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Radioprotective effect of sodium diethyldithiocarbamate (DDC) and S-2-aminoethyl-isothioronicadenosin-5-triphosphate (adeturon) in γ-irradiated Escherichia coli cells
Mutation Research 422 Ž1998. 339–345
Radioprotective effect of sodium diethyldithiocarbamate žDDC / and S-2-aminoethyl-isothioronicadenosin-5-triphosphate žadeturon / in g-irradiated Escherichia coli cells Jorge L. Fuentes a , Norberto Capetillo b, Mirle Ferrer a , Eloy Padron ´ a, Sonia Altanes ´ a, Montserrat Llagostera c,) a
c
Centro de Estudios Aplicados al desarrollo Nuclear (CEADEN), Calle 30 No. 502 e r 5 t a y 7 m a , P.O.Box 6122, Miramar, Playa, HaÕana, Cuba b Empresa GeoCuba, Estudios Marinos, Punta Santa Catalina, Regla, HaÕana, Cuba ), Edifici Cn, 08193 UniÕersitat Autonoma de Barcelona, Department de Genetica ` ` i de Microbiologia, Unitat de Microbiologia (Ciencies ` Bellaterra, Barcelona, Spain Received 16 April 1998; revised 22 July 1998; accepted 24 July 1998
Abstract The effect of sodium diethyldithiocarbamate ŽDDC. and S-2-aminoethyl-isothiouronicadenosin-5-triphosphate Žadeturon. in the induction of Escherichia coli SOS response promoted by g-irradiation was studied by measuring the induction of sulA gene and the induction of lambda prophage. Furthermore, as a way of measure the exonuclease activity in g-irradiated cells in the presence or absence of both compounds, the DNA degradation was determined. Adeturon did not affected DNA degradation, but inhibited the induction of the SOS functions studied. On the contrary, DDC inhibited DNA degradation as well as the induction of the sulA gene, but enhanced lambda induction in E. coli lysogenic strains. These results indicate that both compounds diminish the DNA damage produced by g-irradiation and also suggest that the mechanisms of radioprotection must be different. Thus, radioprotection mediated by DDC should involve free hydroxyl radical scavenging and a minor activity of exonuclease. The enhancement of phage induction in E. coli cells that DDC produces could be attributed to its quelant effect and this would not be not probably directly related to radioprotection. Adeturon, as thiols, may serve also as scavenging agent of free hydroxyl radicals, diminishing indirectly the DNA damage level. In addition, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. However, adeturon do not modify the exonuclease activity. Some topic about the possible clinical application of both compounds are discussed. q 1998 Elsevier Science B.V. All rights reserved. Keywords: g-Irradiation; Radioprotection; SOS response; Escherichia coli
)
Corresponding author. Tel.: q34-3-5812615; Fax: q34-3-5812387; E-mail: [email protected]
0027-5107r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 1 6 6 - 3
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J.L. Fuentes et al.r Mutation Research 422 (1998) 339–345
1. Introduction Ionizing radiation causes DNA damage and chromosome aberrations both directly and indirectly. The indirect mechanism involves the generation of DNA-damaging radicals by the radiolysis of water. Among free radicals, the hydroxyl radical ŽPOH. is the most reactive toward biological molecules and generates a multitude of modifications in DNA such as base damage, sugar damage and DNA–protein cross-links w1,2x. These kinds of DNA lesions may play an important role in biological processes such as mutagenesis, carcinogenesis and ageing in humans. Oxidative DNA damage may be repaired in cells by a variety of repair enzymes. DNA base products are repaired by both base-excision and nucleotide-excision repair, but predominantly by the former. Two well known Escherichia coli enzymes, endonuclease III and formamidopyrimidine-DNA glycosylase account for the excision of most modified bases from DNA w1x. Eukaryotic counterparts of these enzymes exist in human cells w2x. Radioprotectors are agents that reduce toxicity, mutagenicity, or other biological effects of ionizing radiation w3,4x. The ability of some aminothiols to minimize damage caused by ionizing radiation has stimulated research on the possibility of using these compounds to protect non-target tissues in radiotherapy and to diminish secondary toxic effect of drugs in chemotherapy w5–8x. The mechanisms of radioprotection of aminothiol compounds is thought to involve scavenging of POH radicals, transferring hydrogen to DNA radicals, and causing an anoxic state near the DNA target w9–11x. Stabilization of chromatin may also contribute to the radioprotective properties of aminothiols and other compounds that bind to DNA w12x. Different authors have postulated that aminothiol compounds may form noncovalent bonds with their positively charged amino groups interacting electrostatically with the DNA phosphate groups w13–16x. Other authors have suggested that aminothiol compounds gain their protective effect modulating the endonucleolytic activity w17x. Unlike their protective effect on ionizing radiation, aminothiol compounds as 2- wŽaminopropyl. amino x ethanethiol ŽWR-1065. and cysteamine potentiate the induction of micronuclei by bleomycin treatment in Go human lymphocytes w18,19x and the mitotic re-
