Article pubs.acs.org/jnp
Antimutagenic Thio Compounds from Sisymbrium of f icinale Antonella Di Sotto,*,† Silvia Di Giacomo,† Annabella Vitalone,† Marcello Nicoletti,‡ and Gabriela Mazzanti† †
Department of Physiology and Pharmacology “V. Erspamer”, “Sapienza” University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy Department of Environmental Biology, “Sapienza” University of Rome, P.le Aldo Moro 5, 00185 Rome, Italy
‡
S Supporting Information *
ABSTRACT: Glucoputranjivin (1) and isopropyl isothiocyanate (2) were isolated from an aqueous dry extract of Sisymbrium of f icinale and were identified by spectroscopic analysis. The antimutagenic activity of these compounds was evaluated in a bacterial reverse mutation assay using E. coli WP2, WP2uvrA, and WP2uvrA/pKM101 strains, in comparison with the extract. In the absence of the exogenous metabolic activation system S9, the thio compounds exerted antimutagenic activity against the direct-acting mutagen methyl methanesulfonate, in all strains. In the presence of S9, both thio compounds were active against the indirect mutagens 2-aminoanthracene, in WP2uvrA, and 2-aminofluorene, in WP2. The antimutagenicity seems to be due to specific mechanisms, such as the induction of the adaptive response or the excision repair system. Conversely, the inhibition of the CYP450-mediated activation of mutagens was not supported by the present results. An antimutagenic effect was also observed for the S. off icinale aqueous extract against the arylamines 2AA and 2AF, but not against MMS. These results suggest that both thio compounds are involved in the antimutagenicity of S. of ficinale. The antimutagenicity of glucosinolate 1 is reported for the first time.
G
(Euphorbiaceae),7 is the main glucosinolate found in the aerial part of Sisymbrium of ficinale Scop. (Brassicaceae).8 The latter plant, also named Erysimum of f icinale and hedge mustard, is used as a remedy for airway ailments9 and in the past was considered useful to treat certain malignant growths.10 Previously, an aqueous dry extract of S. off icinale was shown to possess strong antimutagenic properties on an E. coli strain, with lesser effects on Salmonella typhimurium TA98 and TA100. It was hypothesized that this activity was due to glucosinolates, especially to compound 1, or to their metabolic derivatives.11 According to these data and in order to verify the contribution of specific compounds to the activity of the whole extract, in the present paper, the potential antimutagenic effect of thio compound 1 and its metabolic derivative isopropyl isothiocyanate (2) was investigated. The thio compounds 1 and 2 were isolated and chemically characterized, and their ability to inhibit or prevent the genotoxicity of known mutagenic agents was studied, using a bacterial reverse mutation assay (Ames test) on different Escherichia coli strains. This experimental design was based on previous evidence that S. of ficinale displayed antimutagenic activity particularly on the E. coli WP2uvrA strain, with lesser effects on S. typhimurium TA98 and TA100.12
lucosinolates are an important class of secondary plant metabolites distributed in several families of angiosperms, especially in the Brassicaceae.1 They are chemically defined as β-thioglucoside N-hydroxysulfates, which are characterized by a side chain and a sulfur-linked β-D-glucopyranose moiety.1 When vegetables are cut or chewed, glucosinolates get converted into various derivatives (isothiocyanates, thiocyanates, indoles, etc.) by the enzyme myrosinase, which is released from the plant tissue.1 The most common products are isothiocyanates, which arise from a “Lossen”-like rearrangement of the intermediate obtained after hydrolysis of glucosinolate.2 Their ecological role is related to defense mechanisms against bacteria, fungi, and herbivorous amphipods and to recognition of the host plant by predators. These compounds possess many biological activities including antimicrobial, antioxidant, and anti-inflammatory effects.3−5 Furthermore, great interest is