Xenobiotica the fate of foreign compounds in biological systems
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Role of free radicals produced during the metabolism of mitomycin C in Escherichia coli inactivation G. F. Schiavano, G. Brandi, L. Salvaggio, F. C. Cattabeni & O. Cantoni To cite this article: G. F. Schiavano, G. Brandi, L. Salvaggio, F. C. Cattabeni & O. Cantoni (1990) Role of free radicals produced during the metabolism of mitomycin C in Escherichia coli inactivation, Xenobiotica, 20:5, 549-554 To link to this article: http://dx.doi.org/10.3109/00498259009046869
Published online: 27 Aug 2009.
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Date: 15 November 2015, At: 06:04
XENOBIOTICA,
1990, VOL. 20, NO. 5 , 549-554
Role of free radicals produced during the metabolism of mitomycin C in Escherichia coli inactivation
Downloaded by [Deakin University Library] at 06:04 15 November 2015
G. F. SCHIAVANOP, G. BRANDIP, L. SALVAGGIO?, F. C. CATTABENIS and 0. CANTONIS
t Istituto di Scienze Tossicologiche, Igienistiche e Ambientali, Universiti degli Studi di Urbino, Urbino, Italy $ Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Universiti degli Studi di Urbino, Urbino, Italy Received 21 June 1989; r e e d 3 November 1989; accepted 22 December 1989 1. The primary objective of this study was to assess whether reactive oxygen species produced during the metabolism of mitomycin C are responsible for, or contribute to, the induction of the cytotoxic response. 2. Escherichia coli wild-type and superoxide dismutase mutants were grown either anoxically or euoxically, in K medium or minimal glucose medium, and treated with mitomycin C in K medium or M9 salts. The cytotoxic response did not vary significantly between the wild-type and the SOD mutants. 3. The toxicity of mitomycin C was not affected by the hydroxyl radical scavengers, dimethyl sulphoxide or ethanol, whereas protection was afforded by thiourea. 4. It is concluded that oxygen radical species do not play a significant role in mediating mitomycin C inactivation of normally growing bacteria.
Introduction Mitomycin C is an antitumour antibiotic derived from Streptomyces caespitosus which is currently being used as a broad-spectrum antineoplastic agent (Crooke 1979). The drug requires metabolic reduction to form species capable of alkylating cellular macromolecules (Kennedy et al. 1982) and the molecular basis of its cytotoxic action has been related to the cross-linking of complementary DNA strands (Iyer and Szybalski 1963). In addition, there is evidence that hypoxic cells are more sensitive to the drug than aerobic cells (Kennedy et al. 1980). It has also been shown, however, that the cyclic reductionloxidation of the quinone group of mitomycin C produces a reactive oxygen cascade potentially capable of inducing DNA single-strand breaks which have been detected by some investigators (Lown et al. 1976) but not by others (Dorr et al. 1985); whether free radicals contribute to the toxicity of the drug has not been definitively established. This problem has been addressed in a number of studies, and the more general question of whether or not reduced quinones are necessary for the antitumor activity of various quinoid drugs has remained largely unanswered (for a review see Biaglow 1981). Despite this premise, however, it is commonly believed that mitomycin C, as well as other antitumour quinones, at physiologically relevant concentrations, produce free radicals which are not involved in the antitumour activity. In recent years the organism E. coli has been successfully employed to demonstrate that superoxide anions mediate the toxicity of paraquat (Hassan and Fridovich 1977,1978). In this study we have used a similar approach in an attempt to 0049-8254/90 83.00 0 1990 Taylor & Francis Ltd.
550
G . F. Schiavano et al.
assess whether or not free radicals participate in mitomycin C-induced E. coli inactivation.
