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(37) Pfau, W., Pool-Zobel, B. L., von der Lieth, C. W., and Wiessler, M. (1990)The structural basis of mutagenic action of aristolochic acid. Cancer Lett. 55, 7-11. (38) Gassman, P. G., and Campbell, G. A. (1972) Thermal rearrangement of N-chloroanilines. Evidence for the intermediacy of nitrenium-ions. J . Am. Chem. SOC.94, 3891-3896. (39) Kadlubar, F. F., Unruh, L. E., Beland, F. A,, Straub, K. M., and Evans, F. E. (1980) In vitro reaction of the carcinogen, Nhydroxy-2-naphthylamine, with DNA at the C-8 and N2-atomsof guanine and adenine. Carcinogenesis 1, 139-150. (40) Galiegue-Zoutina,S., Bailleul, B., Ginot, Y. M., Perly, B., Vigny, P., and Loucheux-Lefebvre, M. H. (1986) N2-Guanyl and N6adenyl arylation of chicken erythrocyte DNA by the ultimate carcinogen of 4-nitroquinolin-1-oxide. Cancer Res. 46,1856-1863. (41) Scribner, J. D., and Naimy, N. K. (1975) Adducts between the carcinogen 2-acetylamidophenanthrene and adenine and guanine of DNA. Cancer Res. 35, 1416-1421. (42) Tillis, D. R., Straub, K. M., and Kadlubar, F. F. (1981) A comparison of the carcinogen-DNA adducts formed in rat liver in vivo after administration of single or multiple doses of Nmethyl-4-aminoazobenzene. Chem.-Biol.Interact. 38, 15-27. (43) Reitz, A. R., Graden, D. W., Jordan, A. D., and Maryanoff, B. E. (1990) Conformational study of N-substituted adenines by dynamic proton NMR relatively high barrier to rotation about C6-N6 in NS,N6-disubstituted adenines. J . Org. Chem. 55, 5761-5766. (44) Massaro, M., McCartney, M., Rosenkranz, E. J., Anders, M.,
McCoy, E. C., Mermelstein, R., and Rosenkranz, H. S. (1983) Evidence that nitroarene metabolites form mutagenic adducts with DNA-adenine as well as with DNA-guanine. Mutat. Res. 122, 243-249. (45) Lasko, D. D., Harvey, S. C., Malaikal, S. B., Kadlubar, F. F., and Essigmann, J. M. (1988) Specificity of mutagenesis by 4aminobiphenyl. J . Biol. Chem. 263, 15429-15435. (46) Fu, P. P., Miller, D. W., von Tungelen, L. S., Bryant, M. S., Lay, J. O., Huang, K., Jones, L., and Evans, F. E. (1991) Formation of C8-modified deoxyguanosine and CS-modified deoxyadenosine as major DNA adducts from 2-nitropyrene metabolism mediated by rat and mouse microsomes and cytosols. Carcinogenesis 12, 609-6 16. (47) Schmeiser, H. H., Janssen, J. W. G., Lyons, J., Scherf, H. R., Pfau, W., Buchmann, A., Bartram, C. R., and Wiessler, M. (1990) Aristolochic acid activates ras genes in rat tumors at desoxyadenosine residues. Cancer Research 50,5464-5467. (48) Hall, M., and Grover, P. L. (1990) Polycyclic aromatic hydrocarbons: Metabolism, activation and tumour initiation. In Chemical Carcinogenesis and Mutagenesis (Cooper, C. S., and Grover, P. L., Eds.) Vol. I, pp 327-372, Springer, Heidelberg, FRG. (49) Roy, A. K., Upadhyaya, P., Evans, F. E., and El-Bayoumy, K. (1991) Structural characterization of the major adducts formed by with DNA. Carreaction of 4,5-epoxy-4,5-dihydro-l-nitropyrene cinogenesis 12, 577-581.
