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The damage-inducible (din) genes of Escherichia coli are induced by various genotoxins in a different way
Microbiol. Res. (1999) 154,179-183 http://www.urbanfischer.de/joumals/microbiolres
The damage-inducible (din) genes of Escherichia coli are induced by various genotoxins in a different way Tae Jeong Oh, Chang Woo Lee, In Gyu Kim Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yusong, Taejon 305-600, Korea Accepted: June 17,1999
Abstract The SOS response of Escherichia coli strains carrying the lacZ gene fused to the polB (dinA), dinB or dinD gene were investigated after treatment with several chemical agents and y-radiation. The induction levels of polE: :lacZ reached levels between 4.0- and 9.0-fold 120 min after treatment with nalidixic acid, Hz02 or ethanol. Pentachlorophenol did not significantly induce any din genes. y-Irradiation is not an inducer of polB and ethanol failed to induce dinB::lacZ and dinD::lacZ. Following irradiation with a dose of 10 Gy the responses of dinB and dinD were induced about 2.5-3.0-fold above non-irradiated dinB and dinD. We found that the responses of din::lacZ fusion genes to these genotoxins are induced in a dose-dependent manner. The polE gene showed antagonistic responses to the simultaneous treatment of nalidixic acid and Hz02 or nalidixic acid and ethanol. In addition, dinB and dinD in the presence of both nalidixic acid and H20 2 at the same time showed no synergistic responses.
Key words: SOS response - din: :lacZ fusion - genotoxin
Introduction Exposure of Escherichia coli to agents or conditions that damage DNA or interfere with DNA replication results in changes of a diverse set of physiological changes known as SOS response (Witkin 1976; Walker 1984). This includes the induction of a number of genes involved in mutagenesis such as recA, umuCD, uvrAB, and several din (damage-inducible) genes whose function is not fully understood (Walker 1997). The din genes were originally identified by selection for Mu Corresponding author: In Gyu Kim, E-mail: [email protected]. Telephone: 82-42-868-8031, Fax: 82-42-861-9560 0944-5013/99/154/02-179
$12.00/0
d(Ap lac) promoter fusions that were induced during the SOS response. Five different genes, dinA, dinB, dinD, dinE, and dinF, were originally identified (Kenyon and Walker 1980). The dinA gene was later shown to be identical with the polB gene, which encodes DNA polymerase II (Chen etal. 1990) and dinE is identical with uvrA (Kenyon and Walker 1980). The use of various transcriptional fusions also allows detection of agents that interact with DNA (Vollmer etal. 1997). In recent years biochemical and microbial studies for the development of bacterial biosensors are carried out extensively because of rapid response, low costs, and improved reproducibility (Belkin etal. 1996). The SOS induction assay by several pharmaceutical and environmental chemicals is extensively used for generation of large data sets cataloguing the genotoxic and mutagenic effects of many substances in Europe, Japan, and the United States (Vollmer et af. 1997). The SOS induction can be monitored conveniently with strains carrying the lactose operon fused to DNA damage-inducible (din) promoters (Lewis etal. 1992). In this report, we describe the characterization of the responses of din genes against various genotoxins. We determined ~-galactosidase production from three representative din::Mu d (Ap lac) fusion strains treated with nalidixic acid, HP2' ethanol, y-ray and the Environmental Protection Agency priority pollutant, pentachlorophenol.