combination in yeast w20x. Potentiation by radioprotectors of the genotoxic effect of bleomycin without increasing other aspects of its toxicity could enhance its therapeutic effectiveness w16x. Sodium diethyldithiocarbamate ŽDDC. is a sulfhydryl compound, which is a structural analogous of the well-known S-2-aminoethylisothiuronic ŽAET.. This compound has been used in the treatment of acute nickel–carbonyl poisoning w21x. The chelating properties of DDC and other derivatives of dithiocarbamate have been widely studied w22x. On the other hand, S-2-aminoethyl-isothiouronicadenosin-5-triphosphate Žadeturon. is an AET related compound, which includes in its structure one adenosin5-triphosphate ŽATP. molecule. It is known that sulfhydryl compounds selectively reduces the indirect effect of ionizing radiation by scavenging hydroxyl radicals, which constitute the principal radical species responsible for DNA damage caused by radiation w23x. In the present work, we have studied the effect of both DDC and adeturon as possible radioprotective agents. For this, we have used bacterial assays, which give an indirect estimate of DNA damage. Among them, the Inductest w24x and SOS Chromotest w25x, which respectively measure phage and SOS gene induction in E. coli cells, have been reported as useful tools for studies of mutagenic effectiveness of g-rays, as well as to study the mechanisms of action of compounds that modify the effect of radiation w26–28x. Furthermore, and because the study of DNA degradation process may bring important information about the mechanisms by mean of which aminothiol compounds operate, we have studied this parameter in E. coli cells treated with g-irradiation in the presence of both DDC and adeturon. 2. Materials and methods 2.1. Bacterial strains and growth conditions The bacterial strains used in this work are listed in Table 1. Cultures were grown on supplemented minimal medium ŽSMM. Ž0.1% NH 4 Cl, 0.3% KH 2 PO4 , 0.6% Na 2 HPO4 , 0.05% NaCl, 0.001% CaCl 2 , 0.003% MgSO4 , 0.2% glucose, 0.1% casein hydrolizate, 0.001% thiamine HCl, pH 7.3., Luria Broth or Nutrient Broth w29x depending on the assay.
J.L. Fuentes et al.r Mutation Research 422 (1998) 339–345 Table 1 Bacterial strains
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2.5. ß-Galactosidase assay
Strain
Relevant characteristics
Source
AB1157 Hfr Ž l . C GC2181
– thi, str R str R sulA
B. Bachmann B. Bachmann B. Bachmann S. Casaregola
2.2. RadioprotectiÕe compounds Sodium diethyldithiocarbamate and S-2aminoethyl-isothiouronicadenosin-5-triphosphate were used. The effective incorporation time Ž30 min. and the effective radioprotective concentration Ž1000 mgrml, DDC and 1500 mgrml, adeturon. for E. coli cells, were previously determined by us w30x. 2.3. Irradiation g-Irradiation Ž60 Co. was accomplished using an MP-g-25 M irradiator ŽRussia.. The overnight culture was diluted 1:25 in fresh medium and shaken at 378C until a cell density of about 10 8 cellsrml was achieved. Then, cells were diluted 20-fold in fresh medium, with and without radioprotectors, and distributed into glass test tubes. Cell suspensions were irradiated at 28C with different doses. After g-irradiation, cells for ß-galactosidase assay were incubated for two hours at 378C with shaking and cells for the assay of lambda induction were incubated overnight. The dose rate was 10 Gyrmin as had been previously determined following Fricke’s method w31x. 2.4. Assay of bacteriophage induction E. coli lysogenic cells treated or untreated with g-irradiation were grown overnight in Nutrient broth and were centrifuged at 5000 = g for 10 min. Samples Ž0.1 ml. of the supernatant Žundiluted and 10– 100 fold diluted. were mixed with 0.1 ml of an overnight culture of the E. coli C strain Žgrown on Nutrient broth, supplement with 200 mgrml of streptomycin., held for 20 min at 378C, mixed with 2.5-ml of R-top agar and poured over R-agar base plates w32x. The presence of plaque-forming units ŽPFU. was determined after overnight incubation of the plates at 378C.
