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Assessment of potential mutagenic activities of a novel benzothiazole MAO-A inhibitor E2011 using Salmonella typhimurium YG1029
Mutation Research 472 (2000) 163–169
Assessment of potential mutagenic activities of a novel benzothiazole MAO-A inhibitor E2011 using Salmonella typhimurium YG1029 Gen Sato a,∗ , Shoji Asakura a , Atsushi Hakura b , Yoshie Tsutsui-Hiyoshi a , Naoki Kobayashi c , Kazuo Tsukidate a b
a Exploratory Safety Assessment Research, Eisai Co. Ltd., Ibaraki 300-2635, Japan Department of Developmental Safety Assessment Research, Eisai Co. Ltd., Gifu 501-6195, Japan c Discovery Technology Research Laboratories, Eisai Co. Ltd., Ibaraki 300-2635, Japan
Received 25 April 2000; received in revised form 3 October 2000; accepted 12 October 2000
Abstract The potential initiation activities of a novel monoamine oxidase type-A (MAO-A) inhibitor E2011, which induced preneoplastic foci in the rat liver, were investigated by comparing the mutagenic activity of E2011, 6-aminobenzothiazole (6-ABT, a structural scaffold of E2011) and its derivatives, which are suggested primary reactive metabolites for E2011-induced hepatotoxicity in the rats in vivo, in the Ames assay system employing two Salmonella tester strains, TA100 and YG1029, a bacterial O-acetyltransferase-overproducing strain of TA100. E2011, a tertiary amine, showed no mutagenic activity both in the Salmonella typhimurium TA100 and YG1029 with and without S9 mix. On the other hand, a secondary aromatic amine ER-174238-00, a typical decarbonated metabolite of E2011, showed weak but significant mutagenicity in YG1029 in the presence of S9 mix, and a primary aromatic amine ER-174237-00, an N-dealkylated derivative of ER-174238-00, exhibited S9-dependent potent mutagenicity in YG1029. Thus, it appears that primary and secondary amino moieties of benzothiazole derivatives at C6 -position are the specific structures contributing to their mutagenic activity. In addition, the alkyl group at C2 -position of E2011, ER-174237-00 and ER-174238-00 is suggested to intensify the mutagenic activity, since the mutagenicity of ER-174237-00 is approximately two-fold higher than that of 6-ABT, which has hydrogen at C2 -position in the place of the alkyl group. These results strongly suggest that E2011 has potential initiation activities in the rat liver in vivo after undergoing decarbonation, one of the metabolic pathways, at the carbonyl moiety of oxazolidinone ring to form mutagenic amine(s). © 2000 Elsevier Science B.V. All rights reserved. Keywords: E2011; Benzothiazole; Salmonella typhimurium; Acetyltransferase; Decarbonation; Aromatic amine
1. Introduction
∗ Corresponding author. Tel.: +81-298-47-5714; fax: +81-298-47-5956. E-mail address: [email protected] (G. Sato).
Benzothiazole is reported to be widely used as a scaffold in many physiologically active compounds, such as an antifungal drug [1], a topical carbonic anhydrase inhibitor [2], an antihypoxic drug [3], an anti-nematode drug [4], a dual inhibitor of thrombox-
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ane A2 synthetase and 5-lipoxygenase [5], a selective and reversible inhibitor of monoamine oxidase type A (MAO-A) [6] and even in chemicals for the rubber and tyre industry [7]. Benzothiazole derivatives with an amino group, e.g. 6-aminobenzothiazole (6-ABT), are classified as aromatic amines and are expected to be characterized as mutagenic compounds, since many of them possess mutagenic potency and/or carcinogenicity [8]. Their capacity to damage DNA leading to mutagenicity/carcinogenicity depends on metabolic activation catalyzed by cytochrome P450 (CYP) 1A1/2 isozymes and subsequently by acetyltransferase (AT) [9,10] or in some cases by sulfotransferase (ST) [10,11]. Mutagenicity test employing modified Ames tester strains YG1024 and YG1029, bacterial O-AToverproducing strains of TA98 and TA100, respectively, has been extensively validated [12]. These strains were shown to have capacity for O-acetylation and N-acetylation of nitroarenes, aromatic amines or hydrazines to form ultimate mutagens. That is, they are sensitive for detecting potential mutagenic activities of these mutagenic/carcinogenic compounds [13–16]. For instance, promutagen in smokers’ urine was more sensitively detected in YG1024 than in TA98 [17], and 4-aminobiphenyl, an aromatic amine present in tobacco smoke, was more sensitively detected in YG1029 than in TA100 [18]. In our recent study, oral administration of E2011 at doses of 30 and/or 100 mg/kg to rats for 13 weeks produced nuclear enlargement of hepatocytes with appearance of altered cell foci in some animals. Moreover, the number and area of glutathione S-transferase placental form (GST-P) positive hepatic foci were significantly higher in E2011-treated female animals [19]. It has been reported that altered cell foci are to some extent indicative of putative preneoplastic lesions and an ongoing carcinogenic process [20]. To explain the mechanism of this hepatotoxicity, we proposed the hypothesis that the aromatic amine metabolite(s) of E2011 obtained by decarbonation may contribute to the toxicity; decarbonation was identified as one of the in vivo metabolic pathways of E2011, through which about 15% of administered E2011 was metabolized [21]. The purpose of this study was to confirm the potential initiation activities of E2011 by comparing the mutagenic activity of E2011 and its structurally
related compounds, i.e. two suggested metabolites (ER-174237-00 and ER-174238-00) and the structural scaffold of E2011 (6-ABT). The specific structures contributing to the mutagenic activity as well as the mechanism for in vivo hepatotoxicity are discussed.
