Leive 1974). This is probably the reason for the relative resistance of these bacteria to MB compared with those that are Gram-positive (Nikaido 1976). On the other hand, it is known that heat markedly affects the permeability of membranes of procaryotic (Mackey 1983, Tsuchido et al. 1985) and eucaryotic cells (Cress et al. 1982, Lepock 1982). To obtain evidence concerning the role of outer membrane permeability in the interaction between heat and MB, experiments were performed with bacterial strain PQ35, a K12 Gram-negative derived rfa mutant, deficient in lipopolysaccharides of the outer membrane, particularly sensitive to deoxycholate (Quillardet and Hofnung 1985) and thus more permeable to MB. Figure 4 shows that this strain is far more sensitive to 6 bg/ml MB at 37°C than wild type ABI 157 is. 3.4. Lethal interaction between heat and MB as a function of repair If cellular DNA was a target of the combined effect of heat and MB, bacterial strains with different DNA repair capacities would be expected to show differences in sensitivity to treatment with these two agents. Figure 5A shows the survival curves of four isogenic strains of E. coli, differing in their repair capacities. ABI 157 (wild type), AB2463 (recAZ3), AB1886 (uvrA6) and double mutant AB2480 (uvrA6 recAl.3) were incubated for 2 h with 6 pg/ml MB at 46°C and their survival determined at 30-min intervals.

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A gradual increase in sensitivity occurs. The wild type is not affected by the treatment, the strain deficient in the recombinational-dependent repair system is more sensitive than the wild type, the strain deficient in excision repair is much more sensitive than the two preceding strains and the double mutant is the most sensitive but not very different from the excision-deficient strain. As DNA polymerase I enzyme participates in both excision and recombinational repair systems, playing a much more important role in excision, experiments were performed to evaluate the role of this enzyme in the repair of lesions induced in DNA by heat and MB treatment. Figure 5B shows the survival curves of two isogenic bacterial strains, W3110 (wild type for repair) and P3478 (polAl), after concomitant treatment with heat (46°C) and MB (6 pg/ml). While the wild type is a little more sensitive than AB 1157, perhaps reflecting differences in membrane structures, the strain deficient in polymerase I enzyme is very sensitive to the treatment. It is worth noting that the curve for the polA1 strain is almost perfectly superimposable on the curve for the strain deficient in excision repair. These results led to the supposition that the lesions induced in DNA by MB and heat are repaired by the excision repair system. To test this hypothesis, bacteria ABll57 (wild type) and ABl886 (excision deficient), whose DNA were previously radiolabelled with 'H-thymidine, were treated for 1 h with heat (46°C) and MB (30 p g h l and 10 pg/ml, respectively, to account for the differences in sensitivity and to obtain approximately the same rates of survival). After this treatment, followed by an evaluation of survival, the cells were centrifuged and washed to eliminate the dye and were then incubated at 37°C for 2 h to allow for the action of the excision system.

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Synergism between heat and MB

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Figure 5. Lethal interaction between heat and MB as a function of repair. Escherichiu coli cells with different repair capacities were incubated for 2 h with or without MB (6 pg/ml) at 46°C; AB2463 (recA13). survival rates were determined every 30 rnin. (A) ABI 157 (wild type), 0; A; AB1886 (uvrA6), 0 ; AB2480 (uvrA6 recA13). V. (B) W3110 (wild type), 0 ; P3478 (polAl). A. Survival rate of 100%was found in all strains when incubated under the same conditions without MB.

During this period, aliquots were taken at the beginning of incubation (time zero) and subsequently every 30 min to determine survival and DNA degradation (as measured by the loss of radioactivity from the acid-insoluble to the acid-soluble fraction). The results of these experiments are represented in Figure 6. From analysis of this figure, the following conclusions were made. The two strains are almost equally resistant to heat alone, with strain AB1886 slightly more sensitive. After removal of the dye, the rate of survival attained within 1 h of incubation in the dye's presence is maintained for both strains, that is, there is no additional lethality. During the 2 h of incubation after treatment with heat and MB, there is a loss of radioactivity from the acidinsoluble fraction in AB1157, but no detectable loss occurs in AB1886. Moreover, no loss of radioactivity from the acid-insoluble fraction is detected in either strain when they are submitted to heat alone.

3 . 5 . Mutation induction by heat and MB treatment From the results presented in Figure 6, it was expected that treatment with heat and MB would produce more mutants in the strain deficient in the excision repair system than in the wild type. To test this prediction, we treated two strains of tryptophan auxotrophic bacteria (Bh. wild type and Blr hcr, excision deficient) with heat alone (46°C for 1 h), or heat plus MB (10 pg/ml), and scored the revertants to tryptophan prototrophy. Results are shown in Table 2. Incubation with MB (10 pg/ml) for 1 h at 37°C gave results similar to those of incubation without the dye. 4. Discussion Temperatures above 37°C may damage cells proportionally to the duration of exposure. One of the effects of mild hyperthermia is a modification of cellular permeability. Yatvin

