- Email: [email protected]
The structural basis for the mutagenicity of aristolochic acid
Cancer Letters, 55 (1990) 7Elsevier Scientific Publishers
11 Ireland Ltd.
The structural basis for the mutagenicity
of aristolochic
acid
W. Pfau”, * , B.L. Pool-Zobel”, C.-W. von der Liethb and M. Wiessler” OGermon Cancer Research
Neuenheimer
Center,
Feld 280, D-6900
(Received 2 July 1990) (Revision received 4 September (Accepted 4 September 1990)
Institute
Heidelberg
of Toxicology
Molecular orbital calculations with aristolochic acid I (AAl) and the model compounds -naphthoic
acid
(1,8NNA)
and
3-
nitro-Z-naphthoic acid (2,3NNA) confirm a similar conformation of the nitro and carboxyl groups
and bDepartment
of Spectroscopy,
Im
1990)
Summary
8-nitro-1
and Chemotherapy
(Germany)
in these
molecules. The ortho isomer is not mutagenic in the Salmonella strains TA 100 or TA 1537, but the peri-substituted 1,8NNA shows mutagenic actioity similar to AA1 in TA 100, although it is only weakly active in TA 1537. We propose a mechanism of activation uia a cyclic nitrenium ion with an aristolactam structure which is possible only in peri-substituted nitro carboxylic 2,3NNA
acids.
Keywords: aristolochic acid; nitro naphtoic acids; peri substitution; mutagenicity; molecular orbital calculations Introduction Aristolochic acid (AA), a mixture of structurally related nitro phenanthrene derivatives, has been used since antiquity as a herbal drug in obstetrics, the treatment of snake bites, fester‘Present address: The Institute of Cancer Laboratories, 15 Cotswold Road, Belmont,
Research, Haddow Sutton, Surrey SM2
5NG, U.K.
0304-3835/90/$03.50 0 1990 Elsevier Scientific Publishers Published and Printed in Ireland
ing wounds and tumors [2,6]. In 1982 AA was shown independently by Mengs et al. [ 161 and Xing et al. [27] to be a strong carcinogen in rodents. The mutagenic activity of AA was subsequently proven in several short-term tests [14,20]. Using the Ames Salmonella typhimurium mutagenicity assay [15] Schmeiser et al. [23] found the main components of the natural mixture, aristolochic acid I and II (AA1 and AAII), to be direct mutagens in the S. typhimurium tester strains TA 1537 and TA 100 but not in the nitro reductase deficient strain TA 100NR; thus it was postulated that the nitro group in the lo-position is essential for the biological activity of AA; this is supported by earlier studies [26] where the cytotoxic properties of structural analogs of AA were investigated. The main metabolites of AA1 and AA11 are the aristolactams, formed in vitro and excreted in vivo by several animal species including man [8,24]. These aristolactam structures were recently found in DNA adducts formed by AA1 [ 181; this led to the hypothesis that the peculiar molecular structure with carboxyl and nitro groups in a peri position might be a new mutagenic principle. In this paper the mutagenicity of AAI, the peri-substituted model compound 8-nitro- lnaphthoic acid and the ortho isomer 3-nitro-2naphthoic acid are compared. Molecular orbital calculations have been carried out to
Ireland Ltd
confirm the proposed conformation of AAI, aristolactam I and the model compounds.
.
TAlOO
600
Materials and Methods Chemicals
AA1 was isolated from the natural mixture (Madaus, Cologne, F.R.G.) by repeated crystallisation from dimethylformamide as described previously [24]. 8-Nitro-l-naphthoic acid was synthesized according to Eckardt et al. [4] by nitration of 1-naphthoic acid with nitric acid in acetic acid, the by-product 5nitro-1-naphthoic acid was esterified with ethanol and the Snitro-l-naphthoic acid purified by fractional crystallisation from ethanol. 3-Nitro-2-naphthoic acid was prepared by thermolysis of 3-nitro-2- naphthoic acid mag-adduct nesium salt-bis- (hexachloropentadien) (Aldrich, Steinheim, F.R.G.) according to Look [ 121 and recrystallized from ethanol. The compounds were chromatographically pure; MS- and ‘H-NMR-data and melting points were in accordance with published data 17,121.
A0 pg per plate
TA1537
clg per plate
Fig. 1.