Materials and methods Bacterial strains, chemicals and cell growth. All chemicals were analytical grade. Nalidixic acid, H20 2, pentachlorophenol (PCP), o-nitrophenyl-{3-D-galactopyranoMicrobiol. Res. 154 (1999) 2
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side and ethanol were purchased from Sigma (St. Louis, U.S.A.). Stock solutions of nalidixic acid (10 mg/ml, w/v) and H20 2 (35%, v/v) were diluted in distilled water and PCP was dissolved in dimethylsulfoxide (0.1 %, w/v). The E. coli strains used in this study are as follows: GWlOlO [recA441 sulAll Li lacU169 thr-l leu-6 his-4 argE4 ilv (Ts) galK2 rpsL31 dinAl (poIB)::Mu d (Ap lac)], GW1030 [As for GWIOIO, but dinB::Mu d (Ap lac)] and GW1040 [As for GW1010, but dinD::Mu d (Ap lac)] (Kenyon and Walker 1980). Luria-Bertani medium (1 % NaCl, 1% Bacto-tryptone and 0.5 % yeast extract, pH 6.8) was used throughout. Cultures were grown at 30°C under aerobic conditions and were used immediately for SOS induction tests following growth to a cell density of 0.8 x 107 cells/m!. SOS induction assay. When cells were freshly grown to cell density of 0.8 x 107 cells/ml at 30°C in Luria-Bertani medium with shaking, nalidixic acid, H20 2, PCP and ethanol were added direclty to the culture medium at final concentrations of 40 /lg/ml, 40/lg/ml, 100/lg/ml and 4%, respectively. Aliquots of cultures were taken every 30min and the p-galactosidase activity was measured. For y-irradiation, aliquots of 10 ml cultures were divided into several aliquots and immediately irradiated with y-rays (60CO source) with a single exposure of various doses. Irradiated cultures were further incubated at 30°C with shaking for 2 h and the p-galactosidase activity was measured. p-Galactosidase assays were performed according to the method described by Miller (1972). The specific activity of p-galactosidase is expressed with the following formula (Miller units). *A420 - 1.75 x *Asso · = 1000 X -=--:---~ Umts IT X 2y x *A6oo
*A420 and Asso are read from the reaction mixture and A600 reflects on the cell density just before the assay. IT = time of the reaction in minutes. 2y = volume of culture used in the assay, in m!. All induction experiments have been performed at least in triplicate.
Results p-Galactosidase activity in din::lacZ strains challenged with different chemical inducers is shown in Fig. 1. Nalidixic acid, H20 2 and ethanol induce polB::lacZ effectively, but PCP is a less effective inducer (Fig. 1 A). Nalidixic acid and H20 2 are also effective inducers of dinB::lacZ, while ethanol and PCP failed to induce dinB::lacZ (Fig. IB). The dinD::lacZ is significantly induced by nalidixic acid and HP2' but not induced by ethanol or PCP (Fig. 1C). The response to nalidixic acid was found to be the most elevated in all strains. 180
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Time after addition (min) Fig. 1. The response profiles of poLB (A), dinB (B) and dinD (C) following genotoxins treatment. Exponential cultures growing at 30°C in LB medium were exposed to a concentration of nalidixic acid (0), PCP (6), ethanol (\7) and HP2 (0 ). The concentrations of genotoxins in these experiments were 40llg/ml, lOOllg/ml, 4% and 40llg/ml, respectively. Open circles (0) indicate the genotoxin-untreated sample. Samples taken every 30 min were tested for the p-galactosidase activity by the method of Miller.
The dose response curves for each fusion are shown in Fig. 2. polB: :ZacZ responded to nalidixic acid, ethanol and HP2in a dose-dependent manner (Fig. 2 A). The responses of dinB::lacZ and dinD::lacZ to nalidixic acid and HP2 were also dose-dependent in the range of 1 to 10 /lg/ml and 1 to 50 /lg/ml, respectively (Fig. 2B, 2 C). When HP2 concentrations are above 100 )lg/ml, p-galactosidase production is decreased in all strains, probably due to the loss of viability. y-Irradiation significantly induces dinB and dinD but did not induce polB (Fig.2D). To determine whether induction of these din genes by chemical inducers was synergistic or not, we monitored B-galactosidase activity under culture conditions in the presence of combinations of inducers. Unexpectedly, responses in all strains were antagonistic rather than syn-
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Fig. 2. The dose response curves of polB (A), dinB (B) and dinD (C) 120min after treatment with nalidixic acid (0.01 to 10 /lg/ml), H20 2 (0.01 to 100/lg/ml), and ethanol (l to 5 %, v/v). Symbols indicate nalidixic acid (0), H20 2 (D) and ethanol (6). (D) Dose response curves of polB (0), dinB (D) and dinD (6) 120 min after y-irradiation (60eo source) with various doses (0 to 10 Gy). ergistic. The polB gene showed antagonistic responses in the presence of nalidixic acid and HP2 or nalidixic acid and ethanol (Fig. 3A, 3B). In addition, dinB and dinD genes showed no synergistic responses by simultaneous tratment with nalidixic acid and H 20 2 (Fig. 3 C, 3 D).