Materials and methods
Downloaded by [Deakin University Library] at 06:04 15 November 2015
Materials Mitomycin C and most reagent-grade biochemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA) and from Flow Labs (McLean, VA, USA). Cell culture techniques and survival studies E. coli K 12 wild-type and superoxide dismutase (SOD) mutants, originally isolated by Dr D. Touati and co-workers (Institut Jacques Monod, CNRS, Universitk Paris, Paris, France), were a gracious gift from Dr R. Tyrrel of the Institut Suisse de Recherches Experimentales sur le Cancer (Epalinges s/Lausanne, Switzerland). Cells were initially grown overnight (1618 h) at 37°C in K medium (1% glucose, 1%casamino acids, 1 yg/ml thiamine hydrochloride, 25 pg/ml MgSO, . 7 H,O, and 2 yg/ml CaCI, in M9 salts-the composition of M9 salts was 6 g/l Na,HPO,, 3 g/l KH,PO,, 0 5 g/l NaCI, 1g/l (NH,),SO,). Samples were diluted 50-fold in fresh K medium or glucose minimal medium (5 mM glucose in M9 salts) and grown to about 10"cells/ml under aerobic or anaerobic conditions. Aerobic growth was achieved by incubation of 50ml of K medium containing 1-2 x lo7 cells/ml in a 500ml Erlenmeyer flask with 200 rpm of shaking, whereas 40ml of bacterial suspension (approx. 3 x lo7 cells/ml) were incubated in a 50ml graduated conical tube with 150rpm of shaking, in order to obtain anaerobic growth. Cells were then harvested by centrifugation at room temperature, washed once with M9 salts, and resuspended at a density of 5-7 x lo7 cells/ml in prewarmed M9 salts or K medium. Mitomycin C was dissolved in M9 salts and added to the cultures as a 30yl aliquot. Treatments (for 15 min at 37°C) were performed in 3 ml of cell suspension in a 20 ml scintillationvial with shaking at 200 rpm.In some experiments hydroxyl radical scavengers,ethanol or dimethyl sulphoxide, were added to the cultures; thiourea was used as a hydroxyl radical scavenger and was dissolved directly in K medium. The challenge was terminated by dilution in M9 salts. Cells were plated in quadruplicate in LB agar plates and incubated at 37°C for 18-22 h to allow colony formation. The number of surviving clones was defined as the percentage of treated clones that grew into macroscopic colonies as compared to control (untreated cells).
Resu1ts Killing of wild-type and SOD mutants of E. coli by mitomycin C In order to investigate the role of superoxide ions in the bactericidal action of mitomycin C, E. coli wild-type and mutants lacking Mn-containing SOD (Sod A) or Fe-containing SOD (Sod B) were analysed for their sensitivity to increasing concentrations of the antitumour antibiotic. As shown in figure lB, wild-type and SOD mutants exposed to mitomycin C in K medium displayed an approximately equal sensitivity, in contrast to cells treated in M9 salts (figure 1A) where Sod B cells were about twice as susceptible to mitomycin C lethality as wild-type or Sod A cells. By comparing figures 1A and 1B it can also be seen that treatment of the latter two strains in K medium or M9 salts resulted in a similar cytotoxic response. We have also investigated the toxicity of mitomycin C in E. coli wild-type and SOD mutants following anoxic growth and treatment in K medium. It was found that, as shown in figure 2, the three strains were all more sensitive (about 2-5 times) as compared to the cells grown under aerobic conditions (figure 1B). In addition, SOD mutants were slightly more vulnerable to mitomycin C than wild-type cells (figure 2). Since nutritional factors present in the growth medium may affect intracellular levels of various enzymes (like SOD), we have grown wild-type and SOD mutants in a glucose minimal medium and then assessed the cytotoxic response following treatment for 15 min in M9 salts. By comparing the results shown in figure 3 with those displayed in figure lA, it can be seen that growth in glucose minimal medium sensitizes wild-type and Sod A cells about 1.8 times, whereas Sod B cells were killed approximately at the same rate.
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Z P L 0.5. A
B
Yb 20 30 40 MlTOMVClN C (pg/ml) Figure 2. Killing of wild-type and SOD mutants of E. coli grown under anaerobic conditions by mitomycin C. Cells were grown in anaerobiosis,as described in the Methods section, and then treated for 15min with increasingconcentrations ofmitomycin C in K medium. Results are the mean of four separate experiments, each performed in quadruplicate. Standard errors were less than 15%. Symbolsare as in figure 1.
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et
al.
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30 MITOMYCIN C cpg/ml) Figure 3.
40
Killing of wild-type and SOD mutants of E. colt' grown in glucose minimal medium by mitomycin C.