The ß-galactosidase assay was performed as it has been described w25x. Cell membranes of g-irradiated and nonirradiated cells of GC2181 strain were disrupted with SDS 0.1% in Z buffer during 20 min at room temperature. The enzyme reaction was started by adding o-nitrophenyl-ß-D-galactopyranoside ŽONPG. Ž4 mgrml in T buffer pH 8.8.. After 40 min, the enzymatic reaction was stopped by adding Na 2 CO 3 1 M, and absorbances at OD550 and OD420 were measured using a Spekoll-11 spectrometer ŽZeiss, Germany.. Enzyme activity was calculated as described w33x. 2.6. DNA degradation assay An overnight culture of the AB1157 strain was diluted 100-fold into SMM, containing thymine at 100 mgrml and w14 Cx-thymine ŽCENTIS, 30 Cirmmole. at 3 mCirml, and shaken at 378C to an OD650 of 0.2. These radioactive cells were centrifuged, washed and resuspended in the mineral salt portion of SMM, having an OD650 of 0.1, and contained radioprotective compounds, and g-irradiated at D 10 value Ž0.3 kGy.. These cells were mixed with an equal volume of SMM, containing twice the normal concentration of glucose, thiamine–HCl, and supplements, and were incubated. At different times of incubation, duplicate samples of 0.1 ml of culture were placed on Whatman No. 17 paper rectangles, previously saturated with TCA 10%, supported on steel pins and dried. Then, each was moistened with 0.2 ml of thymine at 1 mgrml and dried. At the end of the experiment, the rectangles were washed thrice with TCA 5% and once with ethanol 95%, dried and radioactivity was measured with a liquid scintillation ß-LKB spectrometer. Controls with nonirradiated and irradiated cells without radioprotectors were also included. 2.7. Statistical methods Data were transformed with a power transformation estimated by the methods of Box and Cox w34x. To determine if there were significant differences between treatments, an analysis of variance was
342
J.L. Fuentes et al.r Mutation Research 422 (1998) 339–345
carried out. If a significant value was found Ž p - 0.05., the multiple comparisons, as the Scheff’s method, were used to determine which groups were significantly different Ž p - 0.05. w35x.
exonucleases w37x. Our results indicate that DDC, but not adeturon, should have some effect on exonuclease activity, because it produces inhibition of DNA degradation. These data suggest that mechanisms of radioprotection mediated by these chemicals must be probably different.
3. Results and discussion 3.1. Effect on DNA degradation
3.2. Effect on SOS response in E. coli cells
The effect of DDC and adeturon in DNA degradation processes was determined measuring the remains of 3 H labelled DNA. No significant effect was found with adeturon; however a marked inhibitory effect of DNA degradation processes was observed in the presence of DDC Ž p - 0.05. ŽFig. 1.. Exonuclease activity in E. coli cells is principally undertaken by an inducible enzymatic system w36x. A variety of repair enzymes are capable of detecting oxidative DNA damage caused by ionizing radiation. DNA base products are repaired by both base-excision and nucleotide excision repair, but predominantly by the former w1,2x. It has been reported that aminothiol compounds repress RNA synthesis and inhibit the synthesis of inducible enzymes, including
In E. coli irradiated cells, the SOS regulon is triggered as part of the cellular response to exogenous DNA damage. In this cellular state, endonuclease and exonuclease activities are also enhanced w1x. As consequence of attempting to replicate past DNA lesions, an inducing signal is generated that results in the activation of RecA protein w38x. In its activated state, RecA enhances the self cleavage of the LexA repressor, thus leading to the expression of more than 20 SOS genes, including the sulA gene, and promoting the induction of the l prophage. As indirect markers of the protection mediated by DDC and adeturon of the damage on DNA, the level of induction of both l phage and sulA gene promoted by g-irradiation in E. coli was studied in the presence or absence of both radioprotectors. DDC gave contradictory effects. While a marked reduction of