Urinary Metabolites from F344 Rats and B6C3F1 Mice

1152

Chem. Res. Toxicol. 1997, 10, 1152-1160

Urinary Metabolites from F344 Rats and B6C3F1 Mice Coadministered Acrylamide and Acrylonitrile for 1 or 5 Days Susan C. J. Sumner,* Leena Selvaraj, Sara K. Nauhaus, and Timothy R. Fennell Chemical Industry Institute of Toxicology, 6 Davis Drive, P.O. Box 12137, Research Triangle Park, North Carolina 27709-2137 Received December 31, 1996X

The purpose of this study was to examine the feasibility of using 13C NMR spectroscopy to analyze urinary metabolites produced following coadministration of two structurally similar carbon-13-labeled compounds to rodents. Acrylonitrile (AN) and acrylamide (AM) are used in the chemical industry to manufacture plastics and polymers. These compounds are known to produce carcinogenic, reproductive, or neurotoxic effects in laboratory animals. The potential for human exposure to AN and AM occurs in manufacturing facilities and environmentally. Male F344 rats and B6C3F1 mice were coadministered po [1,2,3-13C]AN (16-17 mg/kg) and [1,2,3-13C]AM (21-22 mg/kg) after 0 or 4 days of administration of unlabeled AN or AM. Urine was collected for 24 h following administration of the 13C-labeled compounds and analyzed by 13C NMR spectroscopy. Rats and mice excreted metabolites derived from glutathione (GSH) conjugation with AM or AN or derived from GSH conjugation with the epoxides cyanoethylene oxide (CEO) or glycidamide (GA). GA and its hydrolysis product were also detected in the urine of rats and mice. For mice, an increased urinary excretion of total AN- and total AMderived metabolites (p < 0.05) on repeated coadministration suggested a possible increase in metabolism via oxidation. In addition, mice had an increased (p < 0.05) percentage of dose excreted as metabolites derived from GSH conjugation with AM, AN, CEO, or GA after five exposures as compared with one exposure that may be related to a significant increase in the synthesis of GSH or an increase in glutathione transferase activity. The only significant (p < 0.05) increase between one and five exposures for the rat was in the percentage of metabolites produced following conversion of AM to GA. The use of 13C NMR spectroscopy has provided a powerful methodology for elucidation of the metabolism of two 13C-labeled chemicals administered simultaneously.

Introduction The separation and identification of small polar metabolites can present a considerable methodological challenge. Techniques such as HPLC and mass spectrometry are not ideally suited to the study of small polar metabolites. The development of appropriate methods for analysis and characterization becomes more challenging when investigating the metabolism of mixtures. Conventional studies therefore generally investigate the metabolism of only one compound (usually 14C-labeled) in a chemical mixture. The purpose of this study was to examine the feasibility of using 13C NMR spectroscopy to analyze urinary metabolites produced following coadministration of two structurally similar carbon-13-labeled compounds to rodents. Acrylonitrile (AN)1 and acrylamide (AM) are used in the chemical industry to manufacture plastics and polymers. Release of acrylonitrile can occur during monomer and polymer production, transportation, and usage (1), with the potential for exposure from acrylonitrile in the air and in water. Acrylamide is used as a grouting agent, in soil, tunnel, and dam stabilization, and in the production of polyacrylamide gels (2). Polyacrylamide is used * Corresponding author. Phone: 919-558-1343. Fax: 919-558-1300. X Abstract published in Advance ACS Abstracts, September 15, 1997. 1 Abbreviations: AM, acrylamide; AN, acrylonitrile; CEO, cyanoethylene oxide; GA, glycidamide; GSH, glutathione.

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in water treatment, and residues of acrylamide may be found in treated water (2). Acrylamide is manufactured by hydration of acrylonitrile. Exposure to both agents can occur during this process (3, 4). AN is carcinogenic in rats, producing tumors in the brain, stomach, and Zymbal’s gland (5). No bioassays have been conducted in other species. Neurotoxic effects have been reported for humans exposed to AM (6). Laboratory animals exposed to AM exhibit neurotoxic, genotoxic, reproductive, and carcinogenic effects (6, 7). The acute toxicity of AN (8) is greater in mice than in rats. Upon administration to rodents, AM (9, 10) and AN (11) are rapidly removed from the blood stream. Within 24 h after administration of carbon-14-labeled AM (10, 12), carbon-13-labeled AM (13), carbon-14-labeled AN (14), or carbon-13-labeled AN (15) to rats and mice, 5060% of the dose is excreted in urine. AM and AN are both metabolized via direct conjugation with glutathione, resulting in the urinary excretion of N-acetyl-S-(2-cyanoethyl)cysteine (15-17), S-(2-cyanoethyl)thioacetic acid (14, 15), and N-acetyl-S-(3-amino3-oxopropyl)cysteine (10, 13). AN is metabolized to the epoxide cyanoethylene oxide (CEO) via cytochrome P450 (18, 19) and has been detected in blood and brain of rats administered AN (20). The epoxide glycidamide, formed presumably via cytochrome P450, has been detected in the urine (13) and blood2 of rodents exposed to AM. Both CEO and GA react with DNA (21-25) and are mutagenic © 1997 American Chemical Society