Cells were grown in minimal glucose medium under aerobic conditions and then treated for 15min with increasing concentrations of mitomycin C in M9 salts. Results represent the mean of three separate experiments, each performed in quadruplicate. Standard errors were less than 15%. Symbols are as in figure 1.
10 20 30 MITOMYCIN C cpg/ml) Figure 4.
40
Effect of hydroxyl radical scavengerson the cytotoxic response of wild-type and SOD mutants of E. coli to mitomycin C.
Aerobically grown cells were exposed for 15min to increasing concentrations of mitomycin C in or 1OOmM ethanol (0-0) or 2 5 0 m ~ the absence ( 0 )or precence of 35 mM thiourea (0-0) DMSO (0....0)and then plated to allow colony formation. Results represent the mean of the three separate experiments, each performed in quadruplicate. Standard errors were less than 15%.
Resistance of E. coli to active oxygen species
553
Eflect of hydroxyl radical
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scavengers on the toxicity of mitomycin C Wild-type E. colicells were exposed to increasing concentrations of mitomycin C either in the absence or presence of 35 mM thiourea, 100mM ethanol or 250 mM DMSO in K medium for 15min at 37°C. The hydroxyl radical scavengers were utilized at the maximum non-toxic concentration. As shown in figure 4, thiourea was able to prevent the cytotoxic response to mitomycin C in contrast to ethanol and DMSO which did not display any protective effect.
Discussion The aim of this paper was to investigate whether oxygen radicals produced during the intracellular metabolism of mitomycin C could contribute to the toxic response of E. coli to this antitumour antibiotic. Indeed, the redox cycling of mitomycin C is known to result in the formation of superoxide ions and hydrogen peroxide (Tomasz 1976). If superoxide ions are responsible for, or contribute to, the induction of the toxic lesions, one should expect a hypersensitivity of those cells lacking the enzyme superoxide dismutase. We have therefore compared the lethality generated by mitomycin C, under different experimental conditions, in wild-type E. coli and in mutants, Sod A and Sod B, lacking the enzymes Mn- or Fe-SOD, respectively. Results have shown that aerobic exposure to the toxin in K medium or M9 salts did not result in striking differences in the cytotoxic response of the three strains. The only exception was Sod B cells treated in M9 salts that were slightly more sensitive as compared to the situation of exposure in K medium and to wildtype or Sod A cells treated under both experimental conditions. These data indicate that superoxide ions do not play a significant role in mediating mitomycin C cytotoxicity or that, at the most, these species may have a marginal role under nonphysiological conditions (treatment of Sod B cells, expressing only Mn-SOD activity, in a salt solution). Since we have recently shown that the toxicity of hydrogen peroxide is higher in E. coli cells treated in K medium, as compared to cells treated in M9 salts (Cantoni et al. 1989),these data also indicate that it is unlikely that hydrogen peroxide mediates cytotoxicity by mitomycin C. This same inference is supported by other data (figure 4) indicating that the toxicity of mitomycin C is not affected by the hydroxyl radical scavengers, ethanol and DMSO. Although thiourea afforded significant protection against mitomycin C killing, it is likely that the action of this agent may not be related to its OH' scavenging ability but may be dependent on non-specific effects, such as interaction with mitomycin C or other active components. It is known that mitomycin C is highly effective in inhibiting the growth of hypoxic tumours (Kennedy et al. 1980) and that E. coli cells grown under anoxic conditions do not produce Mn-SOD (Carlioz and Touati 1986). We have therefore investigated the toxicity of mitomycin C in anoxically grown E. coli, and found that the three strains, although hypersensitive, as compared to situation where cell growth was achieved aerobically, displayed similar inactivation curves. It should be noted that, in these experiments, treatments were not performed in an anoxic atmosphere; therefore it is not possible to discriminate whether hypersensitivity was dependent on an increased rate of metabolism of mitomycin C, as it has been found in hypoxic tumours (Kennedy et al. 1980), or on the reduced defences of anoxically grown cells which, as previously mentioned, are unable to express the gene coding for the Mn-SOD.