the induction level of sulA gene was obtained ŽFig. 2., this compound stimulated phage induction in E. coli Hfr Ž l . cells ŽFig. 3.. The differences between DDC and g-irradiated control data was statistically significant Ž p - 0.05. for the doses from 80 to 240 Gy ŽFig. 2. and for the doses from 400 to 1000 Gy ŽFig. 3.. The fact that DDC clearly diminished the induction level of the sulA gene is an evidence that the level of damage must have been reduced and suggests that the reason of the enhancement of phage induction is not directly related to the SOS response. In relation to this, similar effect on phage induction have been shown by cysteamine w32x. The mechanisms by which DDC potentiates phage induction in E. coli cells are probably not directly related to it being radioprotective. DDC radioprotection, as other sulfhydryl compounds w4x, should involve free hydroxyl radical scavenging. This action would be expected to reduce the phage induction by g-rays but no an enhancement. It is possible to speculate that this enhancement is mediated by its
Fig. 1. Radioprotective effect of DDC ŽI. and adeturon ŽB. on DNA degradation of g-irradiated cells Ž`. of E. coli. DNA degradation of nonirradiated cells Žv . is shown as control. Data are the average of two independent experiments with six replications for each dose. Standard deviations are shown.
J.L. Fuentes et al.r Mutation Research 422 (1998) 339–345
Fig. 2. Effect of DDC ŽI. and adeturon ŽB. on sulA gene induction of g-irradiated cells Ž'. of E. coli. Data are the average of three independent experiments with four replications for each dose. Standard deviations are shown.
quelant effect, perhaps modifying the binding of the cI repressor to the promoters of lytic functions of the phage. On the contrary, adeturon diminished the induction level either sulA gene as phage induction of g-irradiated cells ŽFigs. 2 and 3.. The differences between adeturon and g-irradiated control data was statistically significant Ž p - 0.05. for the doses from 40 to 240 Gy ŽFig. 2. and for the doses from 300 to 1000 Gy ŽFig. 3.. These results clearly show that adeturon diminishes the DNA damage produced by g-irradiation. Different authors have postulated that the mechanism of radioprotection of aminothiol compounds involves scavenging of hydroxyl radicals, donation of hydrogen to sites of DNA damage, and depletion of oxygen w9–11,18x. These also may form non covalent bonds with their positively charged amino groups interacting electrostatically with the DNA phosphate groups, stabilizing the chromatin w12–16x. Two mechanisms may explain the effect of adeturon on SOS functions in E. coli. First, as thiol, this compound may serve as scavenging agent of free hydroxyl radicals, depresses indirectly the DNA damage level. Second, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino
343
groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. Some authors w17x, have suggested that aminothiol compounds are endonuclease repressors that doing more coordinate the DNA repair processes. Nowadays, it is well know that the most of DNA modified base are repaired by both base-excision and nucleotide-excision mechanisms w1x. In our study, we have used the DNA degradative assay as a way of measure the exonuclease activity in g-irradiated cells. The results of this assay suggested that adeturon does not have effect upon exonuclease activity. Other effective radioprotective concentrations of this compound have been also used Ždata not shown.. Radioprotective effect of DDC and adeturon may have clinical implications. There is interest in using aminothiols, to minimize damage to non target tissues in radiotherapy w7,8x. Use of protectors with chemotherapy drugs is also possible, especially if one can exploit a differential protection of normal cells and cancer cells w7,39x, although radioprotectors may reduce the damage caused by cytotoxic drugs in
Fig. 3. Effect of DDC ŽI. and adeturon ŽB. on phage induction of g-irradiated cells Ž'. of E. coli. Data are the average of three independent experiments with six replications for each dose. Standard deviations are shown.
