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On the Nature of the Adaptive Response Induced by Mitomycin C in Vibrio cholerae OGAWA 154 Cells
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220, 509–514 (1996)
0435
On the Nature of the Adaptive Response Induced by Mitomycin C in Vibrio cholerae OGAWA 154 Cells Jayasri Basak Biophysics Laboratory, Saha Institute of Nuclear Physics, 37/1, Belgachia Road, Calcutta 700037, India Received February 20, 1996 The induction of an adaptive response in Vibrio cholerae OGAWA 154 cells was obtained using an alkylating agent, mitomycin C, as both stimulating and challenging agent. Cross-adaptive response was observed in V. cholerae cells when pretreated with a sublethal dose of another alkylating agent, N-methyl-N9nitro-Nnitrosoguanidine, followed by challenging treatment with mitomycin C. The dose of mitomycin C for 50% survival (D50) became almost double for mitomycin C pretreated cells and 1.5 times for N-methyl-N9nitro-Nnitroso guanidine pretreated cells, compared to nonpretreated cells. It was also shown that pretreatment with a sublethal dose of oxidative DNA damaging agents, viz, hydrogen peroxide or nitrofurantoin, did not show any cross-adaptive response against subsequent challenge by mitomycin C. © 1996 Academic Press, Inc.
Mitomycin C is a well-known antibiotic and possesses antibacterial and antitumor properties (1). The bifunctional alkylating agent mitomycin C can react with two nucleotides to link them together, leading to the formation of inter-strand cross-links (2). It inhibits DNA biosynthesis in the treated cell (3). Mitomycin C induces prophage induction in Vibrio cholerae (4) and causes filamentation of the treated Vibrio cholerae cells. Mitomycin C is used for the treatment of human in the cases of advanced malignancy (5). Adaptive survival response is observed when cells become resistant to a certain cytotoxic agent after low dose exposures to that agents (6) and cross adaptive response is defined by the reduction of the effects of an agent by pretreatment with sublethal dose of another agent (7). In our previous communications (8–10), we had already shown that Vibrio cholerae OGAWA 154 cells are capable of producing adaptive responses against both alkylating (MNNG) and oxidative DNA damaging agents (NFT,H2O2). Mitomycin C alkylates at the O6 position of guanine inducing DNA monoadducts and inter-strand biadducts (2). No information is available in the literature as to whether mitomycin C does or does not induce adaptive response in any bacterial cell. This aspect therefore needs to be investigated. In the present work, Vibrio cholerae cell has been used as a model bacterial cell particularly since this organism has already been characterised in this respect in our laboratory. This communication reports the pretreatment with sublethal doses of the alkylating MMC can produce adaptive response in Vibrio cholerae cells. Vibrio cholerae cells further showed crossadaptive responses (CAR) when pretreated with sublethal dose of MNNG followed by challenging treatment with MMC. However, pretreatment of the cells with sublethal doses of H2O2 or NFT does not produce CAR against challenging treatment with MMC. MATERIALS AND METHODS Bacterial strains. The bacterial strains used in this study was 1) Vibrio cholerae (classical) strain OGAWA 154 (wild type), obtained through the kind courtesy of the Director, National Institute for Cholera and Enteric Disease, Calcutta. Culture medium. The culture medium used was nutrient broth (NB) containing i) bacto-peptone (Difco, Michigan, U.S.A., 10 gm); ii) beef extract (Oxoid, Hampshire, England, 10 gm) and iii) NaCl (5 gm) in 1 litre distilled water, pH being adjusted to 8.0. Nutrient agar (NA) plates were prepared by adding 1.3% bacto-agar (Difco) to the NB medium. Abbreviations: MMC, mitomycin C; NFT, nitrofurantoin; H2O2, hydrogen peroxide; MNNG, N-methyl-N9nitro-Nnitrosoguanidine; CAR, cross adaptive response. 509 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Chemicals. Chemically pure nitrofurantoin (NFT), mitomycin C (MMC) and N-methyl-N9-nitro-N-nitrosoguanidine (MNNG) were obtained from Sigma Chemicals Co., St Louis, MO. U.S.A. Hydrogen peroxide (H2O2) used was of analytical grade. Treatment with drug or chemical. 