Urinary Metabolites of Acrylamide and Acrylonitrile

(26-28). These epoxides are believed to be involved in the carcinogenic activity of the parent compounds. A number of products produced from CEO conjugation with glutathione (18) have been detected in urine from rodents administered AN, including: thiocyanate (8, 15), N-acetyl-S-(2-hydroxyethyl)cysteine (15, 17), N-acetyl-S(1-cyano-2-hydroxyethyl)cysteine (15, 29), thiodiglycolic acid (15), thionyldiacetic acid (15), and S-(carboxymethyl)cysteine (15, 30). Rodents administered AM excrete GA, the hydrolysis product of GA (2,3-dihydroxypropionamide), and products derived from glutathione conjugation with GA that include N-acetyl-S-(3-amino-2hydroxy-3-oxopropyl)cysteine and N-acetyl-S-(1-carbamoyl2-hydroxyethyl)cysteine (13). Much of our understanding of the toxic effects of chemicals comes from studies conducted with single chemicals, yet most of the exposures encountered by people will likely be to mixtures of chemicals. One component of assessing the risk of exposures to mixtures is understanding the metabolic interactions of chemicals in competing for enzymes and substrates. In this study, 13C NMR was used to detect and quantitate metabolites in urine of rats and mice coadministered (po) [1,2,3-13C]AN and [1,2,3-13C]AM after 0 or 4 doses of the unlabeled material. The use of 13C NMR with two 13C-labeled chemicals administered simultaneously provides a methodological approach to investigate the metabolism of mixtures.

Materials and Methods Chemicals. [1,2,3-13C]Acrylamide was purchased from ISOTEC Inc. (Miamisburg, OH). [1,2,3-13C]Acrylonitrile was obtained from Cambridge Isotope Ltd. (Cambridge, MA). The 1H and 13C NMR spectra of [13C]AN (CDCl3) and [13C]AM (D2O) were consistent with those previously assigned (13, 15). The chemical purity of [13C]AM was 97.6%, and the isotopic enrichment was 99.5%. The chemical purity of [13C]AN was >98%, and the isotopic enrichment was 99%. Acrylamide (CAS no. 7606-1), acrylonitrile (CAS no. 107-13-1), and 1,4-dioxane (HPLC grade, CAS no. 123-91-1) were purchased from Aldrich Chemical Co. (Milwaukee, WI) and had >99% chemical purities. Animals. Male Fischer F344 rats and male B6C3F1 mice were purchased from Charles River Laboratories (Raleigh, NC) and acclimated for at least 12 days. At the time of dosing, male Fischer F344 rats (165-184 g) and male B6C3F1 mice (24-26 g) were 7-8 weeks old. They were housed according to recommendations listed in the guide for care and use of laboratory animals (DHEW Publication No. NIH 86-23). They were supplied food (NIH 07 diet) and deionized water ad libitum and maintained on a 12-h light-dark cycle at a temperature of 22 ( 2 °C and relative humidity of 55 ( 5%. Dosing and Sample Collection. Four male F344 rats and four male B6C3F1 mice were administered po [1,2,3-13C]AM (21 mg/kg, ∼0.28 mmol/kg) and [1,2,3-13C]AN (16 mg/kg, ∼0.28 mmol/kg). Four male F344 rats and four male B6C3F1 mice were administered po [1,2,3-13C]AM (22 mg/kg) and [1,2,3-13C]AN (17 mg/kg) following 4 days of administration of the unlabeled material (approximately 22 mg/kg AM and 17 mg/kg AN on each day). Four additional rats and mice were administered vehicle for either 1 or 5 days. For all dosing solutions, the acrylamide and acrylonitrile were prepared in distilled water and administered in volumes of 1.0 or 10.0 mL/kg of body weight for rats and mice, respectively. Solutions were administered within 1 h of preparation on each day of dosing, and an aliquot of each dosing solution was analyzed by 1H and 13C NMR spectroscopy to confirm stability. The animals were placed in glass metabolism cages following 2 Cheng, S.-Y., Fennell, T. R., Brown, C. B., and Sumner, S. C. J. (1996) Unpublished results.

Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1153 administration of the labeled material or following administration of the vehicle for 1 or 5 days, and urine was collected for 24 h. NMR Spectroscopy. For characterization of metabolites, samples were prepared for NMR analysis by adding 100 µL of D2O to 400 µL of urine. NMR spectra were acquired with a 5-mm dual proton-multinuclear probe on a Varian VXR-300 spectrometer (Palo Alto, CA). 1H-Decoupled 13C NMR spectra were acquired in the double precision mode with an acquisition time of 0.8 s, 32 K data points, and a relaxation delay of 5 s. Samples were prepared for quantitation of metabolites by adding 100 µL of D2O and 1 µL of 1,4-dioxane to 400 µL of urine. Relaxation times (T1) were determined using the DOT1 experiment from the Varian pulse sequence library (Varian, Palo Alto, CA). The relaxation times (T1) for the labeled methylene or methine carbons in 1,4-dioxane (5 s), all N-acetylcysteine derivatives (1 s), glycidamide (2 s), and the hydrolysis product of glycidamide (2 s) were less than 5 s. The relaxation time of thiocyanate (SCN) was not determined due to the long period required for relaxation (>30 s), prohibiting the quantitation of this compound. The relaxation time of AM was not determined because it appeared in trace quantities and there was signal overlap from endogenous compounds. AN was not detected in urine. 13C NMR spectra were acquired (on two samples of rat urine) for quantitation with a relaxation delay of 60 s and broad-band decoupling only during acquisition. The integral for 1,4-dioxane (added at a known concentration) and a labeled methylene carbon (CH2) from each metabolite were used to calculate the metabolite concentrations reported in this manuscript (taking into account the percentage of 13C enrichment and the number of carbons that give rise to each signal). The best resolved methylene carbon for each metabolite was selected for quantitation. Since the relaxation delay was at least 5 × T1, nearly identical results were obtained when more than one methylene carbon, or a methylene and methine carbon, was used for quantitation of a metabolite. Concentrations of metabolites were also determined by obtaining integrals from spectra acquired with a relaxation delay of 25 s and broad-band decoupling only during acquisition, providing essentially identical results for all urinary products of [13C]AM and [13C]AN. Therefore the 25-s relaxation delay was deemed sufficient for quantitation of urinary products of [13C]AM and [13C]AN (with the exception of SCN), as used in previous investigations of the metabolism of [13C]AM (13) and [13C]AN (15). Metabolites of [13C]AM and [13C]AN at low concentrations (24 h) to obtain sufficient signal-to-noise (S/N) for integration, when using long relaxation delays (g25 s) and broad-band decoupling only during acquisition. In addition, sufficient S/N for integration could not be obtained for many metabolites excreted in mouse urine under these experimental conditions. To circumvent this problem, 13C NMR spectra were acquired with a relaxation delay of 25 s and broad-band decoupling only during acquisition to provide sufficient S/N for integration of metabolites at only the highest concentration (>1.0 mM, rats; >0.5 mM, mice). Concentrations for metabolites in lower concentration were then calculated by comparing the relative intensities (obtained from the 1Hdecoupled 13C NMR spectra acquired with a relaxation delay of 5 s) of carbon signals for high- and low-concentration metabolites. For the two rat samples described above, nearly identical quantitative results were obtained for urinary products of [13C]AM and [13C]AN, demonstrating the utility of using this method. This simplified method was used to analyze four rat and four mouse urine samples from each of the exposure regimens, since it provided results essentially identical to the samples analyzed using each of the quantitative methods described above. Quantitative values are presented in concentration (mM), total amount (µmol), percentage of total AM or AN metabolites excreted, and percentage of the AM or AN dose. Statistics. Statistical analysis was performed using the program JMP 3.1.5. Statistically significant differences were determined using p values obtained from Student’s t-test

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Figure 1. 1H-Decoupled 13C NMR spectra of urine collected from rats administered po (A) distilled water or coadministered po [1,2,3-13C]AM and [1,2,3-13C]AN following (B) 0 or (C) 4 days of coadministration of the unlabeled material. Signals for metabolites of [1,2,3-13C]AM and [1,2,3-13C]AN are labeled in panels B and C, respectively. Each signal is labeled with a metabolite number (Scheme 1) and a letter designating the carbon derived from acrylamide (aCH2dbCH-cCO2H) or acrylonitrile (aCH2dbCH-cCN). analysis. For the purposes of this study, p < 0.05 was assumed to be statistically different.

Results Structural Assignments. The 1H-decoupled 13C NMR spectra of control urine from rats (Figure 1A) and mice (Figure 2A) administered vehicle (single po) have an intense peak for urea, referenced to 162.5 ppm (15). Other peaks present in the spectra are due to endogenous compounds such as creatinine, hippurate, and glucose (31). The 1H-decoupled 13C NMR spectra of control urine from rats and mice administered vehicle po for 5 days (spectra not shown) are qualitatively similar to spectra acquired for urine from rats and mice administered a single po dose of vehicle. The 1H-decoupled 13C NMR spectra of urine obtained from rats (Figure 1B) and mice (Figure 2B) administered (po) a single dose of [1,2,3-13C]AM and [1,2,3-13C]AN and from rats (Figure 1C) and mice (Figure 2C) administered

po [1,2,3-13C]AM and [1,2,3-13C]AN following 4 days of administration of the unlabeled material are qualitatively similar. Metabolites of [1,2,3-13C]AM and [1,2,3-13C]AN are recognized by coupling patterns that are produced by spin-spin interactions between labeled carbons. Here, a 13C nucleus with one adjacent 13C nucleus gives rise to a doublet, while a 13C nucleus with two adjacent 13C nuclei gives rise to a doublet of doublets. A singlet (133 ppm) was present in spectra obtained for urine from rats and mice administered [1,2,3-13C]AM and [1,2,3-13C]AN but not in spectra of control urine. The chemical shifts (Table 1) and coupling constants of signals in the 1H-decoupled 13C NMR spectra are consistent with those previously assigned (13, 15) following administration po of either [1,2,3-13C]AM (50 mg/ kg, rats and mice) or [1,2,3-13C]AN (30 and 10 mg/kg, rats; 10 mg/kg, mice). The signals in the spectra of urine obtained for rats administered the mixture of AM and AN for 1 day (Figure 1B) or 5 days (Figure 1C) are labeled


Chem. Res. Toxicol., Vol. 10, No. 10, 1997 1155

Figure 2. 1H-Decoupled 13C NMR spectra of urine collected from mice administered po (A) distilled water or coadministered po [1,2,3-13C]AM and [1,2,3-13C]AN following (B) 0 or (C) 4 days of coadministration of the unlabeled material. Signals for metabolites of [1,2,3-13C]AM and [1,2,3-13C]AN are labeled in Figure 1B,C, respectively.