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some instances or may increase it in others w40x. Future studies, which will combine molecular techniques and SOS gene induction assay in E. coli, may clarify not skilled topic related to the mode of action of radioprotective compounds.
w12x
w13x
Acknowledgements w14x
We thank the authors cited in the text for the supply of the bacterial strains.
w15x
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Radioprotective effect of sodium diethyldithiocarbamate žDDC / and S-2-aminoethyl-isothioronicadenosin-5-triphosphate žadeturon / in g-irradiated Escherichia coli cells Jorge L. Fuentes a , Norberto Capetillo b, Mirle Ferrer a , Eloy Padron ´ a, Sonia Altanes ´ a, Montserrat Llagostera c,) a
c
Centro de Estudios Aplicados al desarrollo Nuclear (CEADEN), Calle 30 No. 502 e r 5 t a y 7 m a , P.O.Box 6122, Miramar, Playa, HaÕana, Cuba b Empresa GeoCuba, Estudios Marinos, Punta Santa Catalina, Regla, HaÕana, Cuba ), Edifici Cn, 08193 UniÕersitat Autonoma de Barcelona, Department de Genetica ` ` i de Microbiologia, Unitat de Microbiologia (Ciencies ` Bellaterra, Barcelona, Spain Received 16 April 1998; revised 22 July 1998; accepted 24 July 1998
Abstract The effect of sodium diethyldithiocarbamate ŽDDC. and S-2-aminoethyl-isothiouronicadenosin-5-triphosphate Žadeturon. in the induction of Escherichia coli SOS response promoted by g-irradiation was studied by measuring the induction of sulA gene and the induction of lambda prophage. Furthermore, as a way of measure the exonuclease activity in g-irradiated cells in the presence or absence of both compounds, the DNA degradation was determined. Adeturon did not affected DNA degradation, but inhibited the induction of the SOS functions studied. On the contrary, DDC inhibited DNA degradation as well as the induction of the sulA gene, but enhanced lambda induction in E. coli lysogenic strains. These results indicate that both compounds diminish the DNA damage produced by g-irradiation and also suggest that the mechanisms of radioprotection must be different. Thus, radioprotection mediated by DDC should involve free hydroxyl radical scavenging and a minor activity of exonuclease. The enhancement of phage induction in E. coli cells that DDC produces could be attributed to its quelant effect and this would not be not probably directly related to radioprotection. Adeturon, as thiols, may serve also as scavenging agent of free hydroxyl radicals, diminishing indirectly the DNA damage level. In addition, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. However, adeturon do not modify the exonuclease activity. Some topic about the possible clinical application of both compounds are discussed. q 1998 Elsevier Science B.V. All rights reserved. Keywords: g-Irradiation; Radioprotection; SOS response; Escherichia coli
)
Corresponding author. Tel.: q34-3-5812615; Fax: q34-3-5812387; E-mail: [email protected]
0027-5107r98r$ - see front matter q 1998 Elsevier Science B.V. All rights reserved. PII: S 0 0 2 7 - 5 1 0 7 Ž 9 8 . 0 0 1 6 6 - 3
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J.L. Fuentes et al.r Mutation Research 422 (1998) 339–345
1. Introduction Ionizing radiation causes DNA damage and chromosome aberrations both directly and indirectly. The indirect mechanism involves the generation of DNA-damaging radicals by the radiolysis of water. Among free radicals, the hydroxyl radical ŽPOH. is the most reactive toward biological molecules and generates a multitude of modifications in DNA such as base damage, sugar damage and DNA–protein cross-links w1,2x. These kinds of DNA lesions may play an important role in biological processes such as mutagenesis, carcinogenesis and ageing in humans. Oxidative DNA damage may be repaired in cells by a variety of repair enzymes. DNA base products are repaired by both base-excision and nucleotide-excision repair, but predominantly by the former. Two well known Escherichia coli enzymes, endonuclease III and formamidopyrimidine-DNA glycosylase account for the excision of most modified bases from DNA w1x. Eukaryotic counterparts of these enzymes exist in human cells w2x. Radioprotectors are agents that reduce toxicity, mutagenicity, or other biological effects of ionizing radiation w3,4x. The ability of some aminothiols to minimize damage caused by ionizing radiation has stimulated research on the possibility of using these compounds to protect non-target tissues in radiotherapy and to diminish secondary toxic effect of drugs in chemotherapy w5–8x. The mechanisms of radioprotection of aminothiol compounds is thought to involve scavenging of POH radicals, transferring hydrogen to DNA radicals, and causing an anoxic state near the DNA target w9–11x. Stabilization of chromatin may also contribute to the radioprotective properties of aminothiols and other compounds that bind to DNA w12x. Different authors have postulated that aminothiol compounds may form noncovalent bonds with their positively charged amino groups interacting electrostatically with the DNA phosphate groups w13–16x. Other authors have suggested that aminothiol compounds gain their protective effect modulating the endonucleolytic activity w17x. Unlike their protective effect on ionizing radiation, aminothiol compounds as 2- wŽaminopropyl. amino x ethanethiol ŽWR-1065. and cysteamine potentiate the induction of micronuclei by bleomycin treatment in Go human lymphocytes w18,19x and the mitotic re-