1.0 ml aliquots of the log-phase cells with or without any pretreatment were inoculated into Erlenmayer flasks containing 20 ml NB medium and different amounts of mitomycin C/ MNNG/ NFT. The bacteria in each flask were incubated at 37°C in the dark for 1 hour for treatment with mitomycin C and then immediately assayed for viability by the usual pour plate method on the drug or chemical free nutrient agar plates after appropriate serial dilutions. Colonies were counted after at least 24 hours incubation of the nutrient agar plates at 37°C in dark. Pretreatments for adaptation of V.cholera OGAWA 154 cells were performed in the dark at 37°C in the presence of nontoxic concentration of MMC / MNNG / nitrofurantoin for 1 hour or hydrogen peroxide for 30 mins. and then challenged with different concentrations of mitomycin C/MNNG/NFT for 1 hour. The level of any of the compounds used for pretreatment was such as to produce maximum adaptive response as determined by separate and independent experiments. For this purpose from log phase culture of Vibrio cholerae cells 1.0 ml aliquot is incubated to 20 ml fresh nutrient broth containing various sublethal doses of MMC. After completion of 1 hour incubation at 37°C in the dark same amount of challenging doses of MMC were added to each of the sample flask and incubated for another hour at 37°C in the dark. Respective controls which received only the challenged dose were maintained in the similar manner. Percent increase in survival of the pretreated and challenged V.cholerae cells with respect to the respective control cells against each pretreatment dose were determined. Cytotoxicity assay. The cytotoxic effect of the treatment was determined from the relative survival of the colony forming ability of the drug treated cells compared to the untreated control cells.
RESULTS The pretreatment dose of mitomycin C which produced maximum adaptive response of Vibrio cholerae OGAWA 154 cells shown in Fig 1. For Vibrio cholerae cells maximum adaptive response was obtained by a pretreatment with 0.0003 mM mitomycin C for 1 hour. Fig. 2 shows the survival of pretreated or nonpretreated wild type cells of V.cholerae OGAWA 154 after subsequent treatment with different challenging doses of mitomycin C for 1 hour at 37°C in dark. The 50% survival doses (D50) of the cells subjected to a pretreatment with MMC (0.0003 mM) for 1 hour or of the nonpretreated ones were approximately 0.014 mM and 0.007 mM of MMC respectively. On the other hand, when the same cells were subjected to a pretreatment by MNNG (10.0 mM) and subsequently challenged by different doses of MMC, the D50 value was 0.011 mM MMC and was clearly increased from the D50 value of the nonpretreated cells though it was slightly lesser than the D50 value of MMC pretreated cells.
FIG. 1. Change in adaptive response of wild type cells of V. cholerae OGAWA 154 cells with change in mitomycin C dose used for pretreatment. Pretreated or nonpretreated cells were subjected to a challenging dose of 0.03 mM MMC for 1 hour at 37°C in the dark. Percent increase in survival of the pretreated cells with respect to nonpretreated cells (Sc) against different pretreatment doses was determined. For pretreatment cells were incubated at 37°C in the dark for 1 hour with a sublethal dose of mitomycin C. 510
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FIG. 2. Survival of pretreated or nonpretreated wild type cells of V. cholerae OGAWA 154 after subsequent treatment with different challenging doses of mitomycin C for 1 hour at 37°C in the dark. Nonpretreated cells of V. cholerae OGAWA 154 (v—v); V. cholerae OGAWA 154 cells pretreated with 0.0003 mM MMC for 1 hr at 37° C in the dark (V—V); V. cholerae OGAWA 154 cells pretreated with 10.0 mM MNNG for 1 hour at 37°C in the dark (m—m); V. cholerae cells pretreated with 1.6 mM nitrofurantoin for 1 hour at 37°C in the dark (n—n); V. cholerae OGAWA 154 cells pretreated with 30.0 H2O2 for 30 mins. at 37°C in the dark (u—u). Each experimental point in any of the figures presented in this text represents the mean of at least three independent experiments.