with metabolites derived from AM or AN, respectively. The assignments include N-acetyl-S-(2-cyanoethyl)cysteine (metabolite I), N-acetyl-S-(2-hydroxyethyl)cysteine (metabolite II), diastereomers of N-acetyl-S-(1-cyano-2hydroxyethyl)cysteine (metabolites III, III′), S-(carboxymethyl)cysteine (or its N-acetyl derivative) and thiodiglycolic acid (metabolites IV, IV′), thionyldiacetic acid (metabolite V), N-acetyl-S-(3-amino-3-oxopropyl)cysteine (metabolite 1), diastereomers of N-acetyl-S-(3-amino-2hydroxy-3-oxopropyl)cysteine (metabolites 2, 2′), N-acetylS-(1-carbamoyl-2-hydroxyethyl)cysteine (metabolite 3), GA (metabolite 4), and 2,3-dihydroxypropionamide (metabolite 5). The pathways leading to the formation of these described metabolites are shown in Scheme 1. The proposed metabolism is based on previous studies of the

chemical reactions or metabolism determined for AM, AN, GA, or CEO (13, 15). Quantitation of Urinary Metabolites. Quantitative determinations of metabolites in urine from F344 rats and B6C3F1 mice coadministered po [13C]AN and [13C]AM after 0 or 4 days of coadministration of the unlabeled compounds are summarized in Tables 2-5. Following coadministration of [13C]AM and [13C]AN to F344 rats (Table 2), N-acetyl-S-(2-cyanoethyl)cysteine (I, 20% of the administered AN dose) and N-acetyl-S-(3amino-3-oxopropyl)cysteine (1, 23% the administered AM dose), both produced via GSH conjugation with the parent compounds, account for the largest percentage of the excreted AN and AM doses, respectively. N-AcetylS-(2-hydroxyethyl)cysteine (II, 14%) and N-acetyl-S-(1carbamoyl-2-hydroxyethyl)cysteine (2, 2′, 8%), derived from GSH conjugation with the 2-carbon of CEO or GA,

1156 Chem. Res. Toxicol., Vol. 10, No. 10, 1997 Table 1. 13C NMR Chemical Shifts (ppm) for Resonances in Rat and Mouse Urine Produced after Administration po of [13C]A or [13C]AMa chemical shift (ppm)c compd

no.b

I II III III′ IV IV′ V 1 2 2′ 3 4 5

carbon a

carbon b

carbon c

26.89 33.84 61.20 61.28 36.97 37.20 57.85 27.22 35.96 36.14 61.60 46.98 63.18

18.11 60.09 35.33 35.64 177.35 177.35 171.18 34.83 70.22 70.34 49.84 48.61 72.01

120.17 118.91 119.06

177.07 177.55 177.55 175.50 173.78 175.28

a Metabolites that give rise to signals with these chemical shifts were characterized in previous studies in which rodents were administered po [13C]AN (15) or [13C]AM (13). b I, N-acetyl-S-(2cyanoethyl)cysteine; II, N-acetyl-S-(2-hydroxyethyl)cysteine; III, III′, diastereomers of N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine; IV, IV′, S-(carboxymethyl)cysteine (or its N-acetyl derivative) and thiodiglycolic acid; V, thionyldiacetic acid; 1, N-acetyl-S-(3-amino3-oxopropyl)cysteine; 2, 2′, diastereomers of N-acetyl-S-(3-amino2-hydroxy-3-oxopropyl)cysteine; 3, N-acetyl-S-(1-carbamoyl-2hydroxyethyl)cysteine; 4, GA (metabolite 4); 5, 2,3-dihydroxypropionamide. c Chemical shifts are reported for the center of the multiplet patterns.

are also predominant metabolites of AN and AM, respectively. F344 rats coadministered [13C]AM and [13C]AN after 4 days of coadministration of the unlabeled material (Table 3) also excrete I (22%), 1 (27%), II (19%), and 2, 2′ (11%) as predominant metabolites. In mice, metabolites I (12%), IV (14%), and IV′ (10%) account for the largest percentage of the excreted AN dose following one exposure. Metabolites I (21%), IV (20%), and IV′ (14%) also account for the largest percentage of the excreted AN dose following 5 days of exposure. GA (22-27%) accounts for the largest percentage of the AM dose following single or repeated coadministration of AM and AN to B6C3F1 mice (Tables 4 and 5). Following 5 days of exposure, mice excrete a significantly greater percentage (p < 0.05, Table 6) of the AM

Sumner et al.