aroused by the potential chemopreventive properties of both glucosinolates and their breakdown products. Several studies demonstrate that these compounds inhibit the carcinogenic process by several mechanisms, including blocking mutagenesis, inducing apoptosis in cancer cells, and arresting cancer cell progression.4 Epidemiological evidence suggests that dietary intake of cruciferous vegetables reduces the risk of cancer and chronic degenerative diseases, as they are a rich source of glucosinolates, such as glucoraphanin.6 Glucoputranjivin (1), isolated initially from the seeds of Lunaria annua L. (Brassicaceae) and identified as the precursor of isopropyl isothiocyanates from Putranjiva roxburghii Wall. © 2012 American Chemical Society and American Society of Pharmacognosy
Received: March 29, 2012 Published: November 29, 2012 2062
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the latter strain, the susceptibility to mutations is increased by the presence of plasmid pKM101, as this factor enhances errorprone repair. As a consequence, this strain is very sensitive to damage induced by cross-linking and pro-oxidant mutagens.19 Furthermore, since bacteria, unlike mammals, are unable to metabolize chemicals by cytochrome P450 enzyme (CYP450), an exogenous metabolic activation system (S9) was also included in the Ames test, in order to study the antimutagenicity of 1 and 2 when CYP450-mediated biotransformations occur.13 According to strain sensitivity and to the type of mutations induced, the antimutagenicity effects of 1 and 2 were evaluated against three different mutagens, namely, methyl methanesulfonate (MMS), 2-aminoanthracene (2AA), and 2-aminofluorene (2AF). This last compound was used on WP2, as 2AA was not found to be mutagenic for this strain.20 MMS is a direct-acting DNA alkylating agent, which induces mutations both in repair-deficient and in wild strains.21,22 Conversely, the mutagens 2AA and 2AF are pro-carcinogenic compounds that form DNA adducts, as a consequence of metabolic activation by CYP450, especially CYP1A1, CYP1A2, and CYP1B isoenzymes.23−25
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Taking into account that the sensitivity of a bacterial strain to a mutational event is strictly related to its genome, three different E. coli strains, WP2, WP2uvrA, and WP2uvrA/ pKM101 (or WP2uvrA/R), were used in order to study the mechanism of antimutagenicity. These strains all have a common tryptophan dependence, due to an ochre (UAA) nonsense mutation in the trpE65 gene by mechanisms of misreplication or misrepair;14,15 this dependence can be reversed, by a base change at the site of the original alteration or by a change at other sites, so that the original defect is suppressed.16 Moreover, each strain carries additional mutations that make it sensitive to specific mutational damage. In particular, the WP2 strain is a derivative of E. coli B/r, a wild type for DNA repair ability, which possesses a greater resistance to the lethal effects of UV radiation.17 Conversely, WP2uvrA and WP2uvrA/pKM101 are two mutant strains of WP2, in which the uvrA marker induces a DNA-repair deficiency.18 In
RESULTS AND DISCUSSION In the Ames test, the thio compounds 1 and 2 produced neither precipitates nor cytotoxic effects up to 300 μg/plate (110 μg/ mL), so this concentration was chosen as the highest one to use in the following assays. In the range of concentrations tested (10−300 μg/plate), 1 and 2, in a similar manner to the S. of f icinale extract (Table S1, Supporting Information), were devoid of mutagenic effects in all strains tested, in the absence and presence of the metabolic activation system (Table 1), and so were considered suitable to test for their antimutagenic activity against the mutagens MMS, 2AF, and 2AA. Both the thio compounds tested reduced significantly the number of chemically induced revertant colonies, even though with a different spectrum of activity and a different potency. In particular, in the absence of the metabolic activator S9, they