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Resistance of E. coli to active oxygen species
When E. coti cells are allowed to grow in a glucose minimal medium various enzymes, such as SOD, are not efficiently synthesized and cells display hypersensitivity to paraquat, a superoxide-generating compound (Hassan and Fridovich 1978). We have therefore repeated such experiments with either wild-type or SOD mutants and found that, whereas hypersensitivity was apparent in wild-type and Sod A cells, Sod B cells' sensitivity was independent of growth conditions (K medium vs glucose minimal medium). This indicates that Sod B mutants treated in M9 salts are already deficient in whatever defensive function is depressed in wild-type and Sod A cells grown in minimal glucose medium. In conclusion, results presented in this paper are indicative of a marginal role played by superoxide in mediating the killing of E. coli by mitomycin C . In fact, SOD activity of these cells seems to be largely in excess in order to clear the intracellular environment from this reactive oxygen species. In addition, hydrogen peroxide formed by the dismutase reaction seems to be of no toxicological relevance, since no evidence for a contribution of H 2 0 2 in mitomycin C lethality has been collected. The toxicity of mitomycin C to actively growing E. coli is likely to depend solely on its ability to form cross-linking of complementary DNA strands.
Acknowledgements This work was supported by a grant from AIRC.
References BIAGLOW, J. E., 1981, Cellular electron transfer and radical mechanisms for drug metabolism. Radiation Research, 86,212-242. CANTONI, O., BRANDI,G., SCHIAVANO, G. F., ALBANO,A., and CATTABENI, F., 1989, Lethality of hydrogen peroxide in wild-type and superoxide dismutase mutants of Escherichia coli (A hypothesis on the mechanism of HzOz-induced inactivation of Escherichia colz]. ChemicoBiological Interactions, 70, 281-288. CARLIOZ, A., and TOUATI,D., 1986, Isolation of superoxide dismutase mutants in Escherichio coli: is superoxide dismutase necessary for aerobic life? EMBO Journal, 5, 623630. CROOKE, S. T., 1979, Mitomycin C: anoverview. Current StotusandNmDmelopment, edited by J. Carter and S. T. Crooke (New York: Academic Press), pp. 1-4. DORR,R. T., BOWDEN, G. T., ALBERTS, D. S., and LIDDIL,J. D., 1985, Interactions of mitomycin C with mammalian DNA detected by alkaline elution. Cancer Research, 45, 351Cb3516. HASSAN,H. M., and FRIDOVICH, I., 1977, Regulation of the synthesis of superoxide dismutase in Escherichia coli. Journal of Biological Chemistry, 252, 7667-7672. HASSAN, H. M., and FRIDOVICH, I., 1978, Superoxide radical and the oxygen enhancement of the toxicity of paraquat in Escherichia coli. Journol of Biological Chemistry, 253, 8143-8148. IYER,V. H., and SZYBALSKI, W., 1963, A molecular mechanism of mitomycin action: linking of complementary DNA strands. Proceedings of the National Academy of Sciences, USA,50,335-362. KENNEDY, K. A., ROCKWELL, S., and SARTORELLI, A. C., 1980, Preferential activation of mitomycin C to cytotoxic metabolites by hypoxic tumor cells. Cancer Research, 40, 2356-2360. KENNEDY, K. A., SLIGAR, S. G., POLOMSKI, L., and SARTORELLI, A. C., 1982, Metabolic activation of mitomycin C by liver microsomes and nuclei. Biochemical Pharmocology, 31, 201 1-2016. LOWN,J. W., BEGLEITER, A., JOHNSON, D., and MORGAN, A. R., 1976, Studies related to antitumor antibiotics. Part V. Reactions to mitomycin-C with DNA examined by ethidium fluorescence assay. Canadian Journol of Biochemistry, 54, 110-119. TOMASZ, M., 1976, H 2 0 2generation during the redox cycle of mitomycin C and DNA-bound mitomycin C. Chemico-Biological Interactions, 13, 89-97.