(76%) or AN (76%) dose than that excreted following one administration of the mixture (55% of AM dose, 48% of AN dose). The increased excretion of total AM and AN metabolites on repeated exposure suggests a possible increase in metabolism via oxidation, an increase in glutathione transferase activity, and/or an increased synthesis of glutathione. An increase in metabolism was not indicated for rats because the percentage of AM or AN dose excreted was not significantly different (p > 0.12, AN; p > 0.08, AM) following either 1 day (49% AN; 39% AM) or 5 days (60% AN, 48% AM) of coadministration. Previous investigations (15) in which 10 or 30 mg/ kg [1,2,3-13C]AN was administered to mice or rats, respectively, resulted in the excretion of ∼55% of the total dose for either species within 24 h after administration, consistent with the percentage of AN dose excreted (48%) following a single coadministration of AM and AN. Rats and mice administered 50 mg/kg [1,2,3-13C]AM excrete approximately 50% of the dose in the urine within 24 h (13), consistent with that determined for mice (55% of the AM dose) administered a single dose of AM and AN but higher than that determined for rats (39% of the AM dose). Following one administration of the mixture, rats and mice metabolized AN to a similar extent (48% of the dose, Table 6). After five administrations, the percentage of the excreted AN dose was similar (p ) 0.06) for rats (60%) and mice (76%). The percentage of excreted AM dose was significantly higher (p < 0.05) in mice than in rats following either one or five exposures. These data show that mice metabolized AM to a greater extent than rats under both dosing regimens, while mice and rats metabolized AN to a similar extent following one or five administrations of the mixture. To evaluate further species differences in metabolism, the flux through pathways under the two dosing regimens was summarized (Table 6) as the percentage of dose derived from AN or AM, the percentage of dose derived from conjugation of GSH with the parent compound, and the percentage of dose producing metabolites following conversion of the parent compound to CEO or GA. The percentage of the administered dose produced from

Scheme 1. Proposed Metabolism of AM and AN in Rats and Mice following Coadministration of the Carbon-13-Labeled Compoundsa

a Processes that should involve several transformations are indicated by broken arrows. N-AcCys, N-acetylcysteine; Cys, cysteine; GS, glutathione.



Table 2. Quantitative Determination of Metabolites in Urine from F344 Rats Coadministered a Single Dose of [1,2,3-13C]AM and [1,2,3-13C]ANa metabolite no.b

concentration (mM)

total (µmol)

% of total excreted metabolites

% of dose administered

Acrylonitrile I II III IV V

1.4 ( 0.3c 1.0 ( 0.2 0.46 ( 0.07 0.38 ( 0.05 0.22 ( 0.05

11 ( 2 7.8 ( 1.5 3.5 ( 0.5 2.9 ( 0.9 1.7 ( 0.4

1 2, 2′ 3 4 5

1.6 ( 0.3 0.60 ( 0.06 0.25 ( 0.03 0.29 ( 0.02 ndd

12 ( 3 4.5 ( 0.6 1.9 ( 0.2 2.1 ( 0.2

41 ( 2 29 ( 1 13 ( 1 11 ( 2 6.6 ( 2.4

20 ( 4 14 ( 3 6.3 ( 0.8 5.2 ( 1.7 3.1 ( 0.7

59 ( 4 22 ( 2 9.0 ( 0.7 10 ( 2

23 ( 6 8.4 ( 1.1 3.5 ( 0.4 4.0 ( 0.3

Acrylamide

a Metabolites were quantitated in urine from four rats that were administered a dose of 55 ( 1 µmol [1,2,3-13C]AN and 54 ( 1 µmol of [1,2,3-13C]AM. b I, N-acetyl-S-(2-cyanoethyl)cysteine; II, N-acetyl-S-(2-hydroxyethyl)cysteine; III, N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine; IV, IV′, S-(carboxymethyl)cysteine (or its N-acetyl derivative) and thiodiglycolic acid; V, thionyldiacetic acid; 1, N-acetyl-S(3-amino-3-oxopropyl)cysteine; 2, 2′, diastereomers of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine; 3, N-acetyl-S-(1-carbamoyl2-hydroxyethyl)cysteine; 4, GA; 5, 2,3-dihydroxypropionamide. c Mean ( standard deviation (n ) 4). d nd, not detected.

Table 3. Quantitative Determination of Metabolites in Urine from F344 Rats Coadministered [1,2,3-13C]AM and [1,2,3-13C]AN following 4 Days of Administration of the Unlabeled AN and AMa metabolite no.b

concentration (mM)

total (µmol)



Acrylonitrile I II III IV V

1.6 ( 0.5c 1.4 ( 0.5 0.54 ( 0.14 0.71 ( 0.25 0.22 ( 0.17

12 ( 3 11 ( 3 4.1 ( 1.2 5.3 ( 1.6 1.6 ( 1.2

1 2, 2′ 3 4 5

2.0 ( 0.5 0.80 ( 0.21 0.34 ( 0.10 0.31 ( 0.08 0.08 ( 0.05

15 ( 2 6.0 ( 1.3 2.5 ( 0.6 2.4 ( 0.6 0.52 ( 0.36

36 ( 6 32 ( 5 12 ( 2 15 ( 3 5.0 ( 3.9

22 ( 5 19 ( 5 7.3 ( 2.0 9.4 ( 2.9 2.8 ( 2.2

57 ( 2 23 ( 1 9.4 ( 0.7 8.9 ( 1.0 2.1 ( 1.4

27 ( 4 11 ( 2 4.6 ( 1.0 4.3 ( 1.0 0.97 ( 0.67

Acrylamide

a Metabolites were quantitated in urine from four rats that were administered a dose of 56 ( 2 µmol of [1,2,3-13C]AN and 55 ( 3 µmol of [1,2,3-13C]AM after 4 days of dosing with unlabeled AN and AM. b I, N-acetyl-S-(2-cyanoethyl)cysteine; II, N-acetyl-S-(2-hydroxyethyl)cysteine; III, N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine; IV, IV′, S-(carboxymethyl)cysteine (or its N-acetyl derivative) and thiodiglycolic acid; V, thionyldiacetic acid; 1, N-acetyl-S-(3-amino-3-oxopropyl)cysteine; 2, 2′, diastereomers of N-acetyl-S-(3-amino-2-hydroxy-3oxopropyl)cysteine; 3, N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine; 4, GA; 5, 2,3-dihydroxypropionamide. c Mean ( standard deviation (n ) 4).