Mutagenicity assays Reversion to prototrophy using Salmoneh typhimurium histidine auxotrophic strains TA 100 and TA 1537 was measured essentially as described before [ 15,241. Each concentration was assessed in triplicate in the plate incorporation assay. Mutagenesis assays were performed on three different occasions.
Number of revertant colonies of the indicated Salmonella typhimurium strains as a function of the dose of aristolochic acid I (U-El), Snitro- 1-naphthoic acid (A-A) and 3-nitro-2-naphthoic acid ( l - 0) dissolved in DMSO. Each point gives the mean value ( f SD.) of three plates of one representative experiment out of three. TA 100 showed 115 f: 19 spontaneous revertants and 427 f 6 revertants induced by 1 t.q sodium azide per plate. TAB37 showed 6 & 2 spontaneous revertants and 86 zt 15 revertants induced by 1 pg amino acridine per plate.
Computer
calculations The construction of the 3D structures of the molecules was accomplished with the aid of the programs MOLBUILD [ 111 and RINGS [lo]; the molecules were minimized using semi-empirical molecular orbital calculations. Full SCF-calculations for all degrees of freedom were carried out using the AM1 program in AMPAC [3]; the calculations were performed on an IBM 3032 computer. Results and Discussion The results presented
in Fig. 1 show that the
two peri-substituted nitro arene carboxylic acids AA1 and 1,8NNA are directly acting mutagens and exhibit similar dose-dependent mutagenic properties in the Salmonella typhimurium tester strain TA 100, while the isomerit ortho-substituted 2,3NNA has no activity. AA1 shows considerable dose-dependent mutagenicity in the tester strain TA1537, but 1,8NNA gives only a weak response; again 2,3NNA is not active. These results are in agreement with earlier published mutagenicity data from Ames assays performed with AA1 ~41.
9
It is generally accepted that nitro arenes require reductive activation to exhibit their mutagenic potency, a metabolic activation step which procaryotic nitroreductases are capable of [ 131; however differences in the mutagenicity of nitro arenes have been reported in the literature [ 2 11. Fu et al. [5] and Vance et al. [25] have reported the lower mutagenicity of nitro arenes with the nitro group in the peri-position to Hatoms compared to their positional isomers; they suggested perpendicular orientation of the nitro group as a cause for reduced mutagenicity in the Ames assay. Nitro aromatics with the nitro group not coplanar with the aromatic rings might be only poor substrates for the procaryotic nitroreductase and, additionally, the resulting reductive metabolites or ultimate mutagens could not be stabilized by resonance when the functional group is steritally forced out of the molecular plane. In this paper we report on molecular orbital calculations carried out on AA1 and the two isomeric nitro naphtoic acids 1,8NNA and 2,SNNA. The minimized conformations of the nitro compounds as presented in Fig. 2 show that both nitro and carboxyl groups are rotated out of the molecular plane. The diedral angles for the nitro groups are 50.5O for AAI, 45.7O
for 1,8NNA and 31.5O for 2.3 NNA. In accordance with X-ray structural analysis data of comparable peri-substituted naphthalene derivatives [l], the calculation for 1,8NNA showed a planar but considerably distorted aromatic ring system, while the simulated structure of AA1 had a symmetrical phenanthrene backbone, with sterical forces of the peri substituents on one side and the methylene dioxolo-group and H5 on the other keeping the balance. In accordance with the hypothesis of Fu and co-workers [5] the out-of-plane orientation of the nitro group in the three nitro aromatic acids investigated may account for the comparatively low mutagenicity observed in the Ames assay. The so called peri effect has been reviewed [l] and several unusual chemical properties of peri-substituted compounds were reported due to steric hindrance and proximity effects. Chemical reduction of per&substituted nitro aromatic carboxylic acids like AA1 and 1,8NNA leads to lactam compounds like benz[c,d]indol-2(‘H)-one for 1,8NNA or aristolactam for AA (Fig. 3); indeed aristolactams are the major metabolites of AA found in vitro and in vivo [8,24]. The lactam formation after metabolic reduction of AA is the first report to
1
3
/---9
Three dimensional molecular structures of (A) Fig. 2. AAI, (B) aristolactam, (C) Ekitro-1-naphthoic acid and (D) 3-nitro-Z-naphthoic acid as accomplished by minimization of the structures with the program AM1 in AMPAC, showing the out-of-plane orientation of the nitro and carboxyl group in the nitro aromatic carboxylic acids as well as the planar structure of the aristolactam molecule.