2. Materials and methods 2.1. Chemicals and materials (5R)-3-[2-((1S)-3-Cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011) was synthesized at Eisai Chemical Co. (Ibaraki, Japan) [22] and the purity was >99% as determined by TLC and HPLC. 6-ABT (CAS No. 533-30-2, >98% purity) was purchased from Lancaster Synthesis Ltd. (Lancashire, UK). ER-174237-00 and ER-174238-00 were synthesized in our laboratory from (S)-6-amino-2-(5-oxotetrahydrofuran-2-yl)benzothiazole according to the published procedures [22] and their purity determined by HPLC was both 100%. The chemical structures of these four compounds are shown in Fig. 1. S9 fraction of phenobarbital- and -naphthoflavonepretreated male SD rat liver and Cofactor I were purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan). 2.2. In vitro Ames/Salmonella assay Mutagenicity of the four compounds was determined using the Salmonella mutagenicity assay with strains TA100 and/or YG1029. S. typhimurium TA100 and YG1029 were kindly provided by Dr. B.N. Ames (University of California, Berkeley, CA, USA) and by Dr. T. Nohmi (National Institute of Hygienic Sciences, Tokyo, Japan), respectively. The mutagenicity was assayed by the Ames test with a modification of pre-incubation [23,24]. Briefly, a mixture containing the test compound in 0.1 ml of dimethyl sulfoxide (DMSO), 0.1 ml of culture in the early stationary phase of the tester strain, and 0.5 ml of 100 mM sodium phosphate buffer (pH 7.4) or S9 mix was incubated for 20 min at 37◦ C in a test tube with shaking. After incubation, 2 ml of 0.05 mM l-histidine–0.05 mM biotin molten top agar was added to the test tube, and the contents mixed and poured onto a plate of minimal glucose agar medium. The plate was incubated for 48 h
G. Sato et al. / Mutation Research 472 (2000) 163–169
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Fig. 1. Chemical structure of the tested compounds in the Salmonella assay.
at 37◦ C, then revertant colonies were counted. Two plates were used for each dose, and four (TA100) or eight (YG1029) plates were used as negative controls (DMSO). S9 mix (0.5 ml) consisted of 0.05 ml of rat liver S9 fraction (1.2 mg of protein concentration) and 0.45 ml of a cofactor solution (Cofactor I), containing a final concentration of 8 mM MgCl2 , 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADPH and 4 mM NADH, in 100 mM sodium phosphate buffer (pH 7.4). In this study, we employed the tester strains sensitive to chemicals causing base pair-substitution mutation (TA100/YG1029), but not the strains sensitive to frameshift mutation (such as TA98/YG1024), because our preliminary experiment revealed that YG1029 was more sensitive for detecting the 6-ABT mutagenicity than YG1024 (data not shown). 2.3. Statistical analysis Linear regression analyses were made to determine if a linear dose response relationship existed.
3. Results and discussion Fig. 2 shows the mutagenic activity of E2011, ER-174237-00, ER-174238-00, and 6-ABT in S.
typhimurium TA100 and/or YG1029, a bacterial O-AT-overproducing strain of TA100, which is highly sensitive to the mutagenicity of nitroarenes and aromatic amines [12]. E2011, a tertiary amine, showed no mutagenic activity both in TA100 and YG1029 with and without S9 mix up to the doses of 5.0 (TA100) or 2.5 mg (YG1029)/plate without significant decrease in bacterial background lawn. On the other hand, a secondary aromatic amine ER-174238-00, a typical decarbonated metabolite of E2011, showed weak but significant mutagenicity in YG1029 in the presence of S9 mix (linear regression, r 2 = 0.974 in the range of 0.2–2.5 mg/plate). The highest reverse mutation activity of ER-174238-00 (mean ± S.E.; 210 ± 2 revertants/plate) was observed at 2.5 mg/plate, which was apparently higher than that of the negative control (mean ± S.E.; 115 ± 4 revertants/plate). Moreover, a primary aromatic amine ER-174237-00, an N-dealkylated derivative of ER-174238-00, exhibited S9-dependent potent mutagenicity in YG1029 in a dose-dependent manner in the range of 0.1–1.25 mg/plate (linear regression, r 2 = 0.982), with the maximum mutagenic activity of 1972 revertants/plate (17.1-fold increase over control). A primary aromatic amine 6-ABT, which has hydrogen at C2 -position in the place of the alkyl group as in ER-174237-00, exhibited S9-dependent
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Fig. 2. Mutagenicity results of the benzothiazole derivatives in the tester strains TA100 and YG1029. Data are presented as mean ± S.E.