combination in yeast w20x. Potentiation by radioprotectors of the genotoxic effect of bleomycin without increasing other aspects of its toxicity could enhance its therapeutic effectiveness w16x. Sodium diethyldithiocarbamate ŽDDC. is a sulfhydryl compound, which is a structural analogous of the well-known S-2-aminoethylisothiuronic ŽAET.. This compound has been used in the treatment of acute nickel–carbonyl poisoning w21x. The chelating properties of DDC and other derivatives of dithiocarbamate have been widely studied w22x. On the other hand, S-2-aminoethyl-isothiouronicadenosin-5-triphosphate Žadeturon. is an AET related compound, which includes in its structure one adenosin5-triphosphate ŽATP. molecule. It is known that sulfhydryl compounds selectively reduces the indirect effect of ionizing radiation by scavenging hydroxyl radicals, which constitute the principal radical species responsible for DNA damage caused by radiation w23x. In the present work, we have studied the effect of both DDC and adeturon as possible radioprotective agents. For this, we have used bacterial assays, which give an indirect estimate of DNA damage. Among them, the Inductest w24x and SOS Chromotest w25x, which respectively measure phage and SOS gene induction in E. coli cells, have been reported as useful tools for studies of mutagenic effectiveness of g-rays, as well as to study the mechanisms of action of compounds that modify the effect of radiation w26–28x. Furthermore, and because the study of DNA degradation process may bring important information about the mechanisms by mean of which aminothiol compounds operate, we have studied this parameter in E. coli cells treated with g-irradiation in the presence of both DDC and adeturon. 2. Materials and methods 2.1. Bacterial strains and growth conditions The bacterial strains used in this work are listed in Table 1. Cultures were grown on supplemented minimal medium ŽSMM. Ž0.1% NH 4 Cl, 0.3% KH 2 PO4 , 0.6% Na 2 HPO4 , 0.05% NaCl, 0.001% CaCl 2 , 0.003% MgSO4 , 0.2% glucose, 0.1% casein hydrolizate, 0.001% thiamine HCl, pH 7.3., Luria Broth or Nutrient Broth w29x depending on the assay.
J.L. Fuentes et al.r Mutation Research 422 (1998) 339–345 Table 1 Bacterial strains
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2.5. ß-Galactosidase assay
Strain
Relevant characteristics
Source
AB1157 Hfr Ž l . C GC2181
– thi, str R str R sulA
B. Bachmann B. Bachmann B. Bachmann S. Casaregola
2.2. RadioprotectiÕe compounds Sodium diethyldithiocarbamate and S-2aminoethyl-isothiouronicadenosin-5-triphosphate were used. The effective incorporation time Ž30 min. and the effective radioprotective concentration Ž1000 mgrml, DDC and 1500 mgrml, adeturon. for E. coli cells, were previously determined by us w30x. 2.3. Irradiation g-Irradiation Ž60 Co. was accomplished using an MP-g-25 M irradiator ŽRussia.. The overnight culture was diluted 1:25 in fresh medium and shaken at 378C until a cell density of about 10 8 cellsrml was achieved. Then, cells were diluted 20-fold in fresh medium, with and without radioprotectors, and distributed into glass test tubes. Cell suspensions were irradiated at 28C with different doses. After g-irradiation, cells for ß-galactosidase assay were incubated for two hours at 378C with shaking and cells for the assay of lambda induction were incubated overnight. The dose rate was 10 Gyrmin as had been previously determined following Fricke’s method w31x. 2.4. Assay of bacteriophage induction E. coli lysogenic cells treated or untreated with g-irradiation were grown overnight in Nutrient broth and were centrifuged at 5000 = g for 10 min. Samples Ž0.1 ml. of the supernatant Žundiluted and 10– 100 fold diluted. were mixed with 0.1 ml of an overnight culture of the E. coli C strain Žgrown on Nutrient broth, supplement with 200 mgrml of streptomycin., held for 20 min at 378C, mixed with 2.5-ml of R-top agar and poured over R-agar base plates w32x. The presence of plaque-forming units ŽPFU. was determined after overnight incubation of the plates at 378C.