Table 4. Quantitative Determination of Metabolites in Urine from B6C3F1 Mice Coadministered a Single Dose of [1,2,3-13C]AM and [1,2,3-13C]ANa metabolite no.b

concentration (mM)

I II III IV IV′

0.54 ( 0.13c 0.36 ( 0.15 0.19 ( 0.06 0.61 ( 0.21 0.48 ( 0.17

Acrylonitrile 0.92 ( 0.16 0.60 ( 0.21 0.33 ( 0.09 1.0 ( 0.2 0.81 ( 0.22

1 2, 2′ 3 4 5

0.52 ( 0.15 0.61 ( 0.15 0.23 ( 0.05 0.99 ( 0.25 0.11 ( 0.02

0.87 ( 0.19 1.0 ( 0.3 0.40 ( 0.10 1.7 ( 0.3 0.19 ( 0.03

total (µmol)



25 ( 2 16 ( 2 8.8 ( 0.9 28 ( 2 22 ( 1

12 ( 2 7.8 ( 2.3 4.2 ( 1.0 14 ( 3 10 ( 2

21 ( 2 25 ( 3 9.4 ( 1.5 40 ( 2 4.5 ( 0.2

11 ( 2 14 ( 3 5(1 22 ( 3 2.5 ( 0.4

Acrylamide

a Metabolites were quantitated in urine from four mice that were administered a dose of 7.7 ( 0.3 µmol of [1,2,3-13C]AN and 7.5 ( 0.3 µmol of [1,2,3-13C]AM. b I, N-acetyl-S-(2-cyanoethyl)cysteine; II, N-acetyl-S-(2-hydroxyethyl)cysteine; III, N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine; IV, IV′, S-(carboxymethyl)cysteine (or its N-acetyl derivative) and thiodiglycolic acid; 1, N-acetyl-S-(3-amino-3oxopropyl)cysteine; 2, 2′, diastereomers of N-acetyl-S-(3-amino-2-hydroxy-3-oxopropyl)cysteine; 3, N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine; 4, GA; 5, 2,3-dihydroxypropionamide. c Mean ( standard deviation (n ) 4).

conjugation of GSH with the parent compounds was similar for rats coadministered the mixture for 1 or 5 days, while mice had a significant increase (p < 0.05) in the percentage of dose derived from GSH conjugation with AM or AN between single and repeated exposures. Mice (p < 0.05), but not rats (p ) 0.07), had a significant increase in the percentage of metabolites derived from CEO conjugation with GSH between one and five exposures. Rats and mice had a significant increase (p < 0.05) in the percentage of dose derived from GA between 1 and

5 days of coadministration of AM and AN. An increase in the synthesis of GSH or an increase of glutathione transferase activity in mice may account for the increased excreted percentage of dose derived from GSH conjugation with AN-, AM-, or GA-derived metabolites on repeated exposure. A smaller increase in GSH synthesis or glutathione transferase activity may occur in rats and be attributed to the increased (p < 0.05) excreted percentage of dose derived from GSH conjugation only with GA.

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Table 5. Quantitative Determination of Metabolites in Urine from B6C3F1 Mice Coadministered [1,2,3-13C]AM and [1,2,3-13C]AN following 4 Days of Administration of Unlabeled AN and AMa metabolite no.b

concentration (mM)

total (µmol)


I II III IV IV′

0.64 ( 0.18c 0.32 ( 0.04 0.31 ( 0.05 0.59 ( 0.08 0.43 ( 0.06

Acrylonitrile 1.6 ( 0.3 0.83 ( 0.13 0.80 ( 0.17 1.5 ( 0.2 1.1 ( 0.2

1 2, 2′ 3 4 5

0.58 ( 0.14 0.59 ( 0.13 0.15 ( 0.04 0.81 ( 0.15 0.12 ( 0.03

1.5 ( 0.3 1.5 ( 0.3 0.38 ( 0.10 2.1 ( 0.2 0.31 ( 0.05


28 ( 4 14 ( 1 13 ( 2 26 ( 2 19 ( 2

21 ( 4 11 ( 2 10 ( 2 20 ( 2 14 ( 3

26 ( 1 26 ( 2 6.6 ( 0.8 36 ( 2 5.4 ( 0.7

20 ( 3 20 ( 3 5.0 ( 1.2 27 ( 2 4.0 ( 0.6

Acrylamide

a Metabolites were quantitated in urine from four mice that were administered a dose of 7.8 ( 0.2 µmol of [1,2,3-13C]AN and 7.6 ( 0.1 µmol of [1,2,3-13C]AM after 4 days of dosing with unlabeled AN and AM. b I, N-acetyl-S-(2-cyanoethyl)cysteine; II, N-acetyl-S-(2hydroxyethyl)cysteine; III, III′, diastereomers of N-acetyl-S-(1-cyano-2-hydroxyethyl)cysteine; IV, IV′, S-(carboxymethyl)cysteine (or its N-acetyl derivative) and thiodiglycolic acid; 1, N-acetyl-S-(3-amino-3-oxopropyl)cysteine; 2, 2′, diastereomers of N-acetyl-S-(3-amino-2hydroxy-3-oxopropyl)cysteine; 3, N-acetyl-S-(1-carbamoyl-2-hydroxyethyl)cysteine; 4, GA; 5, 2,3-dihydroxypropionamide. c Mean ( standard deviation (n ) 4).

