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Conversion of Aristolochic Acid I into Aristolic Acid by Reaction with Cysteine and Glutathione: Biological Implications Horacio A. Priestap*,† and Manuel A. Barbieri†,‡ †
Department of Biological Sciences, Florida International University, 11200 Southwest 8th Street, Miami, Florida 33199, United States ‡ Fairchild Tropical Botanic Garden, 10901 Old Cutler Road, Coral Gables, Florida 33156, United States S Supporting Information *
ABSTRACT: Aristolochic acid I (AA-I), naturally occurring in Aristolochia plants, is a potent nephrotoxin and carcinogen. Here we report that AA-I suffers hydrogenolysis with loss of the nitro group by reaction with cysteine or glutathione to give aristolic acid. Since the reaction can proceed in aqueous solutions at pH 7.0 and 37 °C, it is inferred that it may also occur in biological systems and contribute to the nephrotoxic effects induced by AA-I.
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Cys and GSH participate in a number of reactions. For example, the free thiol group of Cys and GSH can enter into thiol−disulfide exchange reactions with S−S bridges or can react with α,β-unsaturated enones to give Michael adducts.4,5 It is also known that thiol-bearing compounds (propanedithiol, dithiothreitol, mercaptoethanol, glutathione) reduce aliphatic and aromatic azides to the corresponding amines.6,7 A new type of reaction of Cys and GSH is now described. Preliminary experiments showed that aqueous yellow solutions of AA-I (1a) containing Cys or GSH tend to fade upon standing at room temperature. This behavior is due to conversion of AA-I (1a) into aristolic acid (3a), a compound that does not absorb above 400 nm. HPLC analysis of reaction mixtures containing AA-I (1a) and Cys or GSH at different times showed that the concentrations of AA-I progressively decrease, whereas those of aristolic acid (3a) increase correspondingly (Figure 1). Side products were not observed (Figure 1). Complete conversion of AA-I (1a) into aristolic acid (3a) can be achieved at room temperature, or higher temperatures, in an aqueous solution containing a sufficient excess of Cys or GSH at pH 7.0. A nitrogen atmosphere was used to repress the spontaneous oxidation of Cys to cystine. A typical experiment illustrating the conversion of AA-I (1a) into aristolic acid (3a) with Cys is described in the Experimental Section, and HPLC profiles are shown in Figure 1. GSH is equivalent to Cys in chemical reactivity toward AA-I. However, the reaction is slightly slower than that with Cys. The reactions of Cys and GSH with AA-I are slow as compared with other reactions reported for Cys, such as those with iodoacetate or αmethylene γ-lactones.4 In this work aristolic acid (3a) was
ristolochic acids (AAs) are nitro compounds found in Aristolochia spp. These compounds are nephrotoxic and also display mutagenic and carcinogenic properties in both humans and mammals.1 The chemical reactivity of AAs toward model biological nucleophiles is being investigated, and we now describe the reaction of AA-I (1a) with the amino acid cysteine (Cys) and the tripeptide glutathione (GSH). The nitro group of AAs can be easily reduced. Thus, AA-I (1a) can be converted into the corresponding aristolactam (2) by a variety of reducing agents.2 However, under certain reduction conditions the nitro group of AA-I (1a) can also suffer hydrogenolysis, a reaction in which the nitro group is replaced by hydrogen, to produce the corresponding denitro compound, aristolic acid (3a). Hydrogenolysis of AA-I (1a) to aristolic acid (3a) with sodium borohydride or ammonium sulfide has been reported.2,3 Here we report that the nitro group of AA-I (1a) can be removed to give aristolic acid (3a) by treatment with Cys or GSH, under conditions in which the aristolactam (2) is not formed.