I
NO2
Old0
2
4
Fig. 3. Aristolochic acid I, 1, aristolactam I, 2; ES-nitro1-naphthoic acid, 3; 3-nitro-Z-naphthoic acid, 4.
10 0
DNA binding
1
6
5
Proposed mechanism of metabolic activation of AA: Reduction and ring closure lead to a cyclic hydroxamic Fig. 4. acid 5, which can be dehydrated to form a cyclic nitrenium ion 6 with delocalized positive charge. Binding to DNA occurs at C-7, in ortho position to the N-substitution [ 181.
our knowledge
of a peri reaction
in biological
systems. We propose the reductive formation of an intermediate nitrenium ion, similar to the ultimate carcinogenic species of other nitro substituted carcinogens [21], but with a lactamic structure, which can be formed in the perisubstituted molecules but not in the ortbo-substituted 2,3NNA (Fig. 4). This ring closure can give release to the steric stress and would allow stabilisation of the nitrenium ion by delocalization of the positive charge, thus rationalising the observed mutagenic activity of peri-substituted nitro aromatic carboxylic acids. As an electrophilic species this pentacyclic nitrenium ion could bind to nucleophilic centers in DNA to form adducts [ 181, could be hydrolyzed to form the 7-hydroxyaristolactam [18] or could be further reduced to the aristolactam I. The reductive nature of the metabolic activation of AA has recently been challenged by Peuuto and co-workers [ 171 who proposed an oxidative mechanism based on mutagenic activity in the Salmonella tester strain TA 102. This strain detects oxidative mutagens because of A/T base pairs at the site of mutation [9]. As AA1 forms desoxyadenosine-adducts under reductive conditions [ 181, it seems more likely that the reported mutagenic activity in TA 102 arises from binding to deoxyadenosine; the same lesion has been proposed as responsible
for the activating A/T + T/A transversion mutations detected in H-ras oncogenes in AAIinduced tumors [22]. The data presented here, together with the recently published structures of DNA adducts of AA1 formed in vivo [19] and under reductive conditions in vitro [18], strongly support the proposed pathway of activation for AA1 via a pentacyclic nitrenium ion. References Balasubramaniyan, V. (1966) Pen-interaction ene derivatives. Chem. Rev., 66,567-641.
in naphthal-
Chen, Z.-L. and Zhu, D.-Y. (1987) Aristolochia alkaloids. In: The Alkaloids, Vol. 31, 29-66. Editor: A. Brossi. Dewar, M.J.S. and Thiel, W. (1977) AMPAC (Austin Method 1 Package, IBM version 3090) J. Am. Chem. Sot., 99,4899-4907, QCPE 527. E&strand, J. (1888) Zur Kenntnis der Naphthoesauren. J. prakt. Chem., 38,650-657. Fu, P.P., Chou, M.W., Miller, D.W., White, G.L., Heflich, R.H. and Beland, F.A. (1985) The orientation of the nitro substttuent predicts the direct-acting bacterial mutagenicity of nitrated polycyclic aromatic hydrocarbons. Mutat. Res., 143, 173-181. Hartwell, J.C. (1982) Plants used against cancer. Quaterman Publications, Lawrence, MA, U.S.A. Kienzle, F. (1988) Die Reaktion von Phthalaldehyden mit 3-Nitropropion&reestern . Ein einfacher Zugang zu 3Nitro-2-naphthoe&ren. Helv. Chim. Acta, 63, 23642369. Krumbiegel. G., Hallensleben, J., Mennicke, W. Rittmann, N. and Roth, H.J. (1987) Studies on the metabo-
11
9
10
11
12
13
14
15
16
17
18
lism of aristolochic acid I and II. Xenobiotica, 17, 981991. Levin, D.E., Hollstein, M., Christman, M.F., Schwlers, E.A. and Ames, B.N. (1982) A new Salmonella tester strain (TA10.2) with A/T, base pairs at the site of mutation detects oxidative mutagens. Proc. Natl. Acad. Sci. USA, 79,7445-7449. v.d. Lieth, C.W., Dolata, D.P. and Liljefors, T. (1984) RINGS - a general program to built ring systems. J. Mol. Graph., 2,117-123. Liliefors, T. (1983) MOLBUILD - an interactive computer graphics interface to molecular mechanics. J. Mol. Graph.