Table 1. Effect of Compounds 1 and 2 on the Number of Spontaneous Revertant Colonies in Escherichia coli WP2, WP2uvrA, and WP2uvrA/R Strains with and without the S9 Metabolic Activation System (means ± SEM, n = 6)a number of revertant colonies WP2 [μg/plate] 1
2
vehicle methyl methane sulfonate 2-aminofluorene 2-aminoanthracene
10 30 50 100 300 10 30 50 100 300 500 10 10
− S9 62.7 62.0 62.7 63.0 62.3 51.0 54.0 52.3 54.7 60.3 58.7 190.3
± ± ± ± ± ± ± ± ± ± ± ±
3.3 4.4 7.9 5.5 1.8 4.0 5.6 4.3 3.0 6.7 3.5b 12.2d
WP2uvrA − S9
+ S9 39.7 38.3 37.3 34.3 35.0 33.0 32.0 33.0 33.3 35.3 33.0
± ± ± ± ± ± ± ± ± ± ±
3.0 4.3 5.6 5.0 4.0 5.6 3.2 5.0 3.0 2.6 3.6c
49.3 37.7 47.0 38.3 44.7 48.0 46.7 50.0 45.7 49.3 44.0 167.7
± ± ± ± ± ± ± ± ± ± ± ±
3.0 2.0 8.0 4.4 4.4 3.5 2.0 1.5 3.8 3.0 3.1b 3.6d
WP2uvrA/R + S9
51.7 49.7 51.0 47.3 47.3 45.0 49.7 48.0 54.0 45.7 55.9
± ± ± ± ± ± ± ± ± ± ±
2.6 2.3 2.5 2.0 0.7 1.5 1.4 3.5 3.0 3.0 2.4c
− S9 215.0 221.3 211.3 219.7 220.0 225.3 229.3 230.3 225.7 223.3 234.4 950.1
± ± ± ± ± ± ± ± ± ± ± ±
5.0 5.8 4.3 7.8 6.9 3.9 3.0 6.0 6.2 6.8 10.1b 52.5d
+ S9 347.5 318.5 319.0 322.5 295.5 324.7 325.0 307.0 333.3 331.0 323.2
± ± ± ± ± ± ± ± ± ± ±
4.5 16.5 13.0 2.5 4.5 13.6 16.1 13.6 14.0 11.6 12.6c
80.0 ± 2.3d 168.5 ± 29.7d
994.0 ± 10.5d
a
For each treatment, the number of spontaneous revertant colonies was determined by scoring at least six plates, obtained from two or three experiments. bH2O (25 μL) and DMSO (25 μL). cDMSO (50 μL). dDenotes a significant difference from the vehicle (p < 0.01; Anova + Dunnett’s multiple comparison post-test). 2063
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presence of mutagens, as the viability, with respect to control, was from 80% to 100% for WP2, from 82% to 90% for WP2uvrA, and from 80% to 88% for WP2uvrA/R (Figures 1B and 2B). In the presence of the exogenous metabolic system, 1 and 2 also reduced the number of revertant colonies induced by 2AA for WP2uvrA, with a maximal inhibition of 80.1% and 81.9%, respectively (Figures 3A and 4A), while slight effects were
inhibited the mutagenic effect of MMS in all strains tested. The maximal inhibition exhibited by 1 and 2 was 32.8% and 19.7% for WP2, 44.6% and 45.7% for WP2uvrA, and 38.5% and 32.9% for WP2uvrA/R, respectively (Figures 1A and 2A). According
Figure 1. Effect of compound 1 on the number of revertant colonies induced by methyl methane sulfonate (MMS) in Escherichia coli WP2, WP2uvrA, and WP2uvrA/pKM101 strains. Values are expressed as mean ± SEM (n = 6). (A) Percentage of inhibition. (B) Cell survival. Strong: inhibition >40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%) in WP2uvrA, while moderate or weak (40%; moderate: inhibition between 25% and 40%; weak: inhibition 40%; moderate: inhibition between 25% and 40%; weak: inhibition 96% pure, as determined by HPLC analysis under the conditions already described and using sinigrin as internal standard.11 For biological assays, the substances were dissolved in DMSO by sonication, up to a concentration of 12 mg/mL. Bacterial Strains, Chemicals, and Metabolic Activation System. A set of three tester strains, namely, E. coli WP2 (trpE65), E. coli WP2uvrA (trpE65ΔuvrA), and E. coli WP2uvrA (trpE65 ΔuvrA pKM101), was used. All strains were kindly supplied by the Research Toxicological Centre (Pomezia, Rome, Italy). After confirmation of genotypes by the Strain Check assay,53 the permanent cultures were prepared and then frozen. The working cultures, prepared from the permanent ones, were incubated overnight (16 h) at 37 °C, to reach a concentration of approximately 2 × 109 bacteria/mL. 2066