ISSN: 0049-8254 (Print) 1366-5928 (Online) Journal homepage: http://www.tandfonline.com/loi/ixen20
Role of free radicals produced during the metabolism of mitomycin C in Escherichia coli inactivation G. F. Schiavano, G. Brandi, L. Salvaggio, F. C. Cattabeni & O. Cantoni To cite this article: G. F. Schiavano, G. Brandi, L. Salvaggio, F. C. Cattabeni & O. Cantoni (1990) Role of free radicals produced during the metabolism of mitomycin C in Escherichia coli inactivation, Xenobiotica, 20:5, 549-554 To link to this article: http://dx.doi.org/10.3109/00498259009046869
Published online: 27 Aug 2009.
Submit your article to this journal
Article views: 6
View related articles
Full Terms & Conditions of access and use can be found at http://www.tandfonline.com/action/journalInformation?journalCode=ixen20 Download by: [Deakin University Library]
Date: 15 November 2015, At: 06:04
XENOBIOTICA,
1990, VOL. 20, NO. 5 , 549-554
Role of free radicals produced during the metabolism of mitomycin C in Escherichia coli inactivation
Downloaded by [Deakin University Library] at 06:04 15 November 2015
G. F. SCHIAVANOP, G. BRANDIP, L. SALVAGGIO?, F. C. CATTABENIS and 0. CANTONIS
t Istituto di Scienze Tossicologiche, Igienistiche e Ambientali, Universiti degli Studi di Urbino, Urbino, Italy $ Istituto di Farmacologia e Farmacognosia and Centro di Farmacologia Oncologica Sperimentale, Universiti degli Studi di Urbino, Urbino, Italy Received 21 June 1989; r e e d 3 November 1989; accepted 22 December 1989 1. The primary objective of this study was to assess whether reactive oxygen species produced during the metabolism of mitomycin C are responsible for, or contribute to, the induction of the cytotoxic response. 2. Escherichia coli wild-type and superoxide dismutase mutants were grown either anoxically or euoxically, in K medium or minimal glucose medium, and treated with mitomycin C in K medium or M9 salts. The cytotoxic response did not vary significantly between the wild-type and the SOD mutants. 3. The toxicity of mitomycin C was not affected by the hydroxyl radical scavengers, dimethyl sulphoxide or ethanol, whereas protection was afforded by thiourea. 4. It is concluded that oxygen radical species do not play a significant role in mediating mitomycin C inactivation of normally growing bacteria.
Introduction Mitomycin C is an antitumour antibiotic derived from Streptomyces caespitosus which is currently being used as a broad-spectrum antineoplastic agent (Crooke 1979). The drug requires metabolic reduction to form species capable of alkylating cellular macromolecules (Kennedy et al. 1982) and the molecular basis of its cytotoxic action has been related to the cross-linking of complementary DNA strands (Iyer and Szybalski 1963). In addition, there is evidence that hypoxic cells are more sensitive to the drug than aerobic cells (Kennedy et al. 1980). It has also been shown, however, that the cyclic reductionloxidation of the quinone group of mitomycin C produces a reactive oxygen cascade potentially capable of inducing DNA single-strand breaks which have been detected by some investigators (Lown et al. 1976) but not by others (Dorr et al. 1985); whether free radicals contribute to the toxicity of the drug has not been definitively established. This problem has been addressed in a number of studies, and the more general question of whether or not reduced quinones are necessary for the antitumor activity of various quinoid drugs has remained largely unanswered (for a review see Biaglow 1981). Despite this premise, however, it is commonly believed that mitomycin C, as well as other antitumour quinones, at physiologically relevant concentrations, produce free radicals which are not involved in the antitumour activity. In recent years the organism E. coli has been successfully employed to demonstrate that superoxide anions mediate the toxicity of paraquat (Hassan and Fridovich 1977,1978). In this study we have used a similar approach in an attempt to 0049-8254/90 83.00 0 1990 Taylor & Francis Ltd.
550
G . F. Schiavano et al.
assess whether or not free radicals participate in mitomycin C-induced E. coli inactivation.