potent mutagenicity in YG1029 (linear regression, r 2 = 0.988 in the range of 0.1–1.25 mg/plate, with the maximum mutagenic activity of 956 revertants/plate), but not in TA100 up to the doses of 2.5 mg/plate. Namely, the exocyclic-tertiary amine structure in E2011, i.e. oxazolidinone ring, had no muta-
genic effect. The secondary amine structure as in ER-174238-00, however, acted as S9-dependent weak mutagen in O-AT-overproducing tester strain YG1029, and the primary amine structure as in ER-174237-00 and 6-ABT exhibited its S9-dependent potent mutagenicity in YG1029 in a dose-dependent
G. Sato et al. / Mutation Research 472 (2000) 163–169
manner. These data suggest that primary and secondary amino moieties of benzothiazole derivatives at C6 -position are the specific structures contributing to their S9 mix- and O-AT-dependent mutagenic activity. Furthermore, the alkyl group at C2 -position of E2011,
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ER-174237-00 and ER-174238-00 is suggested to intensify the mutagenic activity, since the mutagenicity of ER-174237-00 is approximately two-fold higher than that of 6-ABT, which has hydrogen at C2 -position in the place of the alkyl group.
Fig. 3. Possible bioactivation pathway of E2011.
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The possible mechanism of bioactivation of E2011 is shown in Fig. 3. In order to exert its potential mutagenic activity, E2011 needs to be initially decarbonated to form a pro-mutagenic secondary amine or possibly a primary amine by following N-dealkylation. They will be subsequently catalyzed by CYPs (in the extracellular S9 mix) to form N-hydroxylamines and then transformed to the ultimate mutagenic species through acetylation (by bacterial intracellular AT) as represented by the well-known mutagenic/carcinogenic aromatic or heterocyclic amines [8–10]. Although it is not clear why E2011 is not mutagenic in TA100 and even in YG1029, a possible explanation for this is the lack of the decarbonation step in the typical Salmonella assay, in contrast to rats, where about 15% of administered E2011 undergoes this in vivo decarbonation pathway [21]. The decarbonation step might require certain cofactors in addition to CYPs’ and/or S9 fraction from extrahepatic tissues such as lung, intestine or kidney, which also participate in drug metabolism [25,26]. For certain chemicals, it is difficult or impossible to detect their potential mutagenicity in routine assays in bacteria [27,28]. E2011 appears to be one of these chemicals that will be classified as a false negative mutagen, in this case, because of the lack of the decarbonation step in the Salmonella assay system. Further work to clarify whether E2011 has promotion activity as well as to identify the mechanisms for the in vivo decarbonation step would be necessary to understand the nature of this chemical. In conclusion, our results strongly suggest that E2011 has potential initiation activities in the rat liver in vivo after undergoing decarbonation, one of the metabolic pathways, at the carbonyl moiety of oxazolidinone ring to form mutagenic amine(s).
Acknowledgements The authors are grateful to Drs. Toyohiko Aoki and Satoru Hosokawa (Department of Developmental Safety Assessment Research, Eisai Co. Ltd.) for their valuable comments and to Ms. Deborah Satoh (Research and Development Planning and Coordination, Eisai Co. Ltd.) for her kind advice in the preparation of this manuscript.
References [1] H. Küçükbay, B. Durmaz, Antifungal activity of organic and organometallic derivatives of benzimidazole and benzothiazole, Arzneim. -Forsch./Drug Res. 47 (1997) 667–670. [2] P.H. Kalina, D.J. Shetlar, R.A. Lewis, L.J. Kullerstrand, R.F. Brubaker, 6-Amino-2-benzothiazolesulfonamide. The effect of a topical carbonic anhydrase inhibitor on aqueous humor formation in the normal human eye, Ophthalmology 95 (1988) 772–777. [3] E. Mohr, N.P.V. Nair, M. Sampson, S. Murtha, G. Belanger, B. Pappas, T. Mendis, Treatment of Alzheimer’s disease with sabeluzole: functional and structural correlates, Clin. Neuropharmacol. 20 (1997) 338–345. [4] J. Surin, Anti-nematode activity of sixteen compounds against Trichinella spiralis in mice — a possible new screen for macrofilaricides, Southeast Asian J. Trop. Med. Public Health 26 (1995) 128–134. [5] Y. Komatsu, N. Minami, Synthesis of a novel dual inhibitor of thromboxane A2 synthetase and 5-lipoxygenase (E3040) via the direct coupling reaction of hydroquinone with 3-pyridinecarboxaldehyde, Chem. Pharm. Bull. 43 (1995) 1614–1616. [6] T. Kagaya, A. Kajiwara, S. Nagato, K. Akasaka, A. Kubota, E2011, a novel, selective and reversible inhibitor of monoamine oxidase type A, J. Pharmacol. Exp. Ther. 278 (1996) 243–251. [7] C.-D. Wacker, B. Spiegelhalder, R. Preussmann, New sulfonamide accelerators derived from ‘safe’ amines for the rubber and tyre industry, in: I.K. O’Neill, J. Chen, H. Bartsch (Eds.), Relevance to Human Cancer of N-Nitroso Compounds, Tobacco Smoke and Mycotoxins, IARC, Lyon, 1991, pp. 592–594. [8] F.A. Beland, F.F. Kadlubar, Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons, in: C.S. Cooper, P.L. Grover (Eds.), Chemical Carcinogenesis and Mutagenesis I, Springer, Berlin, 1990, pp. 267–325. [9] C.M. King, S.J. Land, R.F. Jones, M. Debiec-Rychter, M.-S. Lee, C.Y. Wang, Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines, Mutat. Res. 376 (1997) 123–128. [10] H.-U. Aeschbacher, R.J. Turesky, Mammalian cell mutagenicity and metabolism of heterocyclic aromatic amines, Mutat. Res. 259 (1991) 235–250. [11] H. Glatt, Bioactivation of mutagens via sulfation, FASEB J. 11 (1997) 314–321. [12] P.D. Josephy, P. Gruz, T. Nohmi, Recent advances in the construction of bacterial genotoxicity assays, Mutat. Res. 386 (1997) 1–23. [13] M. Watanabe, M. Ishidate Jr., T. Nohmi, Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels, Mutat. Res. 234 (1990) 337–348. [14] P. Einistö, M. Watanabe, M.T. Nohmi, M. Ishidate Jr., Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities, Mutat. Res. 259 (1991) 95–102.