When the V.cholerae OGAWA 154 cells subjected to a pretreatment with H2O2 (30 mM) for 30 mins. and subsequently challenged by MMC for 1 hr., the D50 value did not increased vis-a-vis nonpretreated cells and was 0.007 mM MMC. When the Vibrio cholerae cells were pretreated with 1.6 mM NFT for 1 hour and subsequently challenged by MMC, the D50 remained same as 0.007 mM MMC. In order to confirm the nature of adaptive responses in Vibrio cholerae cells induced by MMC some relevant cross adaptive response experiments were also done parallely. Fig. 3 shows the survival of Vibrio cholerae cells which were pretreated or nonpretreated with sublethal doses of MNNG or MMC and subsequently challenged by different doses of MNNG. The D50 values of Vibrio cholerae cells subjected to challenging doses of MNNG (without any pretreatment) was 17.0 mM of MNNG. When the cells were both pretreated (10.0 mM) and subsequently challenged with MNNG, the D50 value was much higher and was 129.2 mM. When the cells were pretreated with MMC and (0.0003 mM) subsequently challenged with MNNG, D50 value was 75.0 mM. When the Vibrio cholerae cells were treated with challenging doses of nitrofurantoin (without any pretreatment), the D50 value was 24.0 mM NFT (Fig.4). On pretreatment with sublethal dose of NFT (1.6 mM) and subsequently challenged with different higher doses of NFT, D50 value become 110.0 mM where as pretreatment with sublethal doses of MMC (0.0003 mM), subsequently challenged with NFT decreased the D50 value to 20.0 of mM NFT. The results of all experiments described here have been summarized in Table 1. DISCUSSION Adaptive response in fact a protecting mechanism induced in cells (prokaryotic or eukaryotic) by treatment with sublethal doses of drugs and other agents. Such treatment makes the cells resistant to subsequent challenge by higher doses of the drugs or the agents concerned. The adaptive response originated due to the functioning of one or more genes present in the cells and is basically due to the occurrence of sublethal DNA damage. Accordingly there are genes which response to the alkylating DNA damage and there are others which response to oxidative DNA damage. So study of adaptive response has fundamental importance in that it reveals the presence or absence 511
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FIG. 3. Survival of pretreated or nonpretreated wild type cells of V. cholerae OGAWA 154 after subsequent treatment with different challenging doses of MNNG for 1 hour at 37°C in the dark. Nonpretreated cells of V. cholerae OGAWA 154 (v—v); V. cholerae OGAWA 154 cells pretreated with 10 mM MNNG for 1 hour at 37°C in the dark (V—V); V. cholerae OGAWA 154 cells pretreated with 0.0003 mM MMC for 1 hour at 37°C in the dark (m—m).
of the particular genes within the bacterial cells and also the environmental conditions which regulates the expression of such genes. Such studies have particular importance in that it defines the actual dose necessary for killing the bacteria, thereby eliminating the chance of developing resistant strains particularly in reference to chemotherapy of bacterial disease. In our earlier observation we have shown that Vibrio cholerae cells could induce adaptive response by nitrofurantoin, furazolidone, hydrogen peroxide and MNNG (8–10). Oxidative DNA damaging agent hydrogen peroxide causes single strand breaks (11,12) whereas nitrofurantoin and
FIG. 4. Survival of pretreated or nonpretreated wild type cells of V. cholerae OGAWA 154 after subsequent treatment with different challenging doses of nitrofurantoin for 1 hour at 37°C in the dark. Nonpretreated cells of V. cholerae OGAWA 154 (v—v); V. cholerae OGAWA 154 cells pretreated with 1.6 mM NFT for 1 hour at 37°C in the dark (m—m); V. cholerae OGAWA 154 cells pretreated with 0.0003 mM MMC for 1 hour at 37°C in the dark (n–n). 512
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TABLE 1 Summary of the Results of 11 Different Experiments Carried out for Establishing the Nature of the Adaptive Response Induced in V. cholerae Ogawa 154 (Wild Type) by Mitomycin C
Expt. No.