Materials and methods
Downloaded by [Deakin University Library] at 06:04 15 November 2015
Materials Mitomycin C and most reagent-grade biochemicals were obtained from Sigma Chemical Co. (St Louis, MO, USA) and from Flow Labs (McLean, VA, USA). Cell culture techniques and survival studies E. coli K 12 wild-type and superoxide dismutase (SOD) mutants, originally isolated by Dr D. Touati and co-workers (Institut Jacques Monod, CNRS, Universitk Paris, Paris, France), were a gracious gift from Dr R. Tyrrel of the Institut Suisse de Recherches Experimentales sur le Cancer (Epalinges s/Lausanne, Switzerland). Cells were initially grown overnight (1618 h) at 37°C in K medium (1% glucose, 1%casamino acids, 1 yg/ml thiamine hydrochloride, 25 pg/ml MgSO, . 7 H,O, and 2 yg/ml CaCI, in M9 salts-the composition of M9 salts was 6 g/l Na,HPO,, 3 g/l KH,PO,, 0 5 g/l NaCI, 1g/l (NH,),SO,). Samples were diluted 50-fold in fresh K medium or glucose minimal medium (5 mM glucose in M9 salts) and grown to about 10"cells/ml under aerobic or anaerobic conditions. Aerobic growth was achieved by incubation of 50ml of K medium containing 1-2 x lo7 cells/ml in a 500ml Erlenmeyer flask with 200 rpm of shaking, whereas 40ml of bacterial suspension (approx. 3 x lo7 cells/ml) were incubated in a 50ml graduated conical tube with 150rpm of shaking, in order to obtain anaerobic growth. Cells were then harvested by centrifugation at room temperature, washed once with M9 salts, and resuspended at a density of 5-7 x lo7 cells/ml in prewarmed M9 salts or K medium. Mitomycin C was dissolved in M9 salts and added to the cultures as a 30yl aliquot. Treatments (for 15 min at 37°C) were performed in 3 ml of cell suspension in a 20 ml scintillationvial with shaking at 200 rpm.In some experiments hydroxyl radical scavengers,ethanol or dimethyl sulphoxide, were added to the cultures; thiourea was used as a hydroxyl radical scavenger and was dissolved directly in K medium. The challenge was terminated by dilution in M9 salts. Cells were plated in quadruplicate in LB agar plates and incubated at 37°C for 18-22 h to allow colony formation. The number of surviving clones was defined as the percentage of treated clones that grew into macroscopic colonies as compared to control (untreated cells).
Resu1ts Killing of wild-type and SOD mutants of E. coli by mitomycin C In order to investigate the role of superoxide ions in the bactericidal action of mitomycin C, E. coli wild-type and mutants lacking Mn-containing SOD (Sod A) or Fe-containing SOD (Sod B) were analysed for their sensitivity to increasing concentrations of the antitumour antibiotic. As shown in figure lB, wild-type and SOD mutants exposed to mitomycin C in K medium displayed an approximately equal sensitivity, in contrast to cells treated in M9 salts (figure 1A) where Sod B cells were about twice as susceptible to mitomycin C lethality as wild-type or Sod A cells. By comparing figures 1A and 1B it can also be seen that treatment of the latter two strains in K medium or M9 salts resulted in a similar cytotoxic response. We have also investigated the toxicity of mitomycin C in E. coli wild-type and SOD mutants following anoxic growth and treatment in K medium. It was found that, as shown in figure 2, the three strains were all more sensitive (about 2-5 times) as compared to the cells grown under aerobic conditions (figure 1B). In addition, SOD mutants were slightly more vulnerable to mitomycin C than wild-type cells (figure 2). Since nutritional factors present in the growth medium may affect intracellular levels of various enzymes (like SOD), we have grown wild-type and SOD mutants in a glucose minimal medium and then assessed the cytotoxic response following treatment for 15 min in M9 salts. By comparing the results shown in figure 3 with those displayed in figure lA, it can be seen that growth in glucose minimal medium sensitizes wild-type and Sod A cells about 1.8 times, whereas Sod B cells were killed approximately at the same rate.
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Resistance of E. coli to active oxygen - - species -
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Z P L 0.5. A
B
Yb 20 30 40 MlTOMVClN C (pg/ml) Figure 2. Killing of wild-type and SOD mutants of E. coli grown under anaerobic conditions by mitomycin C. Cells were grown in anaerobiosis,as described in the Methods section, and then treated for 15min with increasingconcentrations ofmitomycin C in K medium. Results are the mean of four separate experiments, each performed in quadruplicate. Standard errors were less than 15%. Symbolsare as in figure 1.