G. Sato et al. / Mutation Research 472 (2000) 163–169 [15] G.J. Stevens, E.J. LaVoie, C.A. McQueen, The role of acetylation in the mutagenicity of the antitumor agent, batracylin, Carcinogenesis 17 (1996) 115–119. [16] L.E. Lemke, C.A. McQueen, Acetylation and its role in the mutagenicity of the antihypertensive agent hydralazine, Drug Metab. Dispos. 23 (1995) 559–565. [17] C. Kuenemann-Migeot, F. Callais, I. Momas, B. Festy, Use of Salmonella typhimurium TA98, YG1024 and YG1021 and deconjugating enzymes for evaluating the mutagenicity from smokers’ urine, Mutat. Res. 390 (1997) 283–291. [18] L.N. Dang, C.A. McQueen, Mutagenicity of 4-aminobiphenyl and 4-acetylaminobiphenyl in Salmonella typhimurium strains expressing different levels of N-acetyltransferase, Toxicol. Appl. Pharmacol. 159 (1999) 77–82. [19] G. Sato, T. Chimoto, T. Aoki, S. Hosokawa, S. Sumigama, K. Tsukidate, F. Sagami, Toxicological response of rats to a novel monoamine oxidase type-A inhibitor, (5R)-3-[2((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011), orally administered for 13 weeks, J. Toxicol. Sci. 24 (1999) 165–175. [20] G.M. Williams, The significance of chemically-induced hepatocellular altered foci in rat liver and application to carcinogen detection, Toxicol. Pathol. 17 (1989) 663–674. [21] T. Naitoh, M. Kakiki, T. Kozaki, M. Mishima, T. Yuzuriha, T. Horie, Identification of the metabolites of a new oxazolidinone MAO-A inhibitor in rat, Xenobiotica 27 (1997) 1071–1089.
169
[22] K. Akasaka, A. Kajiwara, S. Nagato, Y. Iinuma, I. Yoshida, A. Sasaki, M. Mizuno, A. Kubota, T. Kagaya, M. Komatsu, Oxazolidone derivatives, WO 9308179, Patent No. JP 05194440 (1993). [23] D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 (1983) 173–215. [24] A. Hakura, H. Mochida, Y. Tsutsui, K. Yamatsu, Mutagenicity and cytotoxicity of naphthoquinones for Ames Salmonella tester strains, Chem. Res. Toxicol. 7 (1994) 559–567. [25] M. Schwenk, Mucosal biotransformation, Toxicol. Pathol. 16 (1988) 138–146. [26] D.R. Krishna, U. Klotz, Extrahepatic metabolism of drugs in humans, Clin. Pharmacokinet. 26 (1994) 144–160. [27] S. Venitt, R. Baker, H. Liber, T. Matsushima, G. Probst, E. Zeiger, Summary report on the performance of the bacterial mutation assays, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter, M.D. Shelby (Eds.), Progress in Mutation Research, Vol. 5, Evaluation of Short-Term Tests for Carcinogens, Elsevier, Amsterdam, 1985, pp. 11–23. [28] G. Reifferscheid, J. Heil, Validation of the SOS/umu test using test results of 486 chemicals and comparison with the Ames test and carcinogenicity data, Mutat. Res. 369 (1996) 129–145.