Pretreatment by sublethal dose of
Subsequent challenge by
1 2 3 4 5 6 7 8 9 10 11
Nil MMC MNNG NFT H2O2 Nil NFT MMC Nil MNNG MMC
MMC MMC MMC MMC MMC NFT NFT NFT MNNG MNNG MNNG
D50 (mM) 0.007 0.014 0.011 0.007 0.007 24.00 110.0 20.00 17.00 129.2 75.0
Adaptive response N.A.a +veb +ve Nil Nil N.A. +ve −vec N.A. +ve +ve
a
Not applicable since no pretreatment was made. Adaptive response was considered positive where the D50 value increased significantly over the respective value for nonpretreated one. c Adaptive response was considered negative where the D50 value decreased over the respective value for nonpretreated one. b
furazolidone produces inter-strand cross-links in the DNA of treated cells (13–16). Alkylating agent MNNG causes methylation of purine and pyrimidine bases and also forms methylphosphotriesters (mePTE) in the DNA chain of treated cells (17). Mitomycin C generally produces interstrand cross-links by methylating the guanidine base and in addition it has been shown to degrade DNA by making single strand DNA breaks in the DNA chain (18). So it is such an agent that causes both alkylating DNA damage and also oxidative DNA damage. So adaptive response induced by mitomycin C and characterisation of this response is very important. Maximum adaptive dose for NFT, H2O2 and MNNG were determined earlier (9,10). So, only the maximum adaptive dose of MMC was determined here. It has been shown in this study that pretreatment with maximum adaptive dose of mitomycin C could offer significant protection to Vibrio cholerae cells against subsequent challenge by mitomycin C (Fig. 2). It was also confirmed that pretreatment with sublethal doses of MNNG could protect the Vibrio cholerae cells against subsequent challenge by mitomycin C and vice versa (Fig. 2 and Fig. 3). On the other hand pretreatment with nitrofurantoin and hydrogen peroxide could not give any protection to Vibrio cholerae cells against subsequent challenged treatment by mitomycin C (Fig. 2) and it is also true that pretreatment with MMC could not protect the cells against subsequent challenge by NFT (Fig. 4). So far as adaptive response is concerned, from these preliminary experiments it seems that though MMC causes both oxidative and alkylating DNA damages in the DNA of treated cells, adaptive response induced by mitomycin C appeared to be directed towards alkylating and not oxidative adaptive response pathway. In conclusion, it may be stated that pretreatment of sublethal dose of mitomycin C could induce adaptive response in Vibrio cholerae cells. This adaptive response is very weak compared to that induced by nitrofurantoin, furazolidone, hydrogen peroxide and MNNG. Because adaptive response induced by pretreatment of non-toxic doses of mitomycin C could make Vibrio cholerae cells only 2 fold resistant whereas nitrofurantoin and hydrogen peroxide could make Vibrio cholerae cells about 5 fold more resistant and MNNG could make Vibrio cholerae cells about 8–10 fold resistant. 513
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ACKNOWLEDGMENTS J.B. is thankful to the Council of Scientific and Industrial Research, Government of India, for the award of a position of Pool Officer. This work was presented in International Symposium on Trends in Microbiology, Bose Institute, December 1995.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.