Table 6. Urinary Metabolites Derived from AM or AN following Coadministration of [1,2,3-13C]AM and [1,2,3-13C]AN to F344 Rats and B6C3F1 Mice for 1 or 5 Daysa % of AN or AM administered dose B6C3F1 mice metaboliteb total AN metabolites N-AcCys-S-AN (I) CEO-derived (II-V) total AM metabolites N-AcCys-S-AM (1) GA (4) GA-derived (2, 2′, 3, 5) GA and GA-derived (2-5)

F344 rats

1 day

5 days

9c

9c

48 ( 12 ( 2c,d 36 ( 8c 55 ( 9c,d 11 ( 2c,d 22 ( 3c,d 21 ( 5d 44 ( 7c,d

76 ( 21 ( 4c 55 ( 8c,e 76 ( 9c,e 20 ( 3c,e 27 ( 2c,e 29 ( 4e 56 ( 6c,e

1 day

5 days

49 ( 8 20 ( 4d 29 ( 4 39 ( 7d 23 ( 6d 4.0 ( 0.3d 12 ( 1d,f 16 ( 2 d

60 ( 10 22 ( 5 39 ( 8e 48 ( 8e 27 ( 4e 4.3 ( 1.0e 16 ( 3e,f 21 ( 4e

a [1,2,3-13C]AM and [1,2,3-13C]AN were coadministered following 0 or 4 days of coadministration of the unlabeled material. b CEOderived and GA-derived define the sum of metabolites excreted in the urine following P450 conversion of AN or AM, respectively. c Values different in 1- and 5-day mice (p < 0.05). d Values different in 1-day mice compared with 1-day rats (p < 0.05). e Values different in 5-day mice compared with 5-day rats (p < 0.05). f Values different in 1- and 5-day rats (p < 0.05).

Table 7. Urinary Metabolites Derived from GSH Conjugation with CEO or GA following Coadministration of [1,2,3-13C]AM and [1,2,3-13C]AN for 1 or 5 Daysa % of AN or AM administered dose B6C3F1 mice metabolite

1 day

F344 rats 5 day

1 day

5 day

2-carbon conjugate (III, III′) 3-carbon conjugate (II, IV, IV′, V)

Acrylonitrile 4.2 ( 1.0b, c 32 ( 7b,c

10 ( 2c 45 ( 7c,d

6.3 ( 0.8b 23 ( 3b,e

7.3 ( 2.0 31 ( 6d,e

2-carbon conjugate (3) 3-carbon conjugate (2, 2′)

Acrylamide 5.2 ( 1.2b b,c 14 ( 3

5.0 ( 1.2 20 ( 3c,d

3.5 ( 0.4b 8.4 ( 1.1b

4.6 ( 1.0 11 ( 2d

a [1,2,3-13C]AM and [1,2,3-13C]AN were coadministered following 0 or 4 days of coadministration of the unlabeled material. b Values different in 1-day mice compared with 1-day rats (p < 0.05). c Values different in 1- and 5-day mice (p < 0.05). d Values different in 5-day mice compared with 5-day rats (p < 0.05). e Values different in 1- and 5-day rats (p < 0.05).

Discussion

ration of synthetic standards and avoids chromatographic techniques that may alter metabolite structure.

This study has demonstrated the utility of 13C NMR in the detection, identification, and quantitation of 13Clabeled metabolites directly in the urine following coadministration of two 13C-labeled compounds. Although the sensitivity of NMR is much lower than that of HPLC and GC/MS, 13C NMR provides resolution of signals for individual metabolites, including structural isomers and diastereomers, at both low and high concentrations. Metabolites were characterized that accounted for as little as 1% of the administered dose. The simultaneous detection of metabolites directly in urine provides considerable structural information that is useful for prepa-

Following one or five administrations of the mixture to mice, 72-75% of the total excreted AN metabolites (Tables 4 and 5) are derived following conversion of AN to CEO. Mice administered 10 mg/kg [1,2, 3-13C]AN have been shown to excrete 79% of the total urinary metabolites as CEO-derived (15), indicating that neither coadministration, repeated administration, nor dose (10 vs 16 mg/kg) significantly affect the total flux through pathways. For rats, 59-64% of the total excreted metabolites are derived from CEO following one or five administrations of the mixture. This is also consistent with the single-dose studies (15) where 57% of the total