Assessment of potential mutagenic activities of a novel benzothiazole MAO-A inhibitor E2011 using Salmonella typhimurium YG1029 Gen Sato a,∗ , Shoji Asakura a , Atsushi Hakura b , Yoshie Tsutsui-Hiyoshi a , Naoki Kobayashi c , Kazuo Tsukidate a b
a Exploratory Safety Assessment Research, Eisai Co. Ltd., Ibaraki 300-2635, Japan Department of Developmental Safety Assessment Research, Eisai Co. Ltd., Gifu 501-6195, Japan c Discovery Technology Research Laboratories, Eisai Co. Ltd., Ibaraki 300-2635, Japan
Received 25 April 2000; received in revised form 3 October 2000; accepted 12 October 2000
Abstract The potential initiation activities of a novel monoamine oxidase type-A (MAO-A) inhibitor E2011, which induced preneoplastic foci in the rat liver, were investigated by comparing the mutagenic activity of E2011, 6-aminobenzothiazole (6-ABT, a structural scaffold of E2011) and its derivatives, which are suggested primary reactive metabolites for E2011-induced hepatotoxicity in the rats in vivo, in the Ames assay system employing two Salmonella tester strains, TA100 and YG1029, a bacterial O-acetyltransferase-overproducing strain of TA100. E2011, a tertiary amine, showed no mutagenic activity both in the Salmonella typhimurium TA100 and YG1029 with and without S9 mix. On the other hand, a secondary aromatic amine ER-174238-00, a typical decarbonated metabolite of E2011, showed weak but significant mutagenicity in YG1029 in the presence of S9 mix, and a primary aromatic amine ER-174237-00, an N-dealkylated derivative of ER-174238-00, exhibited S9-dependent potent mutagenicity in YG1029. Thus, it appears that primary and secondary amino moieties of benzothiazole derivatives at C6 -position are the specific structures contributing to their mutagenic activity. In addition, the alkyl group at C2 -position of E2011, ER-174237-00 and ER-174238-00 is suggested to intensify the mutagenic activity, since the mutagenicity of ER-174237-00 is approximately two-fold higher than that of 6-ABT, which has hydrogen at C2 -position in the place of the alkyl group. These results strongly suggest that E2011 has potential initiation activities in the rat liver in vivo after undergoing decarbonation, one of the metabolic pathways, at the carbonyl moiety of oxazolidinone ring to form mutagenic amine(s). © 2000 Elsevier Science B.V. All rights reserved. Keywords: E2011; Benzothiazole; Salmonella typhimurium; Acetyltransferase; Decarbonation; Aromatic amine
1. Introduction
∗ Corresponding author. Tel.: +81-298-47-5714; fax: +81-298-47-5956. E-mail address: [email protected] (G. Sato).
Benzothiazole is reported to be widely used as a scaffold in many physiologically active compounds, such as an antifungal drug [1], a topical carbonic anhydrase inhibitor [2], an antihypoxic drug [3], an anti-nematode drug [4], a dual inhibitor of thrombox-
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ane A2 synthetase and 5-lipoxygenase [5], a selective and reversible inhibitor of monoamine oxidase type A (MAO-A) [6] and even in chemicals for the rubber and tyre industry [7]. Benzothiazole derivatives with an amino group, e.g. 6-aminobenzothiazole (6-ABT), are classified as aromatic amines and are expected to be characterized as mutagenic compounds, since many of them possess mutagenic potency and/or carcinogenicity [8]. Their capacity to damage DNA leading to mutagenicity/carcinogenicity depends on metabolic activation catalyzed by cytochrome P450 (CYP) 1A1/2 isozymes and subsequently by acetyltransferase (AT) [9,10] or in some cases by sulfotransferase (ST) [10,11]. Mutagenicity test employing modified Ames tester strains YG1024 and YG1029, bacterial O-AToverproducing strains of TA98 and TA100, respectively, has been extensively validated [12]. These strains were shown to have capacity for O-acetylation and N-acetylation of nitroarenes, aromatic amines or hydrazines to form ultimate mutagens. That is, they are sensitive for detecting potential mutagenic activities of these mutagenic/carcinogenic compounds [13–16]. For instance, promutagen in smokers’ urine was more sensitively detected in YG1024 than in TA98 [17], and 4-aminobiphenyl, an aromatic amine present in tobacco smoke, was more sensitively detected in YG1029 than in TA100 [18]. In our recent study, oral administration of E2011 at doses of 30 and/or 100 mg/kg to rats for 13 weeks produced nuclear enlargement of hepatocytes with appearance of altered cell foci in some animals. Moreover, the number and area of glutathione S-transferase placental form (GST-P) positive hepatic foci were significantly higher in E2011-treated female animals [19]. It has been reported that altered cell foci are to some extent indicative of putative preneoplastic lesions and an ongoing carcinogenic process [20]. To explain the mechanism of this hepatotoxicity, we proposed the hypothesis that the aromatic amine metabolite(s) of E2011 obtained by decarbonation may contribute to the toxicity; decarbonation was identified as one of the in vivo metabolic pathways of E2011, through which about 15% of administered E2011 was metabolized [21]. The purpose of this study was to confirm the potential initiation activities of E2011 by comparing the mutagenic activity of E2011 and its structurally
related compounds, i.e. two suggested metabolites (ER-174237-00 and ER-174238-00) and the structural scaffold of E2011 (6-ABT). The specific structures contributing to the mutagenic activity as well as the mechanism for in vivo hepatotoxicity are discussed.