Tomasz, M., Mercado, C. M., Olson, J., and Chatterjee, N. (1974) Biochemistry 13, 4878–4887. Iyer, V. H., and Szybalski, W. (1963) PNAS (USA) 50, 355–362. Schwartz, H. S., Sternberg, S. S., and Phillips, F. S. (1963) Cancer Res. 23, 1125–1136. Mondal, D., and Chatterjee, S. N. (1987) Ind. J. Biochem. Biophys. 24, 305–307. Conis, R. L., and Carter, S. K. (1974) Cancer 34, 1576–1586. Samson, L., and Cairns, J. (1977) Nature 267, 281–283. Vijayalaxmi, A., and Burkart, W. (1989) Mutat. Res. 211, 1–5. Basak, J., Mukherjee, U., and Chatterjee, S. N. (1991) Ind. J. Phys. 65B (6), 601–607. Basak, J., Mukherjee, U., and Chatterjee, S. N. (1992) Environ. Mol. Mutag. 20, 53–60. Basak, J., and Chatterjee, S. N. (1994) Mutat. Res. 321, 127–132. Christman, M. F., Storz, G., and Ames, B. N. (1989) PNAS (USA) 86, 3484–3488. Demple, B., and Halbrook, J. (1983) Nature 304, 466–468. Mukherjee, U., Basak, J., and Chatterjee, S. N. (1990) Cancer Biochem. Biophy. 11, 275–287. Sengupta, S., Rahman, Md. S., Mukherjee, U., Basak, J., Pal, A. K., and Chatterjee, S. N. (1990) Mutat. Res. 244, 55–60. Chatterjee, S. N., Banerjee, S. K., Pal, A. K., and Basak, J. (1983) Chem. Biol. Interact. 45, 315–326. Basak, J., and Chatterjee, S. N. (1984) Ind. J. Biochem. Biophys. 21, 7–11. Volkert, M. R. (1988) Environ. Mol. Mutagen. 11, 241–255. Lown, S. W., and Weir, G. (1978) Can. J. Biochem. 56, 296–304.
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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS ARTICLE NO.
220, 509–514 (1996)
0435
On the Nature of the Adaptive Response Induced by Mitomycin C in Vibrio cholerae OGAWA 154 Cells Jayasri Basak Biophysics Laboratory, Saha Institute of Nuclear Physics, 37/1, Belgachia Road, Calcutta 700037, India Received February 20, 1996 The induction of an adaptive response in Vibrio cholerae OGAWA 154 cells was obtained using an alkylating agent, mitomycin C, as both stimulating and challenging agent. Cross-adaptive response was observed in V. cholerae cells when pretreated with a sublethal dose of another alkylating agent, N-methyl-N9nitro-Nnitrosoguanidine, followed by challenging treatment with mitomycin C. The dose of mitomycin C for 50% survival (D50) became almost double for mitomycin C pretreated cells and 1.5 times for N-methyl-N9nitro-Nnitroso guanidine pretreated cells, compared to nonpretreated cells. It was also shown that pretreatment with a sublethal dose of oxidative DNA damaging agents, viz, hydrogen peroxide or nitrofurantoin, did not show any cross-adaptive response against subsequent challenge by mitomycin C. © 1996 Academic Press, Inc.
Mitomycin C is a well-known antibiotic and possesses antibacterial and antitumor properties (1). The bifunctional alkylating agent mitomycin C can react with two nucleotides to link them together, leading to the formation of inter-strand cross-links (2). It inhibits DNA biosynthesis in the treated cell (3). Mitomycin C induces prophage induction in Vibrio cholerae (4) and causes filamentation of the treated Vibrio cholerae cells. Mitomycin C is used for the treatment of human in the cases of advanced malignancy (5). Adaptive survival response is observed when cells become resistant to a certain cytotoxic agent after low dose exposures to that agents (6) and cross adaptive response is defined by the reduction of the effects of an agent by pretreatment with sublethal dose of another agent (7). In our previous communications (8–10), we had already shown that Vibrio cholerae OGAWA 154 cells are capable of producing adaptive responses against both alkylating (MNNG) and oxidative DNA damaging agents (NFT,H2O2). Mitomycin C alkylates at the O6 position of guanine inducing DNA monoadducts and inter-strand biadducts (2). No information is available in the literature as to whether mitomycin C does or does not induce adaptive response in any bacterial cell. This aspect therefore needs to be investigated. In the present work, Vibrio cholerae cell has been used as a model bacterial cell particularly since this organism has already been characterised in this respect in our laboratory. This communication reports the pretreatment with sublethal doses of the alkylating MMC can produce adaptive response in Vibrio cholerae cells. Vibrio cholerae cells further showed crossadaptive responses (CAR) when pretreated with sublethal dose of MNNG followed by challenging treatment with MMC. However, pretreatment of the cells with sublethal doses of H2O2 or NFT does not produce CAR against challenging treatment with MMC. MATERIALS AND METHODS Bacterial strains. The bacterial strains used in this study was 1) Vibrio cholerae (classical) strain OGAWA 154 (wild type), obtained through the kind courtesy of the Director, National Institute for Cholera and Enteric Disease, Calcutta. Culture medium. The culture medium used was nutrient broth (NB) containing i) bacto-peptone (Difco, Michigan, U.S.A., 10 gm); ii) beef extract (Oxoid, Hampshire, England, 10 gm) and iii) NaCl (5 gm) in 1 litre distilled water, pH being adjusted to 8.0. Nutrient agar (NA) plates were prepared by adding 1.3% bacto-agar (Difco) to the NB medium. Abbreviations: MMC, mitomycin C; NFT, nitrofurantoin; H2O2, hydrogen peroxide; MNNG, N-methyl-N9nitro-Nnitrosoguanidine; CAR, cross adaptive response. 509 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.