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G. F. Schiavano
10
et
al.
20
30 MITOMYCIN C cpg/ml) Figure 3.
40
Killing of wild-type and SOD mutants of E. colt' grown in glucose minimal medium by mitomycin C.
Cells were grown in minimal glucose medium under aerobic conditions and then treated for 15min with increasing concentrations of mitomycin C in M9 salts. Results represent the mean of three separate experiments, each performed in quadruplicate. Standard errors were less than 15%. Symbols are as in figure 1.
10 20 30 MITOMYCIN C cpg/ml) Figure 4.
40
Effect of hydroxyl radical scavengerson the cytotoxic response of wild-type and SOD mutants of E. coli to mitomycin C.
Aerobically grown cells were exposed for 15min to increasing concentrations of mitomycin C in or 1OOmM ethanol (0-0) or 2 5 0 m ~ the absence ( 0 )or precence of 35 mM thiourea (0-0) DMSO (0....0)and then plated to allow colony formation. Results represent the mean of the three separate experiments, each performed in quadruplicate. Standard errors were less than 15%.
Resistance of E. coli to active oxygen species
553
Eflect of hydroxyl radical
Downloaded by [Deakin University Library] at 06:04 15 November 2015
scavengers on the toxicity of mitomycin C Wild-type E. colicells were exposed to increasing concentrations of mitomycin C either in the absence or presence of 35 mM thiourea, 100mM ethanol or 250 mM DMSO in K medium for 15min at 37°C. The hydroxyl radical scavengers were utilized at the maximum non-toxic concentration. As shown in figure 4, thiourea was able to prevent the cytotoxic response to mitomycin C in contrast to ethanol and DMSO which did not display any protective effect.
Discussion The aim of this paper was to investigate whether oxygen radicals produced during the intracellular metabolism of mitomycin C could contribute to the toxic response of E. coli to this antitumour antibiotic. Indeed, the redox cycling of mitomycin C is known to result in the formation of superoxide ions and hydrogen peroxide (Tomasz 1976). If superoxide ions are responsible for, or contribute to, the induction of the toxic lesions, one should expect a hypersensitivity of those cells lacking the enzyme superoxide dismutase. We have therefore compared the lethality generated by mitomycin C, under different experimental conditions, in wild-type E. coli and in mutants, Sod A and Sod B, lacking the enzymes Mn- or Fe-SOD, respectively. Results have shown that aerobic exposure to the toxin in K medium or M9 salts did not result in striking differences in the cytotoxic response of the three strains. The only exception was Sod B cells treated in M9 salts that were slightly more sensitive as compared to the situation of exposure in K medium and to wildtype or Sod A cells treated under both experimental conditions. These data indicate that superoxide ions do not play a significant role in mediating mitomycin C cytotoxicity or that, at the most, these species may have a marginal role under nonphysiological conditions (treatment of Sod B cells, expressing only Mn-SOD activity, in a salt solution). Since we have recently shown that the toxicity of hydrogen peroxide is higher in E. coli cells treated in K medium, as compared to cells treated in M9 salts (Cantoni et al. 1989),these data also indicate that it is unlikely that hydrogen peroxide mediates cytotoxicity by mitomycin C. This same inference is supported by other data (figure 4) indicating that the toxicity of mitomycin C is not affected by the hydroxyl radical scavengers, ethanol and DMSO. Although thiourea afforded significant protection against mitomycin C killing, it is likely that the action of this agent may not be related to its OH' scavenging ability but may be dependent on non-specific effects, such as interaction with mitomycin C or other active components. It is known that mitomycin C is highly effective in inhibiting the growth of hypoxic tumours (Kennedy et al. 1980) and that E. coli cells grown under anoxic conditions do not produce Mn-SOD (Carlioz and Touati 1986). We have therefore investigated the toxicity of mitomycin C in anoxically grown E. coli, and found that the three strains, although hypersensitive, as compared to situation where cell growth was achieved aerobically, displayed similar inactivation curves. It should be noted that, in these experiments, treatments were not performed in an anoxic atmosphere; therefore it is not possible to discriminate whether hypersensitivity was dependent on an increased rate of metabolism of mitomycin C, as it has been found in hypoxic tumours (Kennedy et al. 1980), or on the reduced defences of anoxically grown cells which, as previously mentioned, are unable to express the gene coding for the Mn-SOD.