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Methyl methanesulfonate (CAS 66-27-3; purity 99%), 2-aminoanthracene (CAS 613-13-8; purity 96%), and 2-aminofluorene (CAS 153-78-6; purity 98%) were used as known mutagens. Sinigrin was obtained from Jimes S.p.a. (Milan, Italy). Ethyl acetate (CAS 141-786), n-butanol (CAS 71-36-3), and n-propanol (CAS 71-23-8), used for chromatographic isolation, were of analytical grade and were purchased from Merck KGaA (Darmstadt, Germany). The nutrient broth, bacteriological agar, and nutrient agar were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). All other chemicals were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). The metabolic activation system S9 was prepared just before use by adding phosphate buffer (0.2 M; 500 μL), deionized water (130 μL), KCl (0.33 M in sterile deionized water; 100 μL), MgCl2 (0.1 M in sterile deionized water; 80 μL), S9 fraction (100 μL), glucose-6phosphate (0.1 M in sterile deionized water; 50 μL), and NADP (0.1 M in sterile deionized water; 40 μL); this mixture was kept on ice during testing. S9 fraction (the liver postmitochondrial supernatant of rats treated with the mixture phenobarbital/β-naphthoflavone to induce the hepatic microsomal enzymes) was purchased from Trinova Biochem GmbH (Giessen, Germany). When manipulating mutagens in the laboratory, the following safety precautions were taken. All workers needed personal protective equipment (masks, disposable coat, double gloves, etc.): this protective clothing was not worn outside the area designated for handling mutagens. A deep cleaning of all possibly contaminated surfaces and areas was done. Finally, mutagens were safely stored in closed containers that were clearly labeled and marked with visible hazard and warning signs. Preliminary Assays. Preliminary tests were performed to determine the nontoxic concentration of test substances on bacterial strains and to exclude a mutagenic effect. Initially, the solubility of samples in the final mixture was assessed to establish the highest concentration to use in the assays. Insolubility was considered as being the formation of a precipitate under the test conditions and evident to the unaided eye.53 Starting from the highest soluble concentrations, solutions of 1 and 2 were prepared in DMSO by serial dilution (dilution factor of about 1:2 and/or 1:3). Cytotoxicity was evaluated as a reduction in the number of revertant colonies and as a change of the auxotrophic background growth (background lawn) in comparison with control plates.54 Toward this aim, an overnight culture (100 μL) was added to a solution of the test substance (50 μL) and the S9 mixture or phosphate buffer (0.1 M; 500 μL). The mixture was preincubated with shaking at 37 °C for 30 min and then added with top agar (2 mL) containing 10% tryptophan (0.5 mM); then, it was poured onto a minimal agar plate. After incubation at 37 °C for 48 h, the plates were examined, with tryptophanindependent revertant colonies and viable cells scored and the bacterial background lawn observed. Mutagenicity of the substances was assayed using the preincubation method, as reported by Maron and Ames54 and Green and Muriel.55 The vehicle DMSO (2% v/v) was used as the negative control. In turn, the mutagens MMS (500 μg/plate) without S9 and 2AA (10 μg/plate) and 2AF (10 μg/plate) with S9 were used as positive controls, in order to verify the bacterial sensitivity to genotoxic damage. These concentrations of mutagens, obtained from the linear part of the concentration−response curve, were chosen, as they increased the number of revertant colonies at least 2-fold above the control value. The experiments were repeated at least twice, and each concentration was tested in triplicate. To perform these tests, an overnight culture (100 μL) was added to the test solutions (50 μL) and to the S9 mixture or phosphate buffer (0.1 M; 500 μL). Each mixture was gently vortexed in a sterile tube; then it was preincubated under shaking at 37 °C for 30 min. After preincubation, the tubes were added with top agar (2 mL) containing 10% tryptophan (0.5 mM), gently vortexed, and poured onto a minimal agar plate. The plates were then incubated at 37 °C for 72 h and then examined. The tryptophan-independent revertant colonies and the viable cells were scored, and the bacterial background lawn was observed. A positive response in the mutagenicity assay was defined as an increase