19
20
21
22
l, lll-117. Look, M. (1974) Hexachlorocyclopentadiene adducts of aromatic compounds and their reactions. Aldrichim. Acta, 7,23-29.
23
McCoy, E.C., (1981) Evidence tases capable of Environ. Mutat.,
24
Rosenkranz, H.S. and Mermelstein, R. for the existence of a family of nitroreducactivating nitrated polycyclics to mutagens. 3,421-427.
Maier, P., Schawalder, H. and Weibel, B. (1987) Low oxygen tension as found in tissues in vivo, alters the mutagenic activity of artstolochic acid I and II. Environ. Mol. Mutat., 10, 275-284. Maron, D.M. and Ames, B.N. (1983) Revised methods for the Salmonella mutagenicity test. Mutat. Res., 113, 173215. Mengs, U., Lang, W. and Poch, J.A. (1982), The carcinogenic action of aristolochic acid. Arch. Toxicol., 51, 107119. Pwuto, J.M., Swanson, S.M., Mar, W., Che, C., Cordell, G.A. and Fong, H.H.S. (1988) Evaluation of the mutagenic and cytostatic potential of aristolochic acid and several of its derivatives. Mutat. Res., 206.447-454. Pfau, W., Schmeiser. H.H. and Wiessler, M. (1990) Aristolochic acid binds covalently to the exocyclic amino group of purine nucleotides 319.
in DNA. Carcinogenesis,
12, 313-
25
26
27
Pfau, W., Schmeiser, postlabelllng analysis
H.H. and Wiessler, M. (1990) “Pof DNA adducts formed by aristo-
lochic acid I and II. Carcinogenesis, in press. Robisch, G., Schimmer, 0. and Goggelmann, W. (1982) Aristolochic acid is a direct mutagen in Salmonella typhimu&m. Mutat. Res., 105,201-203. Rosenkranz, H.S. and Mermelstein, R. (1983) Mutagenicity and genotoxicity of nitroarenes. All nitro-containing chemicals were not created equal. Mutat. Res., 114, 217-267. Schmeiser, H.H., Lyons, J., Janssen, J.W.G., Bartram, C.R., Pfau, W., Buchmann, A. and Wiessler, M. (1989) Aristolochic acid I induced tumors in Wistar rats contain AT + TA transversion mutations in codon 61 of the H-ras oncogene. J. Cancer Res. Clin. Oncol., 115 (Suppl.) S22. Schmeiser, H.H., Pool, B.L. and Wiessler, M. (1984) Mutagenicity of the two main components of commercially available carcinogenic aristolochic acid in Solmonello typhimurium. Cancer Lett. 23,97-98. Schmetser, H.H., Pool, B.L. and Wiessler, M. (1986) Identification and mutagenicity of metabolites of aristolochic acid formed by rat liver. Carcinogenesis, 7.59-63. Vance, W.A., Okamoto, H.S. and Wang, Y.Y. (1988) Structural features of nitroaromatics and their reduction products that affect mutagenic potency in the Ames Salmonella assay. In King, C.M., Romano, L.J. and Schuetsle, D. (eds.) Carcinogenic and mutagenic responses to aromatic amines and nitroarenes. Elsevier, New York, p. 191 -302. Viel, C. and Do&, J.C. (1972) Nouveaux cytotoxiques et antitumoraux de synthhse au dipart de l’acide aristolochique, acide nitroph&anthr&ique d action tumorale, extrait des Aristiolochiacges. II Farmaco, 27, 259-312. Xing, B., Chen, L., Zhou, L., Jing, L. and Liu, F. (1982) Experimental study on the carcinogenic& of aristolochic acid. Guangxi Yixue 4, 118-121. cited in Chem. Abstr., 98 (1982) 65165a.