Downloaded by [Deakin University Library] at 06:04 15 November 2015
554
Resistance of E. coli to active oxygen species
When E. coti cells are allowed to grow in a glucose minimal medium various enzymes, such as SOD, are not efficiently synthesized and cells display hypersensitivity to paraquat, a superoxide-generating compound (Hassan and Fridovich 1978). We have therefore repeated such experiments with either wild-type or SOD mutants and found that, whereas hypersensitivity was apparent in wild-type and Sod A cells, Sod B cells' sensitivity was independent of growth conditions (K medium vs glucose minimal medium). This indicates that Sod B mutants treated in M9 salts are already deficient in whatever defensive function is depressed in wild-type and Sod A cells grown in minimal glucose medium. In conclusion, results presented in this paper are indicative of a marginal role played by superoxide in mediating the killing of E. coli by mitomycin C . In fact, SOD activity of these cells seems to be largely in excess in order to clear the intracellular environment from this reactive oxygen species. In addition, hydrogen peroxide formed by the dismutase reaction seems to be of no toxicological relevance, since no evidence for a contribution of H 2 0 2 in mitomycin C lethality has been collected. The toxicity of mitomycin C to actively growing E. coli is likely to depend solely on its ability to form cross-linking of complementary DNA strands.
Acknowledgements This work was supported by a grant from AIRC.
References BIAGLOW, J. E., 1981, Cellular electron transfer and radical mechanisms for drug metabolism. Radiation Research, 86,212-242. CANTONI, O., BRANDI,G., SCHIAVANO, G. F., ALBANO,A., and CATTABENI, F., 1989, Lethality of hydrogen peroxide in wild-type and superoxide dismutase mutants of Escherichia coli (A hypothesis on the mechanism of HzOz-induced inactivation of Escherichia colz]. ChemicoBiological Interactions, 70, 281-288. CARLIOZ, A., and TOUATI,D., 1986, Isolation of superoxide dismutase mutants in Escherichio coli: is superoxide dismutase necessary for aerobic life? EMBO Journal, 5, 623630. CROOKE, S. T., 1979, Mitomycin C: anoverview. Current StotusandNmDmelopment, edited by J. Carter and S. T. Crooke (New York: Academic Press), pp. 1-4. DORR,R. T., BOWDEN, G. T., ALBERTS, D. S., and LIDDIL,J. D., 1985, Interactions of mitomycin C with mammalian DNA detected by alkaline elution. Cancer Research, 45, 351Cb3516. HASSAN,H. M., and FRIDOVICH, I., 1977, Regulation of the synthesis of superoxide dismutase in Escherichia coli. Journal of Biological Chemistry, 252, 7667-7672. HASSAN, H. M., and FRIDOVICH, I., 1978, Superoxide radical and the oxygen enhancement of the toxicity of paraquat in Escherichia coli. Journol of Biological Chemistry, 253, 8143-8148. IYER,V. H., and SZYBALSKI, W., 1963, A molecular mechanism of mitomycin action: linking of complementary DNA strands. Proceedings of the National Academy of Sciences, USA,50,335-362. KENNEDY, K. A., ROCKWELL, S., and SARTORELLI, A. C., 1980, Preferential activation of mitomycin C to cytotoxic metabolites by hypoxic tumor cells. Cancer Research, 40, 2356-2360. KENNEDY, K. A., SLIGAR, S. G., POLOMSKI, L., and SARTORELLI, A. C., 1982, Metabolic activation of mitomycin C by liver microsomes and nuclei. Biochemical Pharmocology, 31, 201 1-2016. LOWN,J. W., BEGLEITER, A., JOHNSON, D., and MORGAN, A. R., 1976, Studies related to antitumor antibiotics. Part V. Reactions to mitomycin-C with DNA examined by ethidium fluorescence assay. Canadian Journol of Biochemistry, 54, 110-119. TOMASZ, M., 1976, H 2 0 2generation during the redox cycle of mitomycin C and DNA-bound mitomycin C. Chemico-Biological Interactions, 13, 89-97.