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Figure 6. Survival and DNA degradation of Escherichiu coli ABI 157 (wild type) and AB1$86 (uvrA6) after treatment with heat and MB. Cells with radioactively labelled DNA ( ' H thymidine) were incubated for 60 min at 46°C with 30 pg/ml (AB1157). 10 pg/ml (AB1886) MB or without the dye (both strains). Cells were then centrifuged, washed, resuspended in complete medium and incubated at 37°C for an additional period of 2 h. Survival rates and radioactivity of the acid-insoluble fraction were determined at the intervals indicated. Open symbols: survival; closed symbols: radioactivity. Without MB, 0,0;with MB, A, A.( A ) ABI 157; (B) AB1886. (1977) showed that disorganization of the outer membrane in E. coli, as a consequence of increase in temperature, could be the mechanism of hyperthermic killing. The same author and his collaborators have also shown that the greater heat sensitivity of E. cofi strains in the midlog growing stage, relative to stationary-phase cells, is due to higher membrane microviscosity in stationary-phase cells (Yatvin et a f . 1986). More recently, Barker and Bowler (1991) demonstrated that the lipid composition of the membranes of cells from two thermosensitive rat tumours (hepatoma and sarcoma) was different from that of normal liver cells of the same animal, and showed that the components within the membranes of these tumour cells were significantly less ordered than those of normal cells. The surface of the outer membrane of Gram-negative bacteria is hydrophylic due to one of its components (lipopolysaccharides), which enables hydrophylic molecules of low molecular weight, such as MB, to diffuse through the membrane into the cell, also acting as a barrier to hydrophobic molecules (Nikaido 1976, Sheu and Freese 1973, Leive 1974).

~~

~~

Strain B/r B/r hcr

Table 2. Revertants to tryptophan prototrophy. Revertants/ lo6 survivors Spontaneous After incubation for 1 h (1 h, 37°C) with MB (10 bg/ml) at 46°C 8&2 40* 10 9*2 560 f 70

Afer incubation for 1 h at 46°C 28*6 26*7

Results are the mean of three experiments (10 plates to each point) with standard errors.

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Synergism between heat and

MB

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In darkness, concentrations of MB up to 6 pg/ml did not succeed in impairing the colonyforming ability of a Gram-negative bacteria (E. coli ABl157) when incubation was kept at 37"C, as shown in Figures 1 and 2. However, at this same temperature, it was possible for 6 pg/ml MB in darkness to inactivate the Gram-positive Staphylococcus epidermidis (unpublished data) and a mutant of E. coli K12 (PQ35), which is deficient in some lipopolysaccharides of the outer membrane, making this membrane much more permeable to exogenous molecules (Figure 4). This observation has led to the hypothesis that MB alone, independent of light, can inactivate cells when it penetrates into them and becomes intercalated in the DNA. Indeed. it has recently been shown that MB in contact with E. coli DNA produces single-strand breaks or alkali labilizations (Menezes et al. 1990). Figure 1 shows that 6 pg/ml MB, after 2 h of incubation at 25, 37 and 46"C, did not inactivate the bacteria, while at 48 and 50°C this concentration of MB was lethal to the cells. This shows that there is a lethal interaction between heat and MB that is dependent on temperature and time. On the other hand, Figure 2 shows that for the same duration of exposure, lethality is exponentially proportional to the concentration of the dye and temperature of incubation. From 30 to 46°C. the slopes of the curves rise slowly, with each 1°C increase in incubation temperature resulting in an augmentation of only 0.006 in the angular coefficient of the curves. It seems that 46°C is a point of inflection. Above this temperature, each 1 "C increase in the temperature of incubation results in an augmentation of 0.14 in the angular coefficient, that is, the rate of augmentation is 20-fold that of the first range of temperatures. This could signify that, in E. coli ABll57, the structures responsible for the barrier of permeability were able to resist temperatures up to 46°C before disrupting. At 37°C. the incorporation of the dye is proportional to its concentration (data not shown). However, when the cells are incubated with 6 pg/ml MB for 2 h at different temperatures (from 25 to 50"C), the incorporation of the dye into the cells is almost the same up to 40°C after which it falls sharply (Figure 3). This was an unexpected result when compared with the survival experiments. Our supposition is that the total amount of dye incorporated into the cells may be partially adsorbed by the membrane and partially internalized. The partial internalization is probably responsible for the lethality induced by the dye. Increasing the temperature would increase the amount of dye that penetrates into the cells, thus increasing lethality, but would diminish the dye adsorbed by the membranes due, for instance, to increased motility of the molecules in which dye adsorption occurs. Alternatively, temperature could facilitate the intercalation of MB molecules into DNA or make more efficient the damaging effects of intercalated MB. The result presented in Figure 4 reinforces the idea that the penetration of the dye into the cells is the event sine qua non that triggers the lethal effects of MB, since the strain used in this experiment is a mutant (isolated from a Gram-negative bacterium)'defective in lipopolysaccharides of the outer membrane and, therefore, a more permeable cell. This strain is inactivated by 6 pg/ml MB at 37"C, which does not occur in the wild type bacteria. The mechanism of the action that leads to inactivation of cells treated with MB and heat is still unknown, but it is clear from Figures 5 and 6 that damage to DNA is involved. It seems that once there is MB intercalated into DNA, damage (distortion, for instance) is induced that is lethal to the cell if not repaired. Indeed, Kelly et al. (1987) showed that MB intercalates in DNA, chiefly between the G-C base-pairs. This intercalation would lead to distortions of the DNA structure (Hogan et al. 1979, 1983) that could eventually be recognized by endonucleases of the excision repair system (Sancar and Rupp 1983). The results shown in Figure 5 seem to confirm the hypothesis raised by Sancar and Rupp. as they demonstrate that strains deficient in excision repair and in polymerase I are much more sensitive to heat and MB treatment than are wild types. Figure 6 shows without doubt that excision