11 Ireland Ltd.
The structural basis for the mutagenicity
of aristolochic
acid
W. Pfau”, * , B.L. Pool-Zobel”, C.-W. von der Liethb and M. Wiessler” OGermon Cancer Research
Neuenheimer
Center,
Feld 280, D-6900
(Received 2 July 1990) (Revision received 4 September (Accepted 4 September 1990)
Institute
Heidelberg
of Toxicology
Molecular orbital calculations with aristolochic acid I (AAl) and the model compounds -naphthoic
acid
(1,8NNA)
and
3-
nitro-Z-naphthoic acid (2,3NNA) confirm a similar conformation of the nitro and carboxyl groups
and bDepartment
of Spectroscopy,
Im
1990)
Summary
8-nitro-1
and Chemotherapy
(Germany)
in these
molecules. The ortho isomer is not mutagenic in the Salmonella strains TA 100 or TA 1537, but the peri-substituted 1,8NNA shows mutagenic actioity similar to AA1 in TA 100, although it is only weakly active in TA 1537. We propose a mechanism of activation uia a cyclic nitrenium ion with an aristolactam structure which is possible only in peri-substituted nitro carboxylic 2,3NNA
acids.
Keywords: aristolochic acid; nitro naphtoic acids; peri substitution; mutagenicity; molecular orbital calculations Introduction Aristolochic acid (AA), a mixture of structurally related nitro phenanthrene derivatives, has been used since antiquity as a herbal drug in obstetrics, the treatment of snake bites, fester‘Present address: The Institute of Cancer Laboratories, 15 Cotswold Road, Belmont,
Research, Haddow Sutton, Surrey SM2
5NG, U.K.
0304-3835/90/$03.50 0 1990 Elsevier Scientific Publishers Published and Printed in Ireland
ing wounds and tumors [2,6]. In 1982 AA was shown independently by Mengs et al. [ 161 and Xing et al. [27] to be a strong carcinogen in rodents. The mutagenic activity of AA was subsequently proven in several short-term tests [14,20]. Using the Ames Salmonella typhimurium mutagenicity assay [15] Schmeiser et al. [23] found the main components of the natural mixture, aristolochic acid I and II (AA1 and AAII), to be direct mutagens in the S. typhimurium tester strains TA 1537 and TA 100 but not in the nitro reductase deficient strain TA 100NR; thus it was postulated that the nitro group in the lo-position is essential for the biological activity of AA; this is supported by earlier studies [26] where the cytotoxic properties of structural analogs of AA were investigated. The main metabolites of AA1 and AA11 are the aristolactams, formed in vitro and excreted in vivo by several animal species including man [8,24]. These aristolactam structures were recently found in DNA adducts formed by AA1 [ 181; this led to the hypothesis that the peculiar molecular structure with carboxyl and nitro groups in a peri position might be a new mutagenic principle. In this paper the mutagenicity of AAI, the peri-substituted model compound 8-nitro- lnaphthoic acid and the ortho isomer 3-nitro-2naphthoic acid are compared. Molecular orbital calculations have been carried out to
Ireland Ltd
confirm the proposed conformation of AAI, aristolactam I and the model compounds.
.
TAlOO
600
Materials and Methods Chemicals
AA1 was isolated from the natural mixture (Madaus, Cologne, F.R.G.) by repeated crystallisation from dimethylformamide as described previously [24]. 8-Nitro-l-naphthoic acid was synthesized according to Eckardt et al. [4] by nitration of 1-naphthoic acid with nitric acid in acetic acid, the by-product 5nitro-1-naphthoic acid was esterified with ethanol and the Snitro-l-naphthoic acid purified by fractional crystallisation from ethanol. 3-Nitro-2-naphthoic acid was prepared by thermolysis of 3-nitro-2- naphthoic acid mag-adduct nesium salt-bis- (hexachloropentadien) (Aldrich, Steinheim, F.R.G.) according to Look [ 121 and recrystallized from ethanol. The compounds were chromatographically pure; MS- and ‘H-NMR-data and melting points were in accordance with published data 17,121.
A0 pg per plate
TA1537
clg per plate
Fig. 1.
Mutagenicity assays Reversion to prototrophy using Salmoneh typhimurium histidine auxotrophic strains TA 100 and TA 1537 was measured essentially as described before [ 15,241. Each concentration was assessed in triplicate in the plate incorporation assay. Mutagenesis assays were performed on three different occasions.
Number of revertant colonies of the indicated Salmonella typhimurium strains as a function of the dose of aristolochic acid I (U-El), Snitro- 1-naphthoic acid (A-A) and 3-nitro-2-naphthoic acid ( l - 0) dissolved in DMSO. Each point gives the mean value ( f SD.) of three plates of one representative experiment out of three. TA 100 showed 115 f: 19 spontaneous revertants and 427 f 6 revertants induced by 1 t.q sodium azide per plate. TAB37 showed 6 & 2 spontaneous revertants and 86 zt 15 revertants induced by 1 pg amino acridine per plate.