Received: November 23, 2012 © XXXX American Chemical Society and American Society of Pharmacognosy
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Figure 1. Reaction of AA-I (4.8 mM) and Cys (37.5 mM) in aqueous solution at pH 7.0 and 50 °C. Typical HPLC profiles showing decay of AA-I (1a) concentrations and increase of aristolic acid (3a) concentrations as a function of the reaction time. About 50% of AA-I was converted into aristolic acid in 300 min. Under the same conditions but incubating the reaction mixture at 37 °C about 10% of AA-I was converted into aristolic acid in 300 min.
In order to get information about the mechanism, the AA-I− Cys reaction was performed in D2O. It was found that a deuterium atom is introduced at C-10 of aristolic acid, as evidenced by 1H NMR analysis. The 1H NMR spectrum of aristolic acid (3a) shows H-9 and H-10 as two doublets (J = 7.6 Hz) centered at δ 8.02 and 8.79, respectively (the relative positions of H-9 and H-10 resonances were established by NOE experiments in related denitroAAs).13 When the reaction is carried out in D2O, the 1H NMR spectrum of compound 3b was found to be similar to 3a, except for the signals of H-9 and H-10. The signal of H-10 (doublet, J = 7.6 Hz) was drastically reduced, whereas the H-9 signal was transformed into a singlet (accompanied by two small satellite signals; a doublet corresponding to the 9,10-diH species 3a; Supporting Information, Figure S1), consistent with the introduction of a deuterium atom at C-10 of aristolic acid (3b). Signal intensities showed that the reaction product in the presence of D2O consisted of 8% of the 9,10-diH species (3a) and 92% of the 9H-10-D species (3b). The above findings suggest that this hydrogenolysis reaction involves direct transfer of two electrons and a proton, or H−, from the thiol group (SH) of Cys to the C-10−NO2 position of AA-I, hence resulting in the elimination of the nitro group to give aristolic acid (3a). If the AA-I−Cys reaction proceeds through single electron transfer, it may be assumed that the transfer of the first electron creates a radicalion pair, within a solvent cage, that is immediately stabilized by the second electron (and proton) transfer, so that DTBN cannot interact with the transient radical species formed during
isolated and characterized by spectrometric methods. This compound (3a) naturally occurs in Aristolochia plants.8 In studies performed to evaluate the nephrotoxic potential of AA analogues with LLC-PK1 cells, aristolic acid (3a) was nontoxic, indicating that the nitro group is important for the activity and that this acid would not be involved in the AA-induced apoptosis of renal tubule cells.9 The reaction of AA-I with Cys or GSH generates aristolic acid and presumably nitrous acid and an oxidized form of Cys or GSH. Cystine is not a product from the reaction of AA-I (1a) with Cys, so disulfides may not be the oxidation products of Cys and GSH. Cys occurs in various ionic species as a function of the pH.10 At pH 7 the ionic form H3N+CH(CH2SH)COO− is largely predominant, and it could be the species that reacts with AA-I. Reductive replacement of the nitro group by hydrogen in aliphatic and aromatic compounds has been reported.11,12 A mechanism involving electron transfers and radical species was postulated by Kornblum et al. (1979)11 for the hydrogenolysis of the nitro group of tertiary nitroparaffins with methyl mercaptan (CH3SH). A proof in favor of this free radical chain mechanism is that in the presence of 10 mol % of di-tert-butylnitroxide (DTBN; free radical scavenger) the replacement of the nitro group by hydrogen is completely inhibited.11 However, the AA-I−Cys reaction proceeds by a different pathway since 10% mol of DTBN (with respect to AA-I) in the medium does not affect the formation of aristolic acid (3a). B
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modifying key proteins or enzymes essential for cell function. The reaction of AA-I with thiol-containing molecules is slow, the biological impact of which remains to be determined. However, the above evidence suggests that it may have some implication in vivo and that thiol depletion induced by AA-I could be one of the primary events in the ethiology of AAN. In summary, a new type of reaction, which occurs under physiological conditions, is described for Cys and GSH, namely, the hydrogenolysis of the nitro group in an aromatic molecule. The AA-I−thiol reaction may serve as a plausible explanation for the appearance of aristolic acid (3a) and its demethylated derivative (3c) in urine and feces of AA-I-administered rats and probably also contributes to the observed depletion of GSH in AA-I-treated cells. If this AA-I−thiol reaction occurs in vivo and induces adverse effects, a diet supplemented with thiol-bearing agents such as N-acetylcysteine could display protective effects against AAN and other ailments caused by AAs.