excreted metabolites are derived from CEO following administration of 30 mg/kg [1,2,3-13C]AN to rats. Previous investigations (13) in which rats and mice were administered a single dose of 50 mg/kg [1,2,3-13C]AM showed that a greater percentage of the total excreted metabolites was produced following conversion of AM to GA for mice (59%) than for rats (33%). In the present study, 41-43% of the total excreted AM metabolites were derived following conversion of the parent compound to GA for rats administered the mixture of AM and AN for 1 or 5 days. Mice excreted 74-79% of the total AM metabolites as those derived following conversion of AM to GA after 1 or 5 days of administration of the mixture. A higher contribution of the GA-derived metabolites (as a percentage of total excreted metabolites) in the present study could be due to differences in dose level or an effect of coadministration; however, it does not appear to result from repeated administration. For rats and mice, the major percentage of the excreted AN dose following one or five administrations of the mixture is derived from metabolites produced following conversion of AN to CEO. The ratio of metabolites produced via conversion of AN to CEO to metabolites produced from nonoxidative metabolism of the parent compound is 3:1 for mice and 1.5:1 for rats following one exposure. On repeated exposure, the metabolism via the epoxide is still more extensive for mice (2.6:1) than rats (1.8:1) and is similar to the ratio obtained following a single administration of the mixture. Previous studies did not observe an induction in P450 epoxidation of AN in rats (19 ) or mice3 following administration of 10 mg/ kg AN for 3 days. The major percentage of the excreted AM dose following one or five administrations is derived from metabolites produced following conversion of AM to GA for mice. Rats, on the other hand, excrete the majority of the AM dose as a metabolite (1) derived from AM conjugation with GSH. The ratio of metabolites derived following conversion of AM to GA to the metabolite (1) produced after direct GSH conjugation with AM is greater in mice (4:1) than rats (0.7:1) following one administration of the mixture. On repeated administration, the metabolism via the epoxide is still more extensive for mice (2.8:1) than rats (0.8:1). Following one administration (15) of 10 or 30 mg/kg [1,2,3-13C]AN to mice or rats, respectively, comparison of the ratios of the percentage of total metabolites derived from conjugation at the 2- and 3-positions of CEO (metabolites III, III′ vs II, IV, IV′, VI) suggested a different site specificity for conjugation between rats (0.43) and mice (0.21). In the present study, the ratio of metabolites derived from conjugation at the 2- and 3-positions (Table 7) was similar between 1 day (0.27) or 5 days (0.24) of coadministration of AM and AN to rats. Mice had a significantly lower percentage of 2-carbon CEO metabolites following 1 coadministration (0.13), as compared with 5 days of administration (0.22). The greater acute toxicity of AN in the mouse than in the rat has been attributed in part to the greater production of metabolites via conjugation of the 3-position of CEO in the mouse, a pathway that results in the release of cyanide (15). The fact that mice have a decrease in metabolites produced via GSH conjugation at the 2-position of CEO on administration of the AM and AN mixture suggests that the acute toxicity of the mixture in mice would be higher than that obtained for administration 3

Kedderis, G. L. Personal communications.


of the individual compound. In addition, the increase in the ratio of production of 2-carbon CEO metabolites on repeated exposure may indicate a protective mechanism against cyanide toxicity for the mouse. In summary, the increase in total excreted AM and AN metabolites in mice and the increase in metabolites produced after conversion of AN and AM to CEO and GA, respectively, suggest an increase in P450 oxidation of AM and AN, an increase in glutathione transferase activity, and/or an increased synthesis of glutathione. An alteration in metabolism is not as apparent for the rat, where coadministration of AM and AN for 1 or 5 days results in little change in the distribution of metabolites.

Acknowledgment. We are grateful to Mr. Paul Ross, Mr. Tim Shepard, and the animal care staff at CIIT. We thank Dr. Barbara Kuyper for her excellent editorial review of this paper.

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1160 Chem. Res. Toxicol., Vol. 10, No. 10, 1997 (17) van Bladeren, P. J., Delbressine, L. P. C., Hoogerterp, J. J., Beaumont, A. H. G. M., Briemer, D. D., Seutter-Berlage, F., and van der Gen, A. (1981) Formation of mercapturic acids from acrylonitrile, crotononitrile, and cinnamonitrile by direct conjugation and via epoxide intermediate oxidation process. Drug Metab. Dispos. 9, 246-249. (18) Guengerich, F. P., Geiger, L. E., Hogy, L. L., and Wright, P. L. (1981) In vitro metabolism of acrylonitrile to cyanoethylene oxide, reaction with glutathione, and irreversible binding to proteins and nucleic acids. Cancer Res. 41, 4925-4933. (19) Kedderris, G. L., Batra, B., and Koop, D. R. (1993) Epoxidation of acrylonitrile by rats and human cytochrome P450. Chem. Res. Toxicol. 6, 866-871. (20) Kedderis, G. L., Batra, B., Held, S. D., Loos, M. A., and Teo, S. K. O. (1993) Rodent tissue distribution of 2-cyanoethylene oxide, the epoxide metabolite of acrylonitrile. Toxicol. Lett. 69, 25-30. (21) Sega, G. A., Generoso, E., and Brimer, P. A. (1990) Acrylamide exposure induces an unscheduled DNA synthesis in germ cells of mice that is correlated with the temporal pattern of adduct formation on DNA. Environ. Mol. Mutagen. 16, 137-142. (22) Segerba¨ck, D., Calleman, C. J., Schroeder, J. L., Costa, L. G., and Faustman, E. M. (1995) Formation of N-7-(2-carbamoyl-2-hydroxethyl)guanine in DNA of the mouse and the rat following intraperitoneal administration of [14C]acrylamide. Carcinogenesis 16, 1161-1165. (23) Yates, J. M., Fennell, T. R., Turner, M. J., Jr., Recio, L., and Sumner, S. C. J. (1994) Characterization of phosphodiester adducts produced by the reaction of cyanoethylene oxide with nucleotides. Carcinogenesis 15, 277-283.

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Urinary Metabolites from F344 Rats and B6C3F1 Mice

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