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Chemicals. Chemically pure nitrofurantoin (NFT), mitomycin C (MMC) and N-methyl-N9-nitro-N-nitrosoguanidine (MNNG) were obtained from Sigma Chemicals Co., St Louis, MO. U.S.A. Hydrogen peroxide (H2O2) used was of analytical grade. Treatment with drug or chemical. 1.0 ml aliquots of the log-phase cells with or without any pretreatment were inoculated into Erlenmayer flasks containing 20 ml NB medium and different amounts of mitomycin C/ MNNG/ NFT. The bacteria in each flask were incubated at 37°C in the dark for 1 hour for treatment with mitomycin C and then immediately assayed for viability by the usual pour plate method on the drug or chemical free nutrient agar plates after appropriate serial dilutions. Colonies were counted after at least 24 hours incubation of the nutrient agar plates at 37°C in dark. Pretreatments for adaptation of V.cholera OGAWA 154 cells were performed in the dark at 37°C in the presence of nontoxic concentration of MMC / MNNG / nitrofurantoin for 1 hour or hydrogen peroxide for 30 mins. and then challenged with different concentrations of mitomycin C/MNNG/NFT for 1 hour. The level of any of the compounds used for pretreatment was such as to produce maximum adaptive response as determined by separate and independent experiments. For this purpose from log phase culture of Vibrio cholerae cells 1.0 ml aliquot is incubated to 20 ml fresh nutrient broth containing various sublethal doses of MMC. After completion of 1 hour incubation at 37°C in the dark same amount of challenging doses of MMC were added to each of the sample flask and incubated for another hour at 37°C in the dark. Respective controls which received only the challenged dose were maintained in the similar manner. Percent increase in survival of the pretreated and challenged V.cholerae cells with respect to the respective control cells against each pretreatment dose were determined. Cytotoxicity assay. The cytotoxic effect of the treatment was determined from the relative survival of the colony forming ability of the drug treated cells compared to the untreated control cells.
RESULTS The pretreatment dose of mitomycin C which produced maximum adaptive response of Vibrio cholerae OGAWA 154 cells shown in Fig 1. For Vibrio cholerae cells maximum adaptive response was obtained by a pretreatment with 0.0003 mM mitomycin C for 1 hour. Fig. 2 shows the survival of pretreated or nonpretreated wild type cells of V.cholerae OGAWA 154 after subsequent treatment with different challenging doses of mitomycin C for 1 hour at 37°C in dark. The 50% survival doses (D50) of the cells subjected to a pretreatment with MMC (0.0003 mM) for 1 hour or of the nonpretreated ones were approximately 0.014 mM and 0.007 mM of MMC respectively. On the other hand, when the same cells were subjected to a pretreatment by MNNG (10.0 mM) and subsequently challenged by different doses of MMC, the D50 value was 0.011 mM MMC and was clearly increased from the D50 value of the nonpretreated cells though it was slightly lesser than the D50 value of MMC pretreated cells.