Discussion This study presents the induction characteristics of three din::lacZ fusions by several genotoxins with monitoring of p-galactosidase production. To assess the din::lacZ responses, we employed a set of strains containing the lac operon fused to the promoter or the damage-inducible (din) genes with a single chromosomal copy. These strains have a low basal level of p-galactosidase activity and the ratio of its induced/uninduced p-galactosidase level is high, unlike some high-copy number plasmids such as pUCI8. The responses of cells containing these lacZ fusions to known genotoxins appear to be distinct from one another. The responses of the three fusions described in this study are found to have different patterns according to genotoxins and it may be dependent on the degree of damage by each genotoxin.
Fig. 3. Antagonistic responses to nalidixic acid (20/lg/ml) on polB (A, B), dinB (e), and dinD (D) fusion genes in the presence of H20 2 (20/lg/ml), and ethanol (2 %, v/v). Each data is the representative of three independent experiments with parallel cultures. Symbols indicate A: no addition (0), nalidixic acid (D), HP2 (6), nalidixic acid plus H 20 2 (\7). B: no addition (0), nalidixic acid (D), ethanol (6), nalidixic acid plus ethanol (\7). e: no addition (0), nalidixic acid (D), H20 2 (6), nalidixic acid plus H20 2 (\7). D: no addition (0), nalidixic acid (D), H20 2 (6), nalidixic acid plus H20 2 (\7).
Nalidixic acid inhibits DNA gyrase (Gellert et al. 1977; Sugino et a!. 1977) and causes double-strand breaks in DNA (Drlica eta!' 1980; Snyder and Drlica 1979). The hydroxyl radical (-OH) generated by Fentontype reactions may be commonly invoked as the ultimate DNA damaging species in H 20 z-induced genotoxicity (Maneghini 1988). Nalidixic acid and H20 2 affect all fusion genes, and nalidixic acid is a more effective inducer than H 20 2 in all fusions. These results indicate that the DNA damage is induced to a higher degree by nalidixic acid than by H 20 Z' Ethanol is an effective inducer of polB, but an ineffective inducer of dinB and dinD. On the other hand, the environmental pollutant PCP was not an effective inducer of any fusion gene. PCP has been known to be carcinogenic for mice or rats (McConell etal. 1991). However, PCP has not been shown to be mutagenic in some bacterial test systems, whereas weak mutagenicity has been reported in other systems (Seiler 1991; Kim etal. 1997; Yoon etat. 1997). It has been proposed that tetrachlorohydroquinone (TCHQ), a major metabolite of PCP in mice and rats, induced DNA damage by two possible mechanisms (Witte et al. 1985; Van Ommen etal. 1986). Therefore possible reasons why PCP was not an effective inducer are that PCP did not penetrate Microbiol. Res. 154 (1999) 2
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into the cells or PCP did not convert into TCHQ to induce DNA damage. When E. coli is exposed to ionizing radiation, DNA double-strand breaks were induced (Krasin and Hutchinson 1977; Sargenti and Smith 1986). The ionizing radiation generates ·OH in an aqueous solution, therefore we expected that the induction patterns of the three fusions by H20 2 and y-irradiation were identical. However, the response patterns among the three fusion genes to y-rays were different from those by H20 2. The responses of dinB and dinD to ionizing radiation were found to be low and the polB gene was not inducible, whereas HP2 is an effective inducer of all fusion genes. These results provide important clues that the in vivo action of y-ray and H20 2 to damage DNA may be different from each other and the responses of each fusion gene to y-rays and H20 2 are controlled by different mechanisms. To determine whether the responses of these din genes by chemical inducers is synergistic or not, we analyzed the synergistic effect using compounds including nalidixic acid, HP2 and ethanol. However, unexpected results were obtained. It is generally known that SOS induction by simultaneous treatments of chemical genotoxins is synergistic. In our experiments, induction by simultaneous treatment with these chemicals showed antagonistic rather than synergistic responses. Gene activation by these inducers may have been subjected to complicated controls under these experimental conditions. It is not yet determined what mechanisms are involved in the antagonistic responses. In conclusion several important results were obtained from the studies of din::lacZ fusion genes' response to nalidixic acid, HP2' ethanol, PCP and y-irradiation. All din::lacZ fusions examined in this work are differently induced in an inducer-specific manner. Second, the responses of these fusion genes by simultaneous treatment