Computer
calculations The construction of the 3D structures of the molecules was accomplished with the aid of the programs MOLBUILD [ 111 and RINGS [lo]; the molecules were minimized using semi-empirical molecular orbital calculations. Full SCF-calculations for all degrees of freedom were carried out using the AM1 program in AMPAC [3]; the calculations were performed on an IBM 3032 computer. Results and Discussion The results presented
in Fig. 1 show that the
two peri-substituted nitro arene carboxylic acids AA1 and 1,8NNA are directly acting mutagens and exhibit similar dose-dependent mutagenic properties in the Salmonella typhimurium tester strain TA 100, while the isomerit ortho-substituted 2,3NNA has no activity. AA1 shows considerable dose-dependent mutagenicity in the tester strain TA1537, but 1,8NNA gives only a weak response; again 2,3NNA is not active. These results are in agreement with earlier published mutagenicity data from Ames assays performed with AA1 ~41.
9
It is generally accepted that nitro arenes require reductive activation to exhibit their mutagenic potency, a metabolic activation step which procaryotic nitroreductases are capable of [ 131; however differences in the mutagenicity of nitro arenes have been reported in the literature [ 2 11. Fu et al. [5] and Vance et al. [25] have reported the lower mutagenicity of nitro arenes with the nitro group in the peri-position to Hatoms compared to their positional isomers; they suggested perpendicular orientation of the nitro group as a cause for reduced mutagenicity in the Ames assay. Nitro aromatics with the nitro group not coplanar with the aromatic rings might be only poor substrates for the procaryotic nitroreductase and, additionally, the resulting reductive metabolites or ultimate mutagens could not be stabilized by resonance when the functional group is steritally forced out of the molecular plane. In this paper we report on molecular orbital calculations carried out on AA1 and the two isomeric nitro naphtoic acids 1,8NNA and 2,SNNA. The minimized conformations of the nitro compounds as presented in Fig. 2 show that both nitro and carboxyl groups are rotated out of the molecular plane. The diedral angles for the nitro groups are 50.5O for AAI, 45.7O
for 1,8NNA and 31.5O for 2.3 NNA. In accordance with X-ray structural analysis data of comparable peri-substituted naphthalene derivatives [l], the calculation for 1,8NNA showed a planar but considerably distorted aromatic ring system, while the simulated structure of AA1 had a symmetrical phenanthrene backbone, with sterical forces of the peri substituents on one side and the methylene dioxolo-group and H5 on the other keeping the balance. In accordance with the hypothesis of Fu and co-workers [5] the out-of-plane orientation of the nitro group in the three nitro aromatic acids investigated may account for the comparatively low mutagenicity observed in the Ames assay. The so called peri effect has been reviewed [l] and several unusual chemical properties of peri-substituted compounds were reported due to steric hindrance and proximity effects. Chemical reduction of per&substituted nitro aromatic carboxylic acids like AA1 and 1,8NNA leads to lactam compounds like benz[c,d]indol-2(‘H)-one for 1,8NNA or aristolactam for AA (Fig. 3); indeed aristolactams are the major metabolites of AA found in vitro and in vivo [8,24]. The lactam formation after metabolic reduction of AA is the first report to
1
3
/---9
Three dimensional molecular structures of (A) Fig. 2. AAI, (B) aristolactam, (C) Ekitro-1-naphthoic acid and (D) 3-nitro-Z-naphthoic acid as accomplished by minimization of the structures with the program AM1 in AMPAC, showing the out-of-plane orientation of the nitro and carboxyl group in the nitro aromatic carboxylic acids as well as the planar structure of the aristolactam molecule.
I
NO2
Old0
2
4
Fig. 3. Aristolochic acid I, 1, aristolactam I, 2; ES-nitro1-naphthoic acid, 3; 3-nitro-Z-naphthoic acid, 4.