FIG. 1. Change in adaptive response of wild type cells of V. cholerae OGAWA 154 cells with change in mitomycin C dose used for pretreatment. Pretreated or nonpretreated cells were subjected to a challenging dose of 0.03 mM MMC for 1 hour at 37°C in the dark. Percent increase in survival of the pretreated cells with respect to nonpretreated cells (Sc) against different pretreatment doses was determined. For pretreatment cells were incubated at 37°C in the dark for 1 hour with a sublethal dose of mitomycin C. 510
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FIG. 2. Survival of pretreated or nonpretreated wild type cells of V. cholerae OGAWA 154 after subsequent treatment with different challenging doses of mitomycin C for 1 hour at 37°C in the dark. Nonpretreated cells of V. cholerae OGAWA 154 (v—v); V. cholerae OGAWA 154 cells pretreated with 0.0003 mM MMC for 1 hr at 37° C in the dark (V—V); V. cholerae OGAWA 154 cells pretreated with 10.0 mM MNNG for 1 hour at 37°C in the dark (m—m); V. cholerae cells pretreated with 1.6 mM nitrofurantoin for 1 hour at 37°C in the dark (n—n); V. cholerae OGAWA 154 cells pretreated with 30.0 H2O2 for 30 mins. at 37°C in the dark (u—u). Each experimental point in any of the figures presented in this text represents the mean of at least three independent experiments.
When the V.cholerae OGAWA 154 cells subjected to a pretreatment with H2O2 (30 mM) for 30 mins. and subsequently challenged by MMC for 1 hr., the D50 value did not increased vis-a-vis nonpretreated cells and was 0.007 mM MMC. When the Vibrio cholerae cells were pretreated with 1.6 mM NFT for 1 hour and subsequently challenged by MMC, the D50 remained same as 0.007 mM MMC. In order to confirm the nature of adaptive responses in Vibrio cholerae cells induced by MMC some relevant cross adaptive response experiments were also done parallely. Fig. 3 shows the survival of Vibrio cholerae cells which were pretreated or nonpretreated with sublethal doses of MNNG or MMC and subsequently challenged by different doses of MNNG. The D50 values of Vibrio cholerae cells subjected to challenging doses of MNNG (without any pretreatment) was 17.0 mM of MNNG. When the cells were both pretreated (10.0 mM) and subsequently challenged with MNNG, the D50 value was much higher and was 129.2 mM. When the cells were pretreated with MMC and (0.0003 mM) subsequently challenged with MNNG, D50 value was 75.0 mM. When the Vibrio cholerae cells were treated with challenging doses of nitrofurantoin (without any pretreatment), the D50 value was 24.0 mM NFT (Fig.4). On pretreatment with sublethal dose of NFT (1.6 mM) and subsequently challenged with different higher doses of NFT, D50 value become 110.0 mM where as pretreatment with sublethal doses of MMC (0.0003 mM), subsequently challenged with NFT decreased the D50 value to 20.0 of mM NFT. The results of all experiments described here have been summarized in Table 1. DISCUSSION Adaptive response in fact a protecting mechanism induced in cells (prokaryotic or eukaryotic) by treatment with sublethal doses of drugs and other agents. Such treatment makes the cells resistant to subsequent challenge by higher doses of the drugs or the agents concerned. The adaptive response originated due to the functioning of one or more genes present in the cells and is basically due to the occurrence of sublethal DNA damage. Accordingly there are genes which response to the alkylating DNA damage and there are others which response to oxidative DNA damage. So study of adaptive response has fundamental importance in that it reveals the presence or absence 511
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FIG. 3. Survival of pretreated or nonpretreated wild type cells of V. cholerae OGAWA 154 after subsequent treatment with different challenging doses of MNNG for 1 hour at 37°C in the dark. Nonpretreated cells of V. cholerae OGAWA 154 (v—v); V. cholerae OGAWA 154 cells pretreated with 10 mM MNNG for 1 hour at 37°C in the dark (V—V); V. cholerae OGAWA 154 cells pretreated with 0.0003 mM MMC for 1 hour at 37°C in the dark (m—m).