of chemical inducers are antagonistic rather than synergistic. Finally, the environmental pollutant PCP is not an effective inducer of these SOS fusion strains. To satisfy future experimental and diagnostic needs, the development of biosensors that differentially respond to genotoxins with increased sensitivity is required. However, these fusion strains may be used as potent tools for assessing and discriminating genotoxicity in environmental samples including drinking water, wastewater, soil, and air using isolated batch samples in the future studies.
Acknowledgement The authors wish to acknowledge the financial support (the Nuclear R&D Program) of the Ministry of Science and Technology (MOST) of the Republic of Korea. 182
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References Belkin, S., Smulski, D. R., Vollmer, A. C., Van Dyk, T. K., LaRossa, R. A. (1996): Oxidative stress detection with Escherichia coli harboring a katG': :lux fusion. Appl. Environ. Microbiol. 62,2252-2256. Chen, H., Sun, Y., Stark, T., Beattie, w., Moses, R. E. (1990): Nucleotide sequence and deletion analysis of the polB gene of Escherichia coli. DNA Cell BioI. 9, 631-635. Drlica, K., Engle, E. C., Manes, S. H. (1980): DNA gyrase on the bacterial chromosome: possibility of two levels of action. Proc. Natl. Acad. Sci. USA 77,6879-6883. Gellert, M., Mizuuchi, K., O'Dea, M. H., Itoh, T., Tomizawa, J. I. (1977): Nalidixic acid resistance: a second genetic character involved in DNA gyrase activity. Proc. Natl. Acad. Sci. USA 74,4772-4776. Kenyon, C. J., Walker, G. C. (1980): DNA-damaging agents stimulate gene expression at specific loci in Escherichia coli. Proc. Natl. Acad. Sci. USA 77,2819-2823. Kim, I. G., Park, S. Y., Yoon, B. S., Cho, M. H., Lee, Y. S. (1997): Comparison of mutant frequencies induced by y-radiation and pentachlorophenol at hprt locus in human T-Iymphocytes. J. Korean Assoc. Radiat. Protect. 22, 15-21. Krasin, E, Hutchinson, E (1977): Repair of DNA doublestrand braks in Escherichia coli, which requires RecA function and the presence of a dublicate genome. J. Mol. BioI. 116,81-98. Lewis, L. K., Jenkins, M. E., Mount, D. W. (1992): Isolation of DNA damage-inducible promoters in Escherichia coli: Regulation of polE (dinA), dinG, and dinH by LexA repressor. J. Bacterio. 174,3377-3385. Maneghini, R. (1988): Genotoxicity of active oxygen species in mammalian cells Mutat. Res. 195,215-230. McConell, E. E., Huff, J. E., Hejtmancik, M., Peters, A. c., Persing, R. (1991): Toxicology and carcinogenesis studies of two grades of pentachlorophenol in B6C3Fl mice. Appl. Toxicol. 17,519-532. Miller, J. H. (1972): Experiments on molecular genetics. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York. 352-355. Sargenti, N. J., Smith, K. C. (1986): Quantitation of the involvement of the recA, recB, recC, reeF, reel, recN, lexA, radA, radB, uvrD, and umuC genes in the repair of X-rayinduced DNA double-strand brads in Escherichia coli. Radiat. Res. 107,58-72. Seiler, J. P. (1991): Pentachlorophenol, Mutation Res. 257, 27-47. Snyder, M., Drlica, K. (1979): DNA gyrase on the bacterial chromosome: DNA cleavage induced by oxolinic acid. J. Mol. BioI. 131,287-302. Sugino, A., Peebles, C. L., Kreuzer, K. N., Cozzarelli, N. R. (1977) : Mechanism of action of nalidixic acid: purification of Escherichi coli nalA gene product and its relationship to DNA gyrase and a novel nicking-closing enzyme. Proc. Natl. Acad. Sci. USA 74, 4767-4771. Van Ommen, B., Adang, A., Muller, E, Van Bladeren, P. J. (1986): The microsomal metabolism of pentachlorophenol and its covalent binding to protein and DNA. Chern. BioI. Interact. 60, 1-11.