Coumarin Mercapturic Acid Isolated from Rat Urine Indicates Metabolic Formation of Coumarin 3,4-Epoxide Thomas Huwer,? Hans-Jurgen Altmann,t Werner Grunow,t Susanne Lenhardt,* Michael Przybylski,* and Gerhard Eisenbrand**s Max-von-Pettenkofer-Institut, Bundesgesundheitsamt, 0-1000 Berlin, FRG, Fakultut fur Chemie der Universitat, D-7750 Konstanz, FRG, and FR Lebensmittelchemie und Umwelttoxikologie der Universitat, 0-6750 Kaiserslautern, FRG Received October 2, 1990
A coumarin mercapturic acid, N-acetyl-S-(3-coumarinyl)cysteine,has been identified in the urine of coumarin-treated rats. [14C]Coumarinwas applied by gavage as a single dose to male Wistar rats (10-150 mg/kg body weight). Twenty-four-hour urine was collected, and the deproteinized concentrate was analyzed for radiolabeled metabolites by HPLC. The new mercapturic acid metabolite is supposed to result from oxidative biotransformation of coumarin to its 3,4-epoxide and subsequent coupling with glutathione. Introduction Coumarin (2H-l-benzopyran-2-one,Figure l), a widely distributed plant and essential oil ingredient, has been used in food, perfumes, and tobacco products. Its use in food has been restricted since it has been reported to be hepatotoxic to rats and dogs (1-4). The metabolism of coumarin has been studied in several laboratory animal species and in man. In contrast to man, where 7hydroxylation is preferred, the major metabolite in the rat was found to be o-hydroxyphenylacetic acid (5).In addition, 3-, 4-, 5-, 6-, 7-, and 8-hydroxycoumarins, as well as ring-opened compounds, such as o-hydroxyphenyllactic acid, 0-coumaric acid, o-hydroxyphenylpropionicacid, and o-hydroxyphenylethanol, have been found in varying amounts (e.g., refs. 6-10). After oral administration to rats, coumarin [ 100-500 mg/kg body weight BW] was found to induce a dose-dependent reduction of nonprotein sulfhydryl groups, mainly Max-von-Pettenkofer-Institut.
* Universitat Konstanz. 8 Universitat
Kaiserslautern.
representing glutathione in liver, concomitant with an increase in urinary thioether excretion (11). Both findings indicate a conjugation reaction between an as yet elusive coumarin metabolite and glutathione in rat liver. The aim of the present investigation was to examine whether as yet unknown mercapturic acid metabolites of coumarin are excreted in the urine of rats, which would imply an initial conjugation with glutathione. If so, this would suggest the intermediate formation of a possibly genotoxic 3,4-epoxide.
Materials and Methods Chemicals. [benzene-14C(U)]Coumarin (specific activity 155 MBq/mmol) was obtained from New England Nuclear, Boston, MA. Purity was found to be greater than 98% as checked by HPLC. Unlabeled coumarin, 7-hydroxycoumarin, 4-hydroxycoumarin, o-hydroxyphenylacetic acid, o-hydroxyphenylethanol, N-acetylcysteine, triethylamine, and solvents were obtained from commercial sources. All reagents were of analytical grade and were used as received. 3-Hydroxycoumarin was synthesized according to Offe and Jatzkewitz (12),6-hydroxycoumarin according to Pechmann and Welsh (13).,o-hydroxyphenyllactic acid according to Plochl and Wolfrum (14), and 4-chlorocoumarin ac-
0893-228~/91/2704-0586$02.50/0 0 1991 American Chemical Society
Coumarin Mercapturic Acid in R a t Urine
fraction 1 2
3 4
5a 5b 6 7
Chem. Res. Toxicol., Vol. 4, No. 5, 1991 587
Table I. Fractionation of Urine and Some Metabolites Identified mobile phase: water/ volume, % of urinary methanol/acetic acid mL radioactivity % of dose metabolites identified* 500 24.4 11.6 conjugates 100:00.1 250 11.5 5.5 OHPLA, OHPAA, 6-OHC 901O:O.l 250 39.7 18.8 OHPAA 80:200.1 250 5.8 2.8 7-OHC, C, 3-OHC 70:300.1 100 14.0 6.6 M 60:40:0.1 100 1.4 0.7 CA, M 60:40:0.1 250 1.0 0.5 CA, 4-OHC 50:500.1 100 0.2 0.1 unidentified metabolites 0:1000.1
In 0-24-h urine samples (dose 150 mg/kg BW). bAbbreviations: OHPLA, o-hydroxyphenyllacticacid; OHPAA, o-hydroxyphenylacetic acid; OHC, hydroxycoumarin; C, coumarin; M, isolated metabolite; CA, o-coumaric acid.