of the particular genes within the bacterial cells and also the environmental conditions which regulates the expression of such genes. Such studies have particular importance in that it defines the actual dose necessary for killing the bacteria, thereby eliminating the chance of developing resistant strains particularly in reference to chemotherapy of bacterial disease. In our earlier observation we have shown that Vibrio cholerae cells could induce adaptive response by nitrofurantoin, furazolidone, hydrogen peroxide and MNNG (8–10). Oxidative DNA damaging agent hydrogen peroxide causes single strand breaks (11,12) whereas nitrofurantoin and
FIG. 4. Survival of pretreated or nonpretreated wild type cells of V. cholerae OGAWA 154 after subsequent treatment with different challenging doses of nitrofurantoin for 1 hour at 37°C in the dark. Nonpretreated cells of V. cholerae OGAWA 154 (v—v); V. cholerae OGAWA 154 cells pretreated with 1.6 mM NFT for 1 hour at 37°C in the dark (m—m); V. cholerae OGAWA 154 cells pretreated with 0.0003 mM MMC for 1 hour at 37°C in the dark (n–n). 512
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TABLE 1 Summary of the Results of 11 Different Experiments Carried out for Establishing the Nature of the Adaptive Response Induced in V. cholerae Ogawa 154 (Wild Type) by Mitomycin C
Expt. No.
Pretreatment by sublethal dose of
Subsequent challenge by
1 2 3 4 5 6 7 8 9 10 11
Nil MMC MNNG NFT H2O2 Nil NFT MMC Nil MNNG MMC
MMC MMC MMC MMC MMC NFT NFT NFT MNNG MNNG MNNG
D50 (mM) 0.007 0.014 0.011 0.007 0.007 24.00 110.0 20.00 17.00 129.2 75.0
Adaptive response N.A.a +veb +ve Nil Nil N.A. +ve −vec N.A. +ve +ve
a
Not applicable since no pretreatment was made. Adaptive response was considered positive where the D50 value increased significantly over the respective value for nonpretreated one. c Adaptive response was considered negative where the D50 value decreased over the respective value for nonpretreated one. b
furazolidone produces inter-strand cross-links in the DNA of treated cells (13–16). Alkylating agent MNNG causes methylation of purine and pyrimidine bases and also forms methylphosphotriesters (mePTE) in the DNA chain of treated cells (17). Mitomycin C generally produces interstrand cross-links by methylating the guanidine base and in addition it has been shown to degrade DNA by making single strand DNA breaks in the DNA chain (18). So it is such an agent that causes both alkylating DNA damage and also oxidative DNA damage. So adaptive response induced by mitomycin C and characterisation of this response is very important. Maximum adaptive dose for NFT, H2O2 and MNNG were determined earlier (9,10). So, only the maximum adaptive dose of MMC was determined here. It has been shown in this study that pretreatment with maximum adaptive dose of mitomycin C could offer significant protection to Vibrio cholerae cells against subsequent challenge by mitomycin C (Fig. 2). It was also confirmed that pretreatment with sublethal doses of MNNG could protect the Vibrio cholerae cells against subsequent challenge by mitomycin C and vice versa (Fig. 2 and Fig. 3). On the other hand pretreatment with nitrofurantoin and hydrogen peroxide could not give any protection to Vibrio cholerae cells against subsequent challenged treatment by mitomycin C (Fig. 2) and it is also true that pretreatment with MMC could not protect the cells against subsequent challenge by NFT (Fig. 4). So far as adaptive response is concerned, from these preliminary experiments it seems that though MMC causes both oxidative and alkylating DNA damages in the DNA of treated cells, adaptive response induced by mitomycin C appeared to be directed towards alkylating and not oxidative adaptive response pathway. In conclusion, it may be stated that pretreatment of sublethal dose of mitomycin C could induce adaptive response in Vibrio cholerae cells. This adaptive response is very weak compared to that induced by nitrofurantoin, furazolidone, hydrogen peroxide and MNNG. Because adaptive response induced by pretreatment of non-toxic doses of mitomycin C could make Vibrio cholerae cells only 2 fold resistant whereas nitrofurantoin and hydrogen peroxide could make Vibrio cholerae cells about 5 fold more resistant and MNNG could make Vibrio cholerae cells about 8–10 fold resistant. 513
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Vol. 220, No. 3, 1996
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ACKNOWLEDGMENTS J.B. is thankful to the Council of Scientific and Industrial Research, Government of India, for the award of a position of Pool Officer. This work was presented in International Symposium on Trends in Microbiology, Bose Institute, December 1995.
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