Vollmer, A. c., Belkin, S., Smulski, D. R., Van Dyk, T. K., LaRossa, R. A. (1997): Detection of DNA damage by use of Escherichia coli carrying recA'::lux, uvrA'::lux, or alkA'::lux reporter plasmids. Appl. Environ. Microbiol. 63, 2566-2571. Walker, G. C. (1984) : Mutagenesis and inducible responses to deoxyribonucleic acid damage in Escherichia coli. MicrobioI. Rev. 48, 60-93. Walker, G. C. (1997): The SOS response of Escherichia coli. In: Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology (Eds: Curtiss, III. R., Ingraham, J. L., Lin, E. C. c., Low, K. B., Magasanik, B., Reznikov, W. S., Riley, M., Schaechter, M., Umbarger, H. E.). American
Society for Microbiology Press, Washington, D.C. (USA), 1400-1416. Witkin, E. M. (1976): Ultraviolet mutagenesis and inducible DNA repair in Escherichia coli. Bacteriol. Rev. 40, 869-907: Witte, I., Juhl, U., Butte, W. (1985): DNA-damaging properties and cytotoxicity in human fibroblasts of tetrachlorohydroquinone, a pentachlorophenol metabolite. Mutation Res. 145, 71-75. Yoon, B. S., Cho, M. H., Kim, I. G., Park, S. Y, Lee, Y S. (1997) : Mutant frequency at the hprt locus in human T-cell exposed to pentachlorophenol. Korean J. Toxicol. 13,
71-78.
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The damage-inducible (din) genes of Escherichia coli are induced by various genotoxins in a different way Tae Jeong Oh, Chang Woo Lee, In Gyu Kim Department of Radiation Biology, Environmental Radiation Research Group, Korea Atomic Energy Research Institute, P.O. Box 105, Yusong, Taejon 305-600, Korea Accepted: June 17,1999
Abstract The SOS response of Escherichia coli strains carrying the lacZ gene fused to the polB (dinA), dinB or dinD gene were investigated after treatment with several chemical agents and y-radiation. The induction levels of polE: :lacZ reached levels between 4.0- and 9.0-fold 120 min after treatment with nalidixic acid, Hz02 or ethanol. Pentachlorophenol did not significantly induce any din genes. y-Irradiation is not an inducer of polB and ethanol failed to induce dinB::lacZ and dinD::lacZ. Following irradiation with a dose of 10 Gy the responses of dinB and dinD were induced about 2.5-3.0-fold above non-irradiated dinB and dinD. We found that the responses of din::lacZ fusion genes to these genotoxins are induced in a dose-dependent manner. The polE gene showed antagonistic responses to the simultaneous treatment of nalidixic acid and Hz02 or nalidixic acid and ethanol. In addition, dinB and dinD in the presence of both nalidixic acid and H20 2 at the same time showed no synergistic responses.