10 0
DNA binding
1
6
5
Proposed mechanism of metabolic activation of AA: Reduction and ring closure lead to a cyclic hydroxamic Fig. 4. acid 5, which can be dehydrated to form a cyclic nitrenium ion 6 with delocalized positive charge. Binding to DNA occurs at C-7, in ortho position to the N-substitution [ 181.
our knowledge
of a peri reaction
in biological
systems. We propose the reductive formation of an intermediate nitrenium ion, similar to the ultimate carcinogenic species of other nitro substituted carcinogens [21], but with a lactamic structure, which can be formed in the perisubstituted molecules but not in the ortbo-substituted 2,3NNA (Fig. 4). This ring closure can give release to the steric stress and would allow stabilisation of the nitrenium ion by delocalization of the positive charge, thus rationalising the observed mutagenic activity of peri-substituted nitro aromatic carboxylic acids. As an electrophilic species this pentacyclic nitrenium ion could bind to nucleophilic centers in DNA to form adducts [ 181, could be hydrolyzed to form the 7-hydroxyaristolactam [18] or could be further reduced to the aristolactam I. The reductive nature of the metabolic activation of AA has recently been challenged by Peuuto and co-workers [ 171 who proposed an oxidative mechanism based on mutagenic activity in the Salmonella tester strain TA 102. This strain detects oxidative mutagens because of A/T base pairs at the site of mutation [9]. As AA1 forms desoxyadenosine-adducts under reductive conditions [ 181, it seems more likely that the reported mutagenic activity in TA 102 arises from binding to deoxyadenosine; the same lesion has been proposed as responsible
for the activating A/T + T/A transversion mutations detected in H-ras oncogenes in AAIinduced tumors [22]. The data presented here, together with the recently published structures of DNA adducts of AA1 formed in vivo [19] and under reductive conditions in vitro [18], strongly support the proposed pathway of activation for AA1 via a pentacyclic nitrenium ion. References Balasubramaniyan, V. (1966) Pen-interaction ene derivatives. Chem. Rev., 66,567-641.
in naphthal-
Chen, Z.-L. and Zhu, D.-Y. (1987) Aristolochia alkaloids. In: The Alkaloids, Vol. 31, 29-66. Editor: A. Brossi. Dewar, M.J.S. and Thiel, W. (1977) AMPAC (Austin Method 1 Package, IBM version 3090) J. Am. Chem. Sot., 99,4899-4907, QCPE 527. E&strand, J. (1888) Zur Kenntnis der Naphthoesauren. J. prakt. Chem., 38,650-657. Fu, P.P., Chou, M.W., Miller, D.W., White, G.L., Heflich, R.H. and Beland, F.A. (1985) The orientation of the nitro substttuent predicts the direct-acting bacterial mutagenicity of nitrated polycyclic aromatic hydrocarbons. Mutat. Res., 143, 173-181. Hartwell, J.C. (1982) Plants used against cancer. Quaterman Publications, Lawrence, MA, U.S.A. Kienzle, F. (1988) Die Reaktion von Phthalaldehyden mit 3-Nitropropion&reestern . Ein einfacher Zugang zu 3Nitro-2-naphthoe&ren. Helv. Chim. Acta, 63, 23642369. Krumbiegel. G., Hallensleben, J., Mennicke, W. Rittmann, N. and Roth, H.J. (1987) Studies on the metabo-
11
9
10
11
12
13
14
15
16
17
18
lism of aristolochic acid I and II. Xenobiotica, 17, 981991. Levin, D.E., Hollstein, M., Christman, M.F., Schwlers, E.A. and Ames, B.N. (1982) A new Salmonella tester strain (TA10.2) with A/T, base pairs at the site of mutation detects oxidative mutagens. Proc. Natl. Acad. Sci. USA, 79,7445-7449. v.d. Lieth, C.W., Dolata, D.P. and Liljefors, T. (1984) RINGS - a general program to built ring systems. J. Mol. Graph., 2,117-123. Liliefors, T. (1983) MOLBUILD - an interactive computer graphics interface to molecular mechanics. J. Mol. Graph.
19
20
21
22
l, lll-117. Look, M. (1974) Hexachlorocyclopentadiene adducts of aromatic compounds and their reactions. Aldrichim. Acta, 7,23-29.
23
McCoy, E.C., (1981) Evidence tases capable of Environ. Mutat.,
24
Rosenkranz, H.S. and Mermelstein, R. for the existence of a family of nitroreducactivating nitrated polycyclics to mutagens. 3,421-427.
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