the reaction. Reactions with a mixture of AAs showed that AA-I (1a) and AA-II (1b) are equally reactive toward Cys and GSH. In contrast, AA-III (1c) and AA-IV (1d) remain unaffected. Thus, the reaction seems to be restricted to AA-I (1a) and AAII (1b). This result indicates that introduction of a methoxy group at C-6 of AAs significantly alters the electron distribution and electrophilic properties of the molecule. There is a correlation between chemical structure and toxicity since in studies with cultured renal epithelial cells AA-I (1a) was found to be the most toxic, followed by AA-II (1b), whereas AA-III (1c), with a 6-OMe group, was nontoxic.9 The facile replacement of the nitro group of AA-I by hydrogen by interaction with thiols seems to be restricted to AAs since related compounds such as 1-nitronaphthalene and 8-nitro-1-naphthoic acid proved to be unreactive toward Cys, even forcing the reaction at higher temperatures. Consequently, it is concluded that this AA-I−thiol reaction is also closely related to the known high reactivity of the C-9−C-10 bond in phenanthrene. The nephropathy caused by AAs has been designated aristolochic acid nephropathy (AAN). Although a number of mechanisms have been postulated to explain this disease, the true pathogenic mechanism leading to AAN is still unclear.14 A number of natural products and synthetic drugs, or their metabolites, are known to induce cytotoxic lesions through depletion of intracellular thiols that result in a variety of toxic effects including nephropathy.15−20 Intracellular thiol depletion produces cell injury and apoptosis due to generation of reactive oxygen species (ROS), DNA damage, mitochondrial dysfunction, and other processes15−20 that have also been observed to be induced by AA-I,13,21−25 thereby suggesting a connection between the mechanisms of toxicity produced by thioldepleting agents and AA-I. It remains to be determined whether the AA-I−thiol reaction described in this study takes place in cells. However, evidence supporting the occurrence of this reaction in vivo is provided by the fact that aristolic acid (3a) and its demethylation product 3,4-methylenedioxy-8hydroxy-1-phenanthrenecarboxylic acid (3c) were identified in urine and feces of AA-I-treated rats.26,27 In these experiments aristolic acid (3a) and its demethylation product (3c) may account for up to 8% of the dose in the urine and feces collected over 72 h after administration of AA-I.26 These results suggest that the metabolites 3a and 3c may arise from the slow interaction of AA-I with intracellular thiol residues. In vitro studies using mice and human cells as models also showed that thiol depletion can be triggered by AA-I.14,25 Thus, it was found that AA-I depletes intracellular GSH in human HL-60 and mice C3H/He cells.14,25 GSH is an intracellular reducing agent whose primary function may be to maintain the SH groups of proteins and also plays a role in antioxidant defense, protecting the cell against reactive electrophilic xenobiotics.28,29 Depletion of cellular GSH can impair cellular defenses against ROS or toxic compounds and lead to cellular injury followed by apoptosis and necrosis.30 Decreased GSH levels in human HL60 and mice C3H/He cells exposed to AA-I are believed to be responsible for the generation of ROS and apoptosis of renal tubular cells.14,25 The reaction of AA-I with GSH described herein may contribute to the total GSH depletion induced by AA-I. The high concentration of GSH in animal cells (0.5−10 mM)30 together with the observed accumulation of AA-I in kidney31,32 may favor the AA-I−GSH interaction so that it may become significant. In addition to the possible depletion of GSH, AA-I can also interact with Cys residues of proteins, thus