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repair is very active in repairing DNA damage produced by heat and MB treatment, repair activity being manifested in the form of a loss of about 30% of the radioactivity from the acid-insoluble to the acid-soluble fraction in the wild type strain, while no loss is detected in the excision deficient strain. This certainly explains why it requires 30 pg/ml MB for the wild type strain to reach a 10% survival rate after 1 h of incubation at 46°C and only 10 vg/ml for the excision repair deficient strain. Also, this explains why 10 pg/ml MB at 46°C is about 14-fold more mutagenic in the excision deficient strain than in the wild type. 5. Conclusions Heat, like other physical and chemical agents used in cancer therapy, has the property of distinguishing normal cells from malignant cells. This seems to be also true o f MB. This article shows that heat sensitizes E. coli cells to the lethal action of MB (or viceversa). In any case, it is clear that a lethal synergism exists between these two agents when they are applied concomitantly to these bacteria. If the same synergistic action exists in mammalian cells, both in v i m and in vivo,this dye could eventually be used as an adjuvant in the therniotherapy of cancer.

Acknowledgements This work was supported by grants from the following Brazilian agencies: Conselho Nacional de Pesquisa (CNPq); Financiadora de Estudos e Projetos (FINEP); Fundacao de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ); Comissao Nacional de Energia Nuclear (CNEN); Conselho de Ensino para Graduados da Universidade Federal do Rio de Janeiro (CEPG-UFRJ). One of the authors (P. T.) is the holder of a long-term postgraduate scholarship from the Coordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES). The authors are grateful to Mrs Esther S. U . Silva for her help in preparing the manuscript. References ANDERSON, E. M., 1946, Growth requirements of virus-resistant mutants of Escherichia coli strain 8. Proceedings of the National Acudemy of Sciences, USA, 31, 120-128. BARKER, C . J . and BOWLER,K . , 1991, Lipid composition of the membranes from cells of two rat tumors and its relationship to tumor thermosensitivity . Radiation Research, 125, 48-55, CRESS,A . E., CUVER,P. S. and GERNER, E. W . , 1982. The correlation between cellular membrane coniponents and sensitivity to hyperthermia in a variety of mammalian cell lines in culture. Cancer Research. 42, I7 16- 172 1 . FUKUI,I., OHWADA,F . , USHYIAMA, T., W A K U IM , . . TOHMA,T . , MITANI.G., YOKOKAWA, M. and YAMADA,T . . 1977, In vivo staining of the bladder cancer with methylene blue. Japane.~e Jourtiril of Clinical Urology, 31, 41-46. FUKLII, I . . YOKOKAWA, M . , MITANI,G., OHWADA,F . , WAKUI,M . , WASHIZUKA, M . , TOHMA. T . , IGARASHI, K. and YAMADA,T., 1983. In vivo staining test with methylene blue for bladder cancer. Journal of Urology, 130, 252-255. HAHN,G. M., 1979, Potential for therapy of drugs and hyperthermia. Cancer Research, 39, 2264-2268. HAHN,G. M . , BRAUN,J. and HAR-KEDAR,I., 1975, Thermochemotherapy: synergism between hyperthermia (42-43°C) and adriamycin (or bleomycin) in mammalian cell inactivation. Proceedings of the National Academy of Sciences, USA, 72, 937-940. HAHN,G. M., LI, G. C . and SHIU,E., 1977, Interaction of amphotericin B and 43°C hyperthermia. Cancer Research, 37, 761-764. HOGAN,M.. DATTAGUPTA, N. and CROTHERS, D . M . , 1979, Transient electric dichroism studies of the structure of the DNA complex with intercalated drugs. Biochemistry, 18, 280-288. HOGAN,M . , LEGRANGE, J . and AUSTIN,B., 1983, Dependence of DNA helix flexibility on base composition. Nature, 304, 752-754. KELLY,J. M . , VANDER PUTTEN.W . J . M. and MCCONNELL.D. J., 1987, Laser flash spectroscopy of methylene blue with nucleic acids. Photochemistry and Photobiology. 45, 167- 175.

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Synergism between heat and MB

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Lethal interaction between heat and methylene blue in Escherichia coli.

Hyperthermia treatment is shown to act synergistically with methylene blue (MB), from the end point of lethality in Gram-negative Escherichia coli bac...
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