Key words: SOS response - din: :lacZ fusion - genotoxin
Introduction Exposure of Escherichia coli to agents or conditions that damage DNA or interfere with DNA replication results in changes of a diverse set of physiological changes known as SOS response (Witkin 1976; Walker 1984). This includes the induction of a number of genes involved in mutagenesis such as recA, umuCD, uvrAB, and several din (damage-inducible) genes whose function is not fully understood (Walker 1997). The din genes were originally identified by selection for Mu Corresponding author: In Gyu Kim, E-mail: [email protected]. Telephone: 82-42-868-8031, Fax: 82-42-861-9560 0944-5013/99/154/02-179
$12.00/0
d(Ap lac) promoter fusions that were induced during the SOS response. Five different genes, dinA, dinB, dinD, dinE, and dinF, were originally identified (Kenyon and Walker 1980). The dinA gene was later shown to be identical with the polB gene, which encodes DNA polymerase II (Chen etal. 1990) and dinE is identical with uvrA (Kenyon and Walker 1980). The use of various transcriptional fusions also allows detection of agents that interact with DNA (Vollmer etal. 1997). In recent years biochemical and microbial studies for the development of bacterial biosensors are carried out extensively because of rapid response, low costs, and improved reproducibility (Belkin etal. 1996). The SOS induction assay by several pharmaceutical and environmental chemicals is extensively used for generation of large data sets cataloguing the genotoxic and mutagenic effects of many substances in Europe, Japan, and the United States (Vollmer et af. 1997). The SOS induction can be monitored conveniently with strains carrying the lactose operon fused to DNA damage-inducible (din) promoters (Lewis etal. 1992). In this report, we describe the characterization of the responses of din genes against various genotoxins. We determined ~-galactosidase production from three representative din::Mu d (Ap lac) fusion strains treated with nalidixic acid, HP2' ethanol, y-ray and the Environmental Protection Agency priority pollutant, pentachlorophenol.
Materials and methods Bacterial strains, chemicals and cell growth. All chemicals were analytical grade. Nalidixic acid, H20 2, pentachlorophenol (PCP), o-nitrophenyl-{3-D-galactopyranoMicrobiol. Res. 154 (1999) 2
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side and ethanol were purchased from Sigma (St. Louis, U.S.A.). Stock solutions of nalidixic acid (10 mg/ml, w/v) and H20 2 (35%, v/v) were diluted in distilled water and PCP was dissolved in dimethylsulfoxide (0.1 %, w/v). The E. coli strains used in this study are as follows: GWlOlO [recA441 sulAll Li lacU169 thr-l leu-6 his-4 argE4 ilv (Ts) galK2 rpsL31 dinAl (poIB)::Mu d (Ap lac)], GW1030 [As for GWIOIO, but dinB::Mu d (Ap lac)] and GW1040 [As for GW1010, but dinD::Mu d (Ap lac)] (Kenyon and Walker 1980). Luria-Bertani medium (1 % NaCl, 1% Bacto-tryptone and 0.5 % yeast extract, pH 6.8) was used throughout. Cultures were grown at 30°C under aerobic conditions and were used immediately for SOS induction tests following growth to a cell density of 0.8 x 107 cells/m!. SOS induction assay. When cells were freshly grown to cell density of 0.8 x 107 cells/ml at 30°C in Luria-Bertani medium with shaking, nalidixic acid, H20 2, PCP and ethanol were added direclty to the culture medium at final concentrations of 40 /lg/ml, 40/lg/ml, 100/lg/ml and 4%, respectively. Aliquots of cultures were taken every 30min and the p-galactosidase activity was measured. For y-irradiation, aliquots of 10 ml cultures were divided into several aliquots and immediately irradiated with y-rays (60CO source) with a single exposure of various doses. Irradiated cultures were further incubated at 30°C with shaking for 2 h and the p-galactosidase activity was measured. p-Galactosidase assays were performed according to the method described by Miller (1972). The specific activity of p-galactosidase is expressed with the following formula (Miller units). *A420 - 1.75 x *Asso · = 1000 X -=--:---~ Umts IT X 2y x *A6oo
*A420 and Asso are read from the reaction mixture and A600 reflects on the cell density just before the assay. IT = time of the reaction in minutes. 2y = volume of culture used in the assay, in m!. All induction experiments have been performed at least in triplicate.