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EXPERIMENTAL SECTION
General Experimental Procedures. Aristolochic acids are from plant origin. Pure AA-I was obtained from a mixture of AAs by preparative HPLC. Cysteine (C-7755), glutathione (G-4251), and ditert-butylnitroxide (300721-1G) were purchased from Sigma. pH measurements were carried out with a Jenco model 60 portable digital pH meter provided with a PHR-146 Micro Combination pH electrode (Lazar Research Laboratories, Los Angeles, CA, USA). NMR spectra were recorded on a Bruker 400 MHz FT-NMR spectrometer in DMSO with TMS as internal standard. ESI mass spectra were acquired on a Thermo Scientific LCQ Deca XP MAX instrument. HPLC analysis was carried out with a Thermo-Finnigan chromatograph (Thermo Electron Corporation, San Jose, CA, USA). The chromatograph consisted of a SpectraSystem SMC1000 solvent delivery system, vacuum membrane degasser, P4000 gradient pumps, and AS3000 autosampler. Column effluent was monitored at 226 or 254 nm with a SpectraSystem UV6000LP variable-wavelength PDA detector and ChromQuest 4.1 software. Analytical separations were performed using a C18 RP Hypersil GOLD column (RP5, 250 × 4.6 mm, pore size 5 μm, Thermo Electron Corporation). The following eluting system was used: A, MeCN; B, 0.1% TFA in H2O, linear gradient 30% to 45% A in 60 min (AA-I, tR 22.06 min; aristolic acid, tR 26.88 min), flow rate 1.0 mL/min at room temperature. Reaction mixtures (20 μL) were injected after 1:100 dilution in DMSO. 3,4-Methylenedioxy-8-methoxy-1-phenanthrenecarboxylic Acid, Aristolic Acid (3a). AA-I (ca. 6 mg, 18 μmol) was suspended in H2O (1 mL). The suspension was treated with NaHCO3 (ca. 20 mg) and heated to dissolve the acid, and the solution cooled at room temperature. Some turbidity was removed by centrifugation. The exact amount of AA-I in the solution was determined by HPLC analysis versus a calibration curve. Cysteine (31 mg, ca. 250 μmol) was added to the solution, and the pH was adjusted to 7.0 with 8.5% phosphoric acid. The reaction mixture was purged with N2 to remove oxygen. The yellow solution was heated at 37 °C overnight with magnetic stirring. Analysis of the reaction mixture by HPLC showed a predominant peak of aristolic acid (3a) and traces of unreacted AA-I (1). The reaction mixture was centrifuged to remove some insoluble material. The supernatant was diluted (×2) with H2O and loaded to a C18 cartridge (SepPack Vac C18 cartridge, 500 mg, WAT036905, Waters; conditioned with MeOH and H2O). The cartridge was washed with H2O (4 mL) and 1% HOAc in CH3CN/H2O 4:6 (4 mL). Aristolic acid was eluted with 1% HOAc in CH3CN/H2O (7:3, 4 mL). The eluate containing aristolic acid was extracted with EtOAc. The EtOAc phase was evaporated to dryness to give aristolic acid, 4 mg: UV λmax 255, 296, 319 sh, 327, 355, 375 nm; 1H NMR δ ppm (DMSO-d6) 4.00 (3H, s, 8-OCH3), 6.39 (2H, s, −OCH2O−), 7.22 (1H, d, J7,6 = 8.0 Hz, 7-H), 7.61 (1H, t, J6,5 = J6,7 = 8.2 Hz, 6-H), 7.80 (1H, s, 2-H), 8.02 (1H, d, J9,10 = 7.6 Hz, 9-H), 8.64 (1H, d, J5,6 = 8.4 Hz, 5-H), and 8.79 (1H, d, J10,9 = 7.6 Hz, 10-H) (coherent with Mukhopadhyay et al., 1983);2 13C NMR δ ppm (DMSO-d6) 168.4 (CO), 154.7 (C-8), C
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145.6 (C-4), 144.3 (C-3), 128.4 (C-4b), 128.0 (C-10a), 127.1 (C-6), 123.9 (C-10), 121.8 (C-8a), 119.5 (C-1), 119.1 (C-9), 118.7 (C-5), 115.5 (C-4a), 111.7 (C-2), 107.3 (C-7), 102.5 (OCH2O), 55.7 (OMe) (assignments were based on ref 33); ESIMS (MeOH) positive mode 297 [M + H]+; 279 [M + H − H2O]+.