F i g u r e 1. Structure of coumarin. cording to Zagorevskii and Zykov (15). Aminoacylase (N-acylamino acid amidohydrolase, EC 3.5.1.14) from hog kidney was obtained from Boehringer Mannheim, FRG. Glutathione Stransferases from rat and horse liver were from Sigma Chemical Co., St. Louis, MO. Treatment of Animals. Male rats (SPF Wistar, Zentrale Versuchstienucht, Bundesgesundheitaamt), weighing 220-280 g, were gavaged with [l"C]coumarin in arachis oil (DAB 8 , 5 mL/kg BW) a t doses of 10,100, and 150 mg/kg BW, corresponding to specific activities from 75 to 110 KBq/mmol. The animals were transferred to metabolism cages and had free access to tap water and standard laboratory diet. Synthesis of N-Acetyl-S-(4-~oumarinyl)cysteine. A mixture of 7.5 mmol of N-acetylcysteine, 7.5 mmol of 4-chlorocoumarin, and 15 mmol of triethylamine was stirred in 80 mL of methanol under nitrogen a t room temperature. After 2 days it was acidified to p H 5 with diluted hydrochloric acid, and the solvent was evaporated under reduced pressure. The white residue was dissolved in 75 mL of sodium hydroxide (0.1 N)and filtrated. The filtrate was titrated with hydrochloric acid to pH 5, and the precipitated product was crystallized from methanol (mp 223-225 "C). The structure was confirmed by lH nuclear magnetic resonance spectroscopy and mass spectrometry. (Anal. Calcd for C14H13NO& C, 54.71; H, 4.26; N, 4.56. Found: C, 54.49; H, 4.18; N, 4.40.) Separation of Urine w i t h Preparative HPLC. Acetone/ methanol (31 v/v, 320 mL) was added slowly to 80 mL of pooled urine (4 animals) a t 4 "C. The precipitate, consisting mostly of proteins and salts, was separated by centrifugation a t 3000 rpm for 10 min, rewashed with 100 mL of acetone/methanol/water (3:l:l v/v), and again centrifuged. The combined supernatants, containing more than 98% of radioactivity, were evaporated under reduced pressure to 40 mL. The concentrate was applied onto a preparative reversed-phase column (Nucleosil100-C 18,5 pm, 250 x 16 mm id.) using a Knauer HPLC pump 64 (flow rate 16 mL/min) equipped with a Knauer variable-wavelength monitor set a t 285 nm. Elution with 0.1% acetic acid (500 mL) gave fraction I; subsequent elution by water/methanol/acetic acid (W10:0.1v/v, 250 mL) gave fraction 11. For the following fractions the methanol content of the eluent was increased in 10% increments until a ratio of 5050 (water/methanol) was obtained. (Table
I).
Fraction 5 (mobile phase: water/methanol/acetic acid, m400.1 v/v) appeared to contain one compound in large excess together with some minor constituents and was therefore divided into subfractions 5a and 5b (see Table I). Fractions were evaporated to 5 mL under reduced pressure, aliquots were taken for determination of distribution of radioactivity, and the rest was stored a t -20 "C until further examination. Isolation of an U n k n o w n Metabolite. Fraction 5a was evaporated to 2 mL and stored for several days a t -20 OC. After thawing, white crystals had deposited, which were separated by filtration. The deposit was dissolved in water and applied onto
a SepPak C-18 minicolumn. After washing with water (2 mL), it was eluted with methanol. Evaporation yielded a white solid (mp 152-153 "C), which was uniform by HPLC. Incubation with Aminoacylase. Fractions 1-7 (0.1 mL) were made up with water to 1 mL and adjusted to pH 6, if necessary. The isolated metabolite and, for comparison, synthetic Nacetyl-S-(4-coumarinyl)cysteine(50 pg each) were dissolved in water (1mL) Aminoacylase (125 units, 0.5 mL) was added to each solution and the suspension incubated in a water bath a t 25 OC for 16 h. After protein precipitation by acetone/methanol (3:l v/v, 4 mL) the supernatant was evaporated to remove organic solvents. The residue was examined by HPLC (column: Nucleosil 100 (2-18, 5 pm, 250 X 4 mm i.d.; mobile phase: water/ methanol/acetic acid, 70:300.1 v/v; flow 1mL/min; W 285 nm). Analytical H P L C a n d Liquid Scintillation Counting. Metabolites in the fractions were determined by HPLC using a Du Pont chromatographic system series 8800, equipped with a chromatographic pump, an 850 absorbance detector (W285 nm), and a reversed-phase column (Nucleosil 100 C-18 or Spherisorb ODS 11)with water/methanol/tetrahydrofuran/acetonitrile/acetic acid (764:10085:50:1 v/v) as the mobile phase (flow rate 1 mL/min). Fractions of 1mL were collected in scintillation vials, mixed with 5 mL of Hionic-Fluor (Packard Instruments Co., Downers Grove, IL), and counted for radioactivity. Metabolites were also quantified by comparing peak areas. Spectroscopic Analysis. Nuclear magnetic resonance spectroscopy was performed on a Bruker AM 270,270 MHz, with DMSO-ds as internal standard, 6 = 2.49 ppm. Mass Spectrometry. Fast atom bombardement (FAB) mass spectra were obtained with a modified Finnigan MAT 312/AMD 5000 mass spectrometer connected to a SS 200 data system. A Cs' ion source (AMD, Intectra GmbH, Beckeln) was used as primary beam source with a heating current of 2.2 A and an acceleration voltage of 12.5 kV. The following instrumental conditions were used: 1800 (10%) resolution, 3 kV acceleration voltage, and 2.0 kV electron multiplier voltage. Spectra were obtained a t a scan rate of 15 s decade-' by the data system. The sample solution had a concentration of 20 pg/pL in dimethyl sulfoxide. One microliter of the solution was mixed with 1pL of matrix (glycerol with 0.1% trifluoroacetic acid) and deposited on the FAB target. The stainless steel target (2 mm2) was held a t 20 "C. E1 mass spectra were obtained with a Finnigan MAT312 mass spectrometer at 100 "C ion source temperature and 70 eV. The materials were introduced by direct probe insertion.