Results p-Galactosidase activity in din::lacZ strains challenged with different chemical inducers is shown in Fig. 1. Nalidixic acid, H20 2 and ethanol induce polB::lacZ effectively, but PCP is a less effective inducer (Fig. 1 A). Nalidixic acid and H20 2 are also effective inducers of dinB::lacZ, while ethanol and PCP failed to induce dinB::lacZ (Fig. IB). The dinD::lacZ is significantly induced by nalidixic acid and HP2' but not induced by ethanol or PCP (Fig. 1C). The response to nalidixic acid was found to be the most elevated in all strains. 180
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Time after addition (min) Fig. 1. The response profiles of poLB (A), dinB (B) and dinD (C) following genotoxins treatment. Exponential cultures growing at 30°C in LB medium were exposed to a concentration of nalidixic acid (0), PCP (6), ethanol (\7) and HP2 (0 ). The concentrations of genotoxins in these experiments were 40llg/ml, lOOllg/ml, 4% and 40llg/ml, respectively. Open circles (0) indicate the genotoxin-untreated sample. Samples taken every 30 min were tested for the p-galactosidase activity by the method of Miller.
The dose response curves for each fusion are shown in Fig. 2. polB: :ZacZ responded to nalidixic acid, ethanol and HP2in a dose-dependent manner (Fig. 2 A). The responses of dinB::lacZ and dinD::lacZ to nalidixic acid and HP2 were also dose-dependent in the range of 1 to 10 /lg/ml and 1 to 50 /lg/ml, respectively (Fig. 2B, 2 C). When HP2 concentrations are above 100 )lg/ml, p-galactosidase production is decreased in all strains, probably due to the loss of viability. y-Irradiation significantly induces dinB and dinD but did not induce polB (Fig.2D). To determine whether induction of these din genes by chemical inducers was synergistic or not, we monitored B-galactosidase activity under culture conditions in the presence of combinations of inducers. Unexpectedly, responses in all strains were antagonistic rather than syn-
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y-ray (Gy)
Fig. 2. The dose response curves of polB (A), dinB (B) and dinD (C) 120min after treatment with nalidixic acid (0.01 to 10 /lg/ml), H20 2 (0.01 to 100/lg/ml), and ethanol (l to 5 %, v/v). Symbols indicate nalidixic acid (0), H20 2 (D) and ethanol (6). (D) Dose response curves of polB (0), dinB (D) and dinD (6) 120 min after y-irradiation (60eo source) with various doses (0 to 10 Gy). ergistic. The polB gene showed antagonistic responses in the presence of nalidixic acid and HP2 or nalidixic acid and ethanol (Fig. 3A, 3B). In addition, dinB and dinD genes showed no synergistic responses by simultaneous tratment with nalidixic acid and H 20 2 (Fig. 3 C, 3 D).
Discussion This study presents the induction characteristics of three din::lacZ fusions by several genotoxins with monitoring of p-galactosidase production. To assess the din::lacZ responses, we employed a set of strains containing the lac operon fused to the promoter or the damage-inducible (din) genes with a single chromosomal copy. These strains have a low basal level of p-galactosidase activity and the ratio of its induced/uninduced p-galactosidase level is high, unlike some high-copy number plasmids such as pUCI8. The responses of cells containing these lacZ fusions to known genotoxins appear to be distinct from one another. The responses of the three fusions described in this study are found to have different patterns according to genotoxins and it may be dependent on the degree of damage by each genotoxin.