2. Materials and methods 2.1. Chemicals and materials (5R)-3-[2-((1S)-3-Cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011) was synthesized at Eisai Chemical Co. (Ibaraki, Japan) [22] and the purity was >99% as determined by TLC and HPLC. 6-ABT (CAS No. 533-30-2, >98% purity) was purchased from Lancaster Synthesis Ltd. (Lancashire, UK). ER-174237-00 and ER-174238-00 were synthesized in our laboratory from (S)-6-amino-2-(5-oxotetrahydrofuran-2-yl)benzothiazole according to the published procedures [22] and their purity determined by HPLC was both 100%. The chemical structures of these four compounds are shown in Fig. 1. S9 fraction of phenobarbital- and -naphthoflavonepretreated male SD rat liver and Cofactor I were purchased from Oriental Yeast Co. Ltd. (Tokyo, Japan). 2.2. In vitro Ames/Salmonella assay Mutagenicity of the four compounds was determined using the Salmonella mutagenicity assay with strains TA100 and/or YG1029. S. typhimurium TA100 and YG1029 were kindly provided by Dr. B.N. Ames (University of California, Berkeley, CA, USA) and by Dr. T. Nohmi (National Institute of Hygienic Sciences, Tokyo, Japan), respectively. The mutagenicity was assayed by the Ames test with a modification of pre-incubation [23,24]. Briefly, a mixture containing the test compound in 0.1 ml of dimethyl sulfoxide (DMSO), 0.1 ml of culture in the early stationary phase of the tester strain, and 0.5 ml of 100 mM sodium phosphate buffer (pH 7.4) or S9 mix was incubated for 20 min at 37◦ C in a test tube with shaking. After incubation, 2 ml of 0.05 mM l-histidine–0.05 mM biotin molten top agar was added to the test tube, and the contents mixed and poured onto a plate of minimal glucose agar medium. The plate was incubated for 48 h
G. Sato et al. / Mutation Research 472 (2000) 163–169
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Fig. 1. Chemical structure of the tested compounds in the Salmonella assay.
at 37◦ C, then revertant colonies were counted. Two plates were used for each dose, and four (TA100) or eight (YG1029) plates were used as negative controls (DMSO). S9 mix (0.5 ml) consisted of 0.05 ml of rat liver S9 fraction (1.2 mg of protein concentration) and 0.45 ml of a cofactor solution (Cofactor I), containing a final concentration of 8 mM MgCl2 , 33 mM KCl, 5 mM glucose-6-phosphate, 4 mM NADPH and 4 mM NADH, in 100 mM sodium phosphate buffer (pH 7.4). In this study, we employed the tester strains sensitive to chemicals causing base pair-substitution mutation (TA100/YG1029), but not the strains sensitive to frameshift mutation (such as TA98/YG1024), because our preliminary experiment revealed that YG1029 was more sensitive for detecting the 6-ABT mutagenicity than YG1024 (data not shown). 2.3. Statistical analysis Linear regression analyses were made to determine if a linear dose response relationship existed.
3. Results and discussion Fig. 2 shows the mutagenic activity of E2011, ER-174237-00, ER-174238-00, and 6-ABT in S.
typhimurium TA100 and/or YG1029, a bacterial O-AT-overproducing strain of TA100, which is highly sensitive to the mutagenicity of nitroarenes and aromatic amines [12]. E2011, a tertiary amine, showed no mutagenic activity both in TA100 and YG1029 with and without S9 mix up to the doses of 5.0 (TA100) or 2.5 mg (YG1029)/plate without significant decrease in bacterial background lawn. On the other hand, a secondary aromatic amine ER-174238-00, a typical decarbonated metabolite of E2011, showed weak but significant mutagenicity in YG1029 in the presence of S9 mix (linear regression, r 2 = 0.974 in the range of 0.2–2.5 mg/plate). The highest reverse mutation activity of ER-174238-00 (mean ± S.E.; 210 ± 2 revertants/plate) was observed at 2.5 mg/plate, which was apparently higher than that of the negative control (mean ± S.E.; 115 ± 4 revertants/plate). Moreover, a primary aromatic amine ER-174237-00, an N-dealkylated derivative of ER-174238-00, exhibited S9-dependent potent mutagenicity in YG1029 in a dose-dependent manner in the range of 0.1–1.25 mg/plate (linear regression, r 2 = 0.982), with the maximum mutagenic activity of 1972 revertants/plate (17.1-fold increase over control). A primary aromatic amine 6-ABT, which has hydrogen at C2 -position in the place of the alkyl group as in ER-174237-00, exhibited S9-dependent
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Fig. 2. Mutagenicity results of the benzothiazole derivatives in the tester strains TA100 and YG1029. Data are presented as mean ± S.E.