Results and Discussion Initially, a Nucleosil 100 (3-18 column was selected to separate and quantify coumarin metabolites. However, separation of coumarin and an unidentified metabolite remained incomplete. C h r o m a t o g r a p h y o n Spherisorb ODS I1 resulted i n a more satisfactory resolution (see o-Hydroxyphenylethanol and o-hydroxyFigure 2). phenylacetic acid, however, had nearly identical retention times and were n o t resolved. Table I shows a typical radioactivity distribution in urine after separation with preparative HPLC (dose 150 m g / k g BW). Fraction 1 contained polar conjugates, which were hydrolyzed b y 4 N H C l (100 "C, 1 h) i n t o o-hydroxy-
588 Chem. Res. Toxicol., Vol. 4, No. 5, 1991
Huwer et al.
6ol
.05
40
A2B5
0
I
h
R
109
B
300
200 0
MH+
Ip 304
I
I
II lo
0
I0
20
0 100
30
R e t e n t i o n time ( m i n l
Figure 2. HPLC profile of authentic standards mixed with the isolated metabolite (arrow) on Spherisorb ODS 11. Retention times: 6.6 min; o-hydroxyphenyllactic acid; 8.4 min, o-hydroxyphenylacetic acid; 9.7 min, 7-hydroxycoumarin; 10.6 min, isolated coumarin 3-mercapturic acid; 12.3 min, coumarin; 20.0 min, 3hydroxycoumarin; 29.6 min, o-coumaric acid. phenylacetic acid (OHPAA), 3-hydroxycoumarin, and ocoumaric acid. Fractions 2-4, 5b, and 6 contained unconjugated metabolites. OHPAA, o-hydroxyphenyllacticacid, 0-coumaric acid, unchanged coumarin, and 3-, 4-, 6-, and 7-hydroxycoumarins could be identified (Table I). It is possible that the fraction containing OHPAA also contained some ohydroxyphenylethanol (OHPE), hidden under the OHPAA peak. OHPE has been reported to represent about 15% of the OHPAA peak (8). Fraction 5a contained an unknown metabolite which could be isolated. In order to elucidate its chemical structure, the potential metabolite N-acetyl-S-(4coumariny1)cysteinewas synthesized. On the basis of their chromatographic behavior, however, both substances were not identical. Treatment of fraction 5a with aminoacylase resulted in a marked shift toward shorter retention time of the major constituent, representing over 90% of radioactivity in that fraction. The pure metabolite isolated from this fraction showed the same behavior by incubation with aminoacylase, indicating the presence of an N-acylated amino acid conjugate. Synthetic N-acetyl-S-(4-~oumarinyl)cysteine, although not identical with the unknown metabolite, proved to be an equally well-accepted substrate for the enzyme. Spectral information useful for structural identification was obtained from comparison of FAB-MS of the isolated metabolite (Figure 3A) and synthetic coumarin 4mercapturic acid (Figure 3B). Both mass spectra showed great similarity with the following significant ions (mlz): 308 (MH+),401 [MH+(glycerol)],493 [MH+(glycerol),],585 [MH+(glycerol),], 615 (M2H+),179 [MH+ - H 2C=C(COOH)NHCOCH,], 130 [ [H,C=C(COOH)NHCOCH,]H+]. Besides, several signals resulted from the glycerol matrix [e.g., 185 [ (gly~erol)~H+]]. These ions indicate a molecular weight of 307 for the unknown metabolite and are best reconciled with a mercapturic acid conjugate of coumarin. Supportive though not conclusive evidence was obtained by EI-MS of both substances. The spectrum of the iso-
400
I " "
200
I
,,.,,,
; , I
30 0
[,,!