Fig. 3. Antagonistic responses to nalidixic acid (20/lg/ml) on polB (A, B), dinB (e), and dinD (D) fusion genes in the presence of H20 2 (20/lg/ml), and ethanol (2 %, v/v). Each data is the representative of three independent experiments with parallel cultures. Symbols indicate A: no addition (0), nalidixic acid (D), HP2 (6), nalidixic acid plus H 20 2 (\7). B: no addition (0), nalidixic acid (D), ethanol (6), nalidixic acid plus ethanol (\7). e: no addition (0), nalidixic acid (D), H20 2 (6), nalidixic acid plus H20 2 (\7). D: no addition (0), nalidixic acid (D), H20 2 (6), nalidixic acid plus H20 2 (\7).
Nalidixic acid inhibits DNA gyrase (Gellert et al. 1977; Sugino et a!. 1977) and causes double-strand breaks in DNA (Drlica eta!' 1980; Snyder and Drlica 1979). The hydroxyl radical (-OH) generated by Fentontype reactions may be commonly invoked as the ultimate DNA damaging species in H 20 z-induced genotoxicity (Maneghini 1988). Nalidixic acid and H20 2 affect all fusion genes, and nalidixic acid is a more effective inducer than H 20 2 in all fusions. These results indicate that the DNA damage is induced to a higher degree by nalidixic acid than by H 20 Z' Ethanol is an effective inducer of polB, but an ineffective inducer of dinB and dinD. On the other hand, the environmental pollutant PCP was not an effective inducer of any fusion gene. PCP has been known to be carcinogenic for mice or rats (McConell etal. 1991). However, PCP has not been shown to be mutagenic in some bacterial test systems, whereas weak mutagenicity has been reported in other systems (Seiler 1991; Kim etal. 1997; Yoon etat. 1997). It has been proposed that tetrachlorohydroquinone (TCHQ), a major metabolite of PCP in mice and rats, induced DNA damage by two possible mechanisms (Witte et al. 1985; Van Ommen etal. 1986). Therefore possible reasons why PCP was not an effective inducer are that PCP did not penetrate Microbiol. Res. 154 (1999) 2
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into the cells or PCP did not convert into TCHQ to induce DNA damage. When E. coli is exposed to ionizing radiation, DNA double-strand breaks were induced (Krasin and Hutchinson 1977; Sargenti and Smith 1986). The ionizing radiation generates ·OH in an aqueous solution, therefore we expected that the induction patterns of the three fusions by H20 2 and y-irradiation were identical. However, the response patterns among the three fusion genes to y-rays were different from those by H20 2. The responses of dinB and dinD to ionizing radiation were found to be low and the polB gene was not inducible, whereas HP2 is an effective inducer of all fusion genes. These results provide important clues that the in vivo action of y-ray and H20 2 to damage DNA may be different from each other and the responses of each fusion gene to y-rays and H20 2 are controlled by different mechanisms. To determine whether the responses of these din genes by chemical inducers is synergistic or not, we analyzed the synergistic effect using compounds including nalidixic acid, HP2 and ethanol. However, unexpected results were obtained. It is generally known that SOS induction by simultaneous treatments of chemical genotoxins is synergistic. In our experiments, induction by simultaneous treatment with these chemicals showed antagonistic rather than synergistic responses. Gene activation by these inducers may have been subjected to complicated controls under these experimental conditions. It is not yet determined what mechanisms are involved in the antagonistic responses. In conclusion several important results were obtained from the studies of din::lacZ fusion genes' response to nalidixic acid, HP2' ethanol, PCP and y-irradiation. All din::lacZ fusions examined in this work are differently induced in an inducer-specific manner. Second, the responses of these fusion genes by simultaneous treatment
of chemical inducers are antagonistic rather than synergistic. Finally, the environmental pollutant PCP is not an effective inducer of these SOS fusion strains. To satisfy future experimental and diagnostic needs, the development of biosensors that differentially respond to genotoxins with increased sensitivity is required. However, these fusion strains may be used as potent tools for assessing and discriminating genotoxicity in environmental samples including drinking water, wastewater, soil, and air using isolated batch samples in the future studies.
Acknowledgement The authors wish to acknowledge the financial support (the Nuclear R&D Program) of the Ministry of Science and Technology (MOST) of the Republic of Korea. 182
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