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(19) Wullner, U.; Seyfried, J.; Groscurth, P.; Beinroth, S.; Winter, S.; Gleichmann, N.; Heneka, M.; Loschmann, P.-A.; Schultz, J. B.; Weller, M.; Klockgether, T. Brain Res. 1999, 826, 53−62. (20) Tukov, F. F.; Anand, S.; Gadepalli, R. S. V. S.; Gunatilaka, A. A. L.; Matthews, J. C.; Rimoldi, J. M. Chem. Res. Toxicol. 2004, 17, 1170− 1176. (21) Qi, X.; Cai, Y.; Gong, L.; Liu, L.; Chen, F.; Xiao, Y.; Wu, F.; Li, Y.; Xue, X.; Ren, J. Toxicol. Appl. Pharmacol. 2007, 222, 105−110. (22) Wang, W.; Zhang, J. Eur. J. Pharmacol. 2008, 588, 135−140. (23) Liu, Q.; Wang, Q.; Yang, X.; Shen, X.; Zhang, B. Toxicol. Lett. 2009, 184, 5−12. (24) Yang, H.; Dou, Y.; Zheng, X.; Tan, Y.; Cheng, J.; Li, L.; Du, Y.; Zhu, D.; Lou, Y. Toxicology 2011, 287, 38−45. (25) Yu, F.-Y; Wu, T.-S.; Chen, T.-W.; Liu, B.-H. Toxicol. in Vitro 2011, 25, 810−816. (26) Krumbiegel, G.; Hallensleben, J.; Mennicke, W. H.; Rittmann, N.; Roth, H. J. Xenobiotica 1987, 17, 981−991. (27) Chan, W.; Cui, L.; Xu, G.; Cai, Z. Rapid Commun. Mass Spectrom. 2006, 20, 1755−1760. (28) Sen, C. K. J. Nutr. Biochem. 1997, 8, 660−672. (29) Lu, S. C. FASEB J. 1999, 13, 1169−1183. (30) Reed, J. D.; Fariss, M. W. Pharmacol. Rev. 1984, 36, 25S−33S. (31) Xiao, I.; Ge, M.; Xue, X.; Wang, C.; Wang, H.; Wu, X.; Li, L.; Liu, L.; Qi, X.; Zhang, Y.; Li, Y.; Lou, H.; Xie, T.; Gu, J.; Ren, G. Kidney Int. 2008, 73, 1231−1239. (32) Priestap, H. A.; Torres, M. C.; Rieger, R. A.; Dickman, K. G.; Freshwater, T.; Taft, D. R.; Barbieri, M. A.; Iden, C. R. Chem. Res. Toxicol. 2011, 25, 130−139. (33) Achari, B.; Bandyopadhyay, S.; Chakravarty, A. K.; Pakrashi, S. Org. Magn. Reson. 1984, 22, 741−746.
ASSOCIATED CONTENT
S Supporting Information *
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H NMR spectrum of deuterated aristolic acid (1b). This information is available free of charge via the Internet at http:// pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: 305-348-0375. Fax: 305-348-1986. E-mail: priestap@ fiu.edu. Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are grateful to Y. L. Hsu and Y. Song (Florida International University) for recording NMR and ESIMS spectra. We specially thank Dr. S. Rose for her support (School of Integrated Science and Humanity, Florida International University).
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REFERENCES
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