of at least 2-fold above the control value in the tryptophanindependent revertant colonies in each strain.56 Antimutagenicity Assay. This test was carried out, as previously described by Edenharder et al.,57 by the preincubation method reported above. Substances were evaluated at the same concentrations used in the mutagenicity testing. Solutions of samples were prepared as for the mutagenicity assay. Antimutagenic activity was determined against the mutagens MMS (1700 μg/plate) without S9 and 2AF (25 μg/plate) and 2AA (25 μg/plate) with S9. These concentrations, obtained from the linear part of the concentration−response curve of mutagens, were chosen, as they induced a submaximal mutagenic effect (about 70%). Plates containing mutagen (100% of mutagenic activity) or vehicle (DMSO 2% v/v; lack of mutagenic activity) were included. Plates containing both strain and test substances were included also, in order to exclude the reduction in the number of revertant colonies observed as being due to a cytotoxic effect. Experiments were repeated at least twice, and each concentration was tested in triplicate. To perform this test, the bacterial overnight culture (100 μL), the mutagen (25 μL), the test substance solution (25 μL), and the S9 mixture or 0.1 M phosphate buffer (500 μL) were preincubated under shaking at 37 °C for 30 min. After preincubation, top agar (2 mL), containing 10% tryptophan (0.5 mM), was added. This mixture then was gently vortexed and poured onto a minimal agar plate. The plates were incubated at 37 °C for 72 h and then examined. The tryptophan-independent revertant colonies and the viable cells were scored, and a bacterial background lawn was observed. The percentage of inhibition of the mutagenic effect was calculated according to the formula 100 − [(T/M) × 100], where T is the number of revertant colonies/plate in the presence of mutagen and test substance, and M is the number of revertant colonies/plate in the presence of the mutagen alone. According to Negi et al.,26 the antimutagenicity effect was considered moderate when the inhibitory effect of the test compounds was in the range 25−40% and strong when the inhibitory effect was higher than 40%. When the inhibitory effect was lower than 25%, this was considered weak, and it was not recognized as a positive result. Cell Survival. In order to exclude that the treatment could reduce cell viability by inducing cytotoxicity, the same experimental protocol used for the antimutagenicity assay was repeated in cell survival studies. Likewise, besides the plates treated with the test substances in the presence of the mutagens MMS (1700 μg/plate), 2AF (25 μg/ plate), and 2AA (25 μg/plate), control plates containing only the mutagen or solvent were set up. To perform this testing, 100 μL of bacterial overnight culture, 25 μL of the mutagen solutions, 25 μL of the test substances, and 500 μL of S9 mix or 0.1 M phosphate buffer were mixed in a sterile tube, gently vortexed, and preincubated under shaking at 37 °C for 30 min. At the end of the preincubation, each mixture was diluted to obtain a concentration of 2 × 103 cells/mL, then added to 2 mL of top agar, and plated onto nutrient agar plates. The positive control was prepared by adding the solvent of the test substance to the mutagen (25 μL + 25 μL), while the negative control was obtained by adding the solvent of the test substance to that of the mutagen (25 μL + 25 μL); then both controls underwent the experimental procedure described above. The resulting plates were incubated at 37 °C for 72 h. After incubation, plates were scored for the colonies originating from viable cells. The reduction of cell viability induced by the treatment was evaluated by comparing the number of viable cells of the negative control and those of each treatment. A treatment was considered cytotoxic when the cell viability was less than 70% with respect to the control. Statistical Analysis. All values are expressed as means ± SEM. The one-way analysis of variance (one-way ANOVA), followed by Dunnett’s multiple comparison post-test, was used to analyze the difference between treatments. A p value of