The ß-galactosidase assay was performed as it has been described w25x. Cell membranes of g-irradiated and nonirradiated cells of GC2181 strain were disrupted with SDS 0.1% in Z buffer during 20 min at room temperature. The enzyme reaction was started by adding o-nitrophenyl-ß-D-galactopyranoside ŽONPG. Ž4 mgrml in T buffer pH 8.8.. After 40 min, the enzymatic reaction was stopped by adding Na 2 CO 3 1 M, and absorbances at OD550 and OD420 were measured using a Spekoll-11 spectrometer ŽZeiss, Germany.. Enzyme activity was calculated as described w33x. 2.6. DNA degradation assay An overnight culture of the AB1157 strain was diluted 100-fold into SMM, containing thymine at 100 mgrml and w14 Cx-thymine ŽCENTIS, 30 Cirmmole. at 3 mCirml, and shaken at 378C to an OD650 of 0.2. These radioactive cells were centrifuged, washed and resuspended in the mineral salt portion of SMM, having an OD650 of 0.1, and contained radioprotective compounds, and g-irradiated at D 10 value Ž0.3 kGy.. These cells were mixed with an equal volume of SMM, containing twice the normal concentration of glucose, thiamine–HCl, and supplements, and were incubated. At different times of incubation, duplicate samples of 0.1 ml of culture were placed on Whatman No. 17 paper rectangles, previously saturated with TCA 10%, supported on steel pins and dried. Then, each was moistened with 0.2 ml of thymine at 1 mgrml and dried. At the end of the experiment, the rectangles were washed thrice with TCA 5% and once with ethanol 95%, dried and radioactivity was measured with a liquid scintillation ß-LKB spectrometer. Controls with nonirradiated and irradiated cells without radioprotectors were also included. 2.7. Statistical methods Data were transformed with a power transformation estimated by the methods of Box and Cox w34x. To determine if there were significant differences between treatments, an analysis of variance was
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carried out. If a significant value was found Ž p - 0.05., the multiple comparisons, as the Scheff’s method, were used to determine which groups were significantly different Ž p - 0.05. w35x.
exonucleases w37x. Our results indicate that DDC, but not adeturon, should have some effect on exonuclease activity, because it produces inhibition of DNA degradation. These data suggest that mechanisms of radioprotection mediated by these chemicals must be probably different.
3. Results and discussion 3.1. Effect on DNA degradation
3.2. Effect on SOS response in E. coli cells
The effect of DDC and adeturon in DNA degradation processes was determined measuring the remains of 3 H labelled DNA. No significant effect was found with adeturon; however a marked inhibitory effect of DNA degradation processes was observed in the presence of DDC Ž p - 0.05. ŽFig. 1.. Exonuclease activity in E. coli cells is principally undertaken by an inducible enzymatic system w36x. A variety of repair enzymes are capable of detecting oxidative DNA damage caused by ionizing radiation. DNA base products are repaired by both base-excision and nucleotide excision repair, but predominantly by the former w1,2x. It has been reported that aminothiol compounds repress RNA synthesis and inhibit the synthesis of inducible enzymes, including
In E. coli irradiated cells, the SOS regulon is triggered as part of the cellular response to exogenous DNA damage. In this cellular state, endonuclease and exonuclease activities are also enhanced w1x. As consequence of attempting to replicate past DNA lesions, an inducing signal is generated that results in the activation of RecA protein w38x. In its activated state, RecA enhances the self cleavage of the LexA repressor, thus leading to the expression of more than 20 SOS genes, including the sulA gene, and promoting the induction of the l prophage. As indirect markers of the protection mediated by DDC and adeturon of the damage on DNA, the level of induction of both l phage and sulA gene promoted by g-irradiation in E. coli was studied in the presence or absence of both radioprotectors. DDC gave contradictory effects. While a marked reduction of the induction level of sulA gene was obtained ŽFig. 2., this compound stimulated phage induction in E. coli Hfr Ž l . cells ŽFig. 3.. The differences between DDC and g-irradiated control data was statistically significant Ž p - 0.05. for the doses from 80 to 240 Gy ŽFig. 2. and for the doses from 400 to 1000 Gy ŽFig. 3.. The fact that DDC clearly diminished the induction level of the sulA gene is an evidence that the level of damage must have been reduced and suggests that the reason of the enhancement of phage induction is not directly related to the SOS response. In relation to this, similar effect on phage induction have been shown by cysteamine w32x. The mechanisms by which DDC potentiates phage induction in E. coli cells are probably not directly related to it being radioprotective. DDC radioprotection, as other sulfhydryl compounds w4x, should involve free hydroxyl radical scavenging. This action would be expected to reduce the phage induction by g-rays but no an enhancement. It is possible to speculate that this enhancement is mediated by its
Fig. 1. Radioprotective effect of DDC ŽI. and adeturon ŽB. on DNA degradation of g-irradiated cells Ž`. of E. coli. DNA degradation of nonirradiated cells Žv . is shown as control. Data are the average of two independent experiments with six replications for each dose. Standard deviations are shown.
