Acrylamide is metabolized to glycidamide in the rat: evidence from

Chem. Res. Toxicol. 1990, 3, 406-412

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Art i d e s Acrylamide Is Metabolized to Glycidamide in the Rat: Evidence from Hemoglobin Adduct Formation' Carl Johan Calleman, Emma Bergmark, and Lucio G. Costa* Department of Environmental Health, SC-34, University of Washington, Seattle, Washington 98195

Downloaded by UNIV OF MANITOBA on September 1, 2015 | http://pubs.acs.org Publication Date: September 1, 1990 | doi: 10.1021/tx00017a004

Received August 17, 1989 Acrylamide is a n important industrial chemical which is neurotoxic to experimental animals as well as humans and recently has been shown to be mutagenic and carcinogenic. Despite much research it is still unclear whether the parent compound or a metabolite is responsible for the observed toxic effects. Contradictory results as to the role of cytochrome P-450mediated metabolism of acrylamide in the induction of neurotoxic effects prompted us to investigate the possible formation of glycidamide, a reactive epoxide metabolite. The formation of this epoxide was strongly indicated by the identification by means of gas chromatography-mass spectrometry of derivatized S-(2-carboxy-2-hydroxyethyl)cysteine in hydrolyzed hemoglobin samples from rats treated with acrylamide in vivo and in microsomal suspensions of acrylamide with cysteine in vitro. This amino acid was found t o be present in uninduced and phenobarbital-induced Sprague-Dawley rats and absent in controls, but occurred in lower amounts than the adduct derived from the parent compound, S-(2-~arboxyethyl)cysteine.This finding suggests that the possible role of glycidamide in the neurotoxicity and carcinogenicity of acrylamide should be evaluated further.

Introduction At room temperature acrylamide is a white crystalline solid with a high solubility in water. The high reactivity of its olefinic bond versus nucleophilic agents in Michael type additions is derived from its conjugation with an amide group. Industrially, acrylamide is produced by catalytic hydration of acrylonitrile and used mainly in the production of polymers such as polyacrylamide. The production of the monomer in the United States has approximately doubled in the past decade and reached 140 million pounds in 1985 ( 1 , 2). Humans are potentially exposed to acrylamide in industrial processes, grouting operations, synthesis of chromatography gels, and leakage of the monomer from polyacrylamide used in the purification of drinking water. According to a survey undertaken by the National Institute of Occupational Safety and Health, some 10 000 workers in 27 occupations are potentially exposed to acrylamide, including about 1000 persons involved in its manufacture and another loo0 licensed to perform grouting operations. The number of laboratory workers exposed to acrylamide in the preparation of chromatography gels was estimated to be as high as 100000-200000 ( 2 ) . Since the early fifties the neurotoxic effects, involving both the central and the peripheral nervous system, have been the primary health concerns associated with human exposure to acrylamide, and close to 150 cases of intoxication have been reported in the past 30 years ( 3 ) . Symptoms of intoxication include excessive tiredness, *Presented in part at the 28th Annual Meeting of the Society of Toxicology, Atlanta, Feb 27-March 3, 1989 (47).

ataxia, dizziness, and in more serious cases confusion and hallucinations. Recovery from light intoxication is usually complete a few months after the onset of symptoms ( 4 ) . These reports have been parallelled by extensive studies in experimental animals, notably aiming at the elucidation of the mechanism of neurotoxic action of acrylamide ( 4 , 5). The molecular mechanisms involved in such action are not known, but the electrophilic nature of acrylamide, Le., its ability to form adducts with glutathione (6),proteins (7), and DNA (8),as well as the cumulative damage to the nervous system it is known to cause, suggests that acrylamide might exert its neurotoxic effect by covalent modification of a critical target macromolecule (9). In more recent years, attention has increasingly focused on the genotoxic and reproductive effects of this compound ( 10). Acrylamide has been shown to be mutagenic in vitro in eukaryotic cells (11) and to give rise to heritable translocations (12) and dominant lethal mutations (13)in rodents. Autoradiographic studies with [14C]acrylamide have indicated high concentrations of radioactivity in the reproductive tract of male mice ( 1 4 ) . Controversy still exists on whether acrylamide induces chromosomal aberrations exclusively in the germ cells (15,16). Unexpectedly for a compound able to produce these types of effects, as well as for having induced tumors in both mice (17) and rats (181, acrylamide has consistently given a negative response in the AmeslSalmonella assay both in the absence and in the presence of S-9 mix (17,19). Its response was negative also in the Klebsiella bacterial test system (I1 ) , and the suggestion has been made that acrylamide induces its genetic effects by reactions with chromosomal proteins rather than with DNA (11,20). Epidemiological studies of mortality patterns in workers employed in the

0S93-228x/90/2703-0406$02.50/0 0 1990 American Chemical Society

Chem. Res, Toxicol., Vol. 3, No. 5, 1990 407


Acrylamide Metabolism to Glycidamide manufacture of the monomer have, however, not demonstrated increased incidences of neoplastic diseases (21). Despite the large number of studies on the toxicity of acrylamide, little is still known on its metabolism, and direct structural evidence for putative active metabolites is missing. As judged by cochromatography on TLC, approximately half the dose of radiolabeled acrylamide administered to rats is excreted in the urine as the mercapturic acid N-acetyl-S-(2-carbamoylethy1)cysteine (22),indicating that the main pathway for detoxification of acrylamide is through conjugation with glutathione. Several reports in the literature have also provided circumstantial evidence that acrylamide undergoes oxidative metabolism mediated by the cytochrome P-450 system. These observations include the following: (1) When acrylamide is labeled in the 1-carbon, 6% of the dose given to rats is exhaled as carbon dioxide (9),whereas when it is labeled in the vinyl carbons, no radiolabeled carbon dioxide is