,;,,
400
Figure 3. FAB mass spectra of the isolated metabolite (A) and synthetic N-acetyl-S-(4-~oumarinyl)cysteine (B) obtained from a glycerol matrix. Table 11. NMR Data (ppm) of Synthetic N-Acetyl-S-(4-~oumarinyl)cysteine and Isolated Metabolite (M)" Identified as N-Acetyl-S-(3-~oumarinyl)cysteine H
R = CHzCHNHCOCH3
I
proton
COOH
CH3
1.84 (s) 1.82 (8) 3.38 (m)and 3.60 (m) ca. 3.30 (m)and 3.50 (m) 4.59 (m) 4.53 (m) 6.38 (9) 7.97 (9) 7.35-7.80 (m) 7.30-7.67 (m) 8.54(d) 8.41 (d)
E 2 3-H 4-H CeH, NH a
Abbreviations: s, singlet; d, doublet; m, multiplet.
lated metabolite showed a fragmentation pattern in the mass range below m/z 200 [178 [M+- H,C=C(COOH)NHCOCH,; loo%], 150 (178 - CO; 50%)] virtually identical with that of the 4-mercapturic acid. In the upper mass range signals at 307 (M+), 289 (M+ - H20), 261 (M+ HCOCH,), 248 (M+ - H2NCOCHJ, 218, and 202 (all below 5%) comply with the mercapturic acid structure. Finally, comparison of the NMR spectra of the metabolite and authentic N-acetyl-S-(4-coumarinyl)cysteine allowed definite structure allocation (Table 11, Figure 4). Signals relevant for identification are the 3-H signal in the spectrum of the 4-adduct (Figure 4, lower panel), which is absent in the spectrum of the isolated metabolite (Figure 4, upper panel), and, vice versa, the 4-H signal in the spectrum of the metabolite, which is absent in the spectrum of the 4-isomer. All other signals comply with the mercapturic acid structure. Taken together, these findings prove the isolated metabolite to be N-acetyl-S-(3coumariny1)cysteine. It is important to note from the data of Table 111that this urinary metabolite is excreted to a major proportion of the doses administered. In the high-dose experiments (100 and 150 mg/kg BW) it proved to be the second prominent metabolite. In the low-dose experiment (10 mg/kg) the mercapturic acid is excreted in about the same amount as 3-hydroxycoumarin. In agreement with pub-
Chem. Res. Toxicol., Vol. 4, No.5, 1991 589
Coumarin Mercapturic Acid in Rat Urine
8 50
I 50
8 00
6 SO
I 00
6 00
5 SO
PP"
5 00
4 SO
4
00
3 50
Z
3 00
50
2 00
Figure 4. 270-MHz NMR spectra of the isolated metabolite (upper panel) and synthetic N-acetyl-S-(4-coumarinyl)cysteine (lower panel) in DMSO-d6. Table 111. Dose-Dependent Urinary Excretion of Some Coumarin Metabolites in the Rat excretion in % for dose, mg/kg BW (% of urinary metabolites) compounda 10 100 150 OHPAA (including OHPE) 25.8 (46.0) 21.0 (42.2) 20.2 (43.0) C 0.8 (1.5) 0.8 (1.6) 1.1 (2.3) 3-OHC 1.2 (2.6) 3.5 (6.2) 1.4 (2.8) CA 0.7 (1.2) 0.5 (1.0) 0.3 (0.7) M 7.5 (15.0) 5.8 (12.4) 2.6 (5.1)
"For abbreviations,see Table I.
+
r
HOAc
- GIU
+ HOAc
+ OH
+I-
- GIu QY
1
.05 R = -CH,-
F-
H NH CO
COOH
-
1
I
CH,
-
Figure 5. Suggested scheme for the formation of coumarin 3-mercapturic acid.
lished results, levels of 4-, 6-, and 7-hydroxycoumarins were below 1% of the urinary activity (5,9). The identification of a mercapturic acid as a metabolite of coumarin provides further evidence for the initial formation of coumarin 3,4-epoxide as postulated by Lake et al., who found conclusive evidence that coumarin hepatotoxicity in the rat was due to its bioactivation by cytochrome P-450 dependent enzymes to toxic metabolites (16). The possibility that coumarin reacts spontaneously with glutathione has been ruled out (11). Likewise, an enzymatically catalyzed direct formation of a glutathione adduct was not observed. After incubation of coumarin (10-1000p M ) with glutathione (5-2000 p M ) in the presence of glutathione S-transferase (30 units, pH 7.2, 37 "C, 60 min) no decrease in glutathione or coumarin concentration was detectable (17). Therefore, we suggest the
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