J.L. Fuentes et al.r Mutation Research 422 (1998) 339–345
Fig. 2. Effect of DDC ŽI. and adeturon ŽB. on sulA gene induction of g-irradiated cells Ž'. of E. coli. Data are the average of three independent experiments with four replications for each dose. Standard deviations are shown.
quelant effect, perhaps modifying the binding of the cI repressor to the promoters of lytic functions of the phage. On the contrary, adeturon diminished the induction level either sulA gene as phage induction of g-irradiated cells ŽFigs. 2 and 3.. The differences between adeturon and g-irradiated control data was statistically significant Ž p - 0.05. for the doses from 40 to 240 Gy ŽFig. 2. and for the doses from 300 to 1000 Gy ŽFig. 3.. These results clearly show that adeturon diminishes the DNA damage produced by g-irradiation. Different authors have postulated that the mechanism of radioprotection of aminothiol compounds involves scavenging of hydroxyl radicals, donation of hydrogen to sites of DNA damage, and depletion of oxygen w9–11,18x. These also may form non covalent bonds with their positively charged amino groups interacting electrostatically with the DNA phosphate groups, stabilizing the chromatin w12–16x. Two mechanisms may explain the effect of adeturon on SOS functions in E. coli. First, as thiol, this compound may serve as scavenging agent of free hydroxyl radicals, depresses indirectly the DNA damage level. Second, adeturon must interact with DNA in the same form that other aminothiol compounds do it. This interaction, mediated by amino
343
groups of adeturon, may serve to concentrate these compounds near of the DNA damage site, increasing the potential for the thiol portion of the molecule to donate hydrogen, decreasing the damage level on DNA molecule. Some authors w17x, have suggested that aminothiol compounds are endonuclease repressors that doing more coordinate the DNA repair processes. Nowadays, it is well know that the most of DNA modified base are repaired by both base-excision and nucleotide-excision mechanisms w1x. In our study, we have used the DNA degradative assay as a way of measure the exonuclease activity in g-irradiated cells. The results of this assay suggested that adeturon does not have effect upon exonuclease activity. Other effective radioprotective concentrations of this compound have been also used Ždata not shown.. Radioprotective effect of DDC and adeturon may have clinical implications. There is interest in using aminothiols, to minimize damage to non target tissues in radiotherapy w7,8x. Use of protectors with chemotherapy drugs is also possible, especially if one can exploit a differential protection of normal cells and cancer cells w7,39x, although radioprotectors may reduce the damage caused by cytotoxic drugs in
Fig. 3. Effect of DDC ŽI. and adeturon ŽB. on phage induction of g-irradiated cells Ž'. of E. coli. Data are the average of three independent experiments with six replications for each dose. Standard deviations are shown.
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some instances or may increase it in others w40x. Future studies, which will combine molecular techniques and SOS gene induction assay in E. coli, may clarify not skilled topic related to the mode of action of radioprotective compounds.
w12x
w13x
Acknowledgements w14x
We thank the authors cited in the text for the supply of the bacterial strains.
w15x
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