potent mutagenicity in YG1029 (linear regression, r 2 = 0.988 in the range of 0.1–1.25 mg/plate, with the maximum mutagenic activity of 956 revertants/plate), but not in TA100 up to the doses of 2.5 mg/plate. Namely, the exocyclic-tertiary amine structure in E2011, i.e. oxazolidinone ring, had no muta-
genic effect. The secondary amine structure as in ER-174238-00, however, acted as S9-dependent weak mutagen in O-AT-overproducing tester strain YG1029, and the primary amine structure as in ER-174237-00 and 6-ABT exhibited its S9-dependent potent mutagenicity in YG1029 in a dose-dependent
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manner. These data suggest that primary and secondary amino moieties of benzothiazole derivatives at C6 -position are the specific structures contributing to their S9 mix- and O-AT-dependent mutagenic activity. Furthermore, the alkyl group at C2 -position of E2011,
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ER-174237-00 and ER-174238-00 is suggested to intensify the mutagenic activity, since the mutagenicity of ER-174237-00 is approximately two-fold higher than that of 6-ABT, which has hydrogen at C2 -position in the place of the alkyl group.
Fig. 3. Possible bioactivation pathway of E2011.
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The possible mechanism of bioactivation of E2011 is shown in Fig. 3. In order to exert its potential mutagenic activity, E2011 needs to be initially decarbonated to form a pro-mutagenic secondary amine or possibly a primary amine by following N-dealkylation. They will be subsequently catalyzed by CYPs (in the extracellular S9 mix) to form N-hydroxylamines and then transformed to the ultimate mutagenic species through acetylation (by bacterial intracellular AT) as represented by the well-known mutagenic/carcinogenic aromatic or heterocyclic amines [8–10]. Although it is not clear why E2011 is not mutagenic in TA100 and even in YG1029, a possible explanation for this is the lack of the decarbonation step in the typical Salmonella assay, in contrast to rats, where about 15% of administered E2011 undergoes this in vivo decarbonation pathway [21]. The decarbonation step might require certain cofactors in addition to CYPs’ and/or S9 fraction from extrahepatic tissues such as lung, intestine or kidney, which also participate in drug metabolism [25,26]. For certain chemicals, it is difficult or impossible to detect their potential mutagenicity in routine assays in bacteria [27,28]. E2011 appears to be one of these chemicals that will be classified as a false negative mutagen, in this case, because of the lack of the decarbonation step in the Salmonella assay system. Further work to clarify whether E2011 has promotion activity as well as to identify the mechanisms for the in vivo decarbonation step would be necessary to understand the nature of this chemical. In conclusion, our results strongly suggest that E2011 has potential initiation activities in the rat liver in vivo after undergoing decarbonation, one of the metabolic pathways, at the carbonyl moiety of oxazolidinone ring to form mutagenic amine(s).
Acknowledgements The authors are grateful to Drs. Toyohiko Aoki and Satoru Hosokawa (Department of Developmental Safety Assessment Research, Eisai Co. Ltd.) for their valuable comments and to Ms. Deborah Satoh (Research and Development Planning and Coordination, Eisai Co. Ltd.) for her kind advice in the preparation of this manuscript.
References [1] H. Küçükbay, B. Durmaz, Antifungal activity of organic and organometallic derivatives of benzimidazole and benzothiazole, Arzneim. -Forsch./Drug Res. 47 (1997) 667–670. [2] P.H. Kalina, D.J. Shetlar, R.A. Lewis, L.J. Kullerstrand, R.F. Brubaker, 6-Amino-2-benzothiazolesulfonamide. The effect of a topical carbonic anhydrase inhibitor on aqueous humor formation in the normal human eye, Ophthalmology 95 (1988) 772–777. [3] E. Mohr, N.P.V. Nair, M. Sampson, S. Murtha, G. Belanger, B. Pappas, T. Mendis, Treatment of Alzheimer’s disease with sabeluzole: functional and structural correlates, Clin. Neuropharmacol. 20 (1997) 338–345. [4] J. Surin, Anti-nematode activity of sixteen compounds against Trichinella spiralis in mice — a possible new screen for macrofilaricides, Southeast Asian J. Trop. Med. Public Health 26 (1995) 128–134. [5] Y. Komatsu, N. Minami, Synthesis of a novel dual inhibitor of thromboxane A2 synthetase and 5-lipoxygenase (E3040) via the direct coupling reaction of hydroquinone with 3-pyridinecarboxaldehyde, Chem. Pharm. Bull. 43 (1995) 1614–1616. [6] T. Kagaya, A. Kajiwara, S. Nagato, K. Akasaka, A. Kubota, E2011, a novel, selective and reversible inhibitor of monoamine oxidase type A, J. Pharmacol. Exp. Ther. 278 (1996) 243–251. [7] C.-D. Wacker, B. Spiegelhalder, R. Preussmann, New sulfonamide accelerators derived from ‘safe’ amines for the rubber and tyre industry, in: I.K. O’Neill, J. Chen, H. Bartsch (Eds.), Relevance to Human Cancer of N-Nitroso Compounds, Tobacco Smoke and Mycotoxins, IARC, Lyon, 1991, pp. 592–594. [8] F.A. Beland, F.F. Kadlubar, Metabolic activation and DNA adducts of aromatic amines and nitroaromatic hydrocarbons, in: C.S. Cooper, P.L. Grover (Eds.), Chemical Carcinogenesis and Mutagenesis I, Springer, Berlin, 1990, pp. 267–325. [9] C.M. King, S.J. Land, R.F. Jones, M. Debiec-Rychter, M.-S. Lee, C.Y. Wang, Role of acetyltransferases in the metabolism and carcinogenicity of aromatic amines, Mutat. Res. 376 (1997) 123–128. [10] H.-U. Aeschbacher, R.J. Turesky, Mammalian cell mutagenicity and metabolism of heterocyclic aromatic amines, Mutat. Res. 259 (1991) 235–250. [11] H. Glatt, Bioactivation of mutagens via sulfation, FASEB J. 11 (1997) 314–321. [12] P.D. Josephy, P. Gruz, T. Nohmi, Recent advances in the construction of bacterial genotoxicity assays, Mutat. Res. 386 (1997) 1–23. [13] M. Watanabe, M. Ishidate Jr., T. Nohmi, Sensitive method for the detection of mutagenic nitroarenes and aromatic amines: new derivatives of Salmonella typhimurium tester strains possessing elevated O-acetyltransferase levels, Mutat. Res. 234 (1990) 337–348. [14] P. Einistö, M. Watanabe, M.T. Nohmi, M. Ishidate Jr., Mutagenicity of 30 chemicals in Salmonella typhimurium strains possessing different nitroreductase or O-acetyltransferase activities, Mutat. Res. 259 (1991) 95–102.
G. Sato et al. / Mutation Research 472 (2000) 163–169 [15] G.J. Stevens, E.J. LaVoie, C.A. McQueen, The role of acetylation in the mutagenicity of the antitumor agent, batracylin, Carcinogenesis 17 (1996) 115–119. [16] L.E. Lemke, C.A. McQueen, Acetylation and its role in the mutagenicity of the antihypertensive agent hydralazine, Drug Metab. Dispos. 23 (1995) 559–565. [17] C. Kuenemann-Migeot, F. Callais, I. Momas, B. Festy, Use of Salmonella typhimurium TA98, YG1024 and YG1021 and deconjugating enzymes for evaluating the mutagenicity from smokers’ urine, Mutat. Res. 390 (1997) 283–291. [18] L.N. Dang, C.A. McQueen, Mutagenicity of 4-aminobiphenyl and 4-acetylaminobiphenyl in Salmonella typhimurium strains expressing different levels of N-acetyltransferase, Toxicol. Appl. Pharmacol. 159 (1999) 77–82. [19] G. Sato, T. Chimoto, T. Aoki, S. Hosokawa, S. Sumigama, K. Tsukidate, F. Sagami, Toxicological response of rats to a novel monoamine oxidase type-A inhibitor, (5R)-3-[2((1S)-3-cyano-1-hydroxypropyl)benzothiazol-6-yl]-5-methoxymethyl-2-oxazolidinone (E2011), orally administered for 13 weeks, J. Toxicol. Sci. 24 (1999) 165–175. [20] G.M. Williams, The significance of chemically-induced hepatocellular altered foci in rat liver and application to carcinogen detection, Toxicol. Pathol. 17 (1989) 663–674. [21] T. Naitoh, M. Kakiki, T. Kozaki, M. Mishima, T. Yuzuriha, T. Horie, Identification of the metabolites of a new oxazolidinone MAO-A inhibitor in rat, Xenobiotica 27 (1997) 1071–1089.
169
[22] K. Akasaka, A. Kajiwara, S. Nagato, Y. Iinuma, I. Yoshida, A. Sasaki, M. Mizuno, A. Kubota, T. Kagaya, M. Komatsu, Oxazolidone derivatives, WO 9308179, Patent No. JP 05194440 (1993). [23] D.M. Maron, B.N. Ames, Revised methods for the Salmonella mutagenicity test, Mutat. Res. 113 (1983) 173–215. [24] A. Hakura, H. Mochida, Y. Tsutsui, K. Yamatsu, Mutagenicity and cytotoxicity of naphthoquinones for Ames Salmonella tester strains, Chem. Res. Toxicol. 7 (1994) 559–567. [25] M. Schwenk, Mucosal biotransformation, Toxicol. Pathol. 16 (1988) 138–146. [26] D.R. Krishna, U. Klotz, Extrahepatic metabolism of drugs in humans, Clin. Pharmacokinet. 26 (1994) 144–160. [27] S. Venitt, R. Baker, H. Liber, T. Matsushima, G. Probst, E. Zeiger, Summary report on the performance of the bacterial mutation assays, in: J. Ashby, F.J. de Serres, M. Draper, M. Ishidate Jr., B.H. Margolin, B.E. Matter, M.D. Shelby (Eds.), Progress in Mutation Research, Vol. 5, Evaluation of Short-Term Tests for Carcinogens, Elsevier, Amsterdam, 1985, pp. 11–23. [28] G. Reifferscheid, J. Heil, Validation of the SOS/umu test using test results of 486 chemicals and comparison with the Ames test and carcinogenicity data, Mutat. Res. 369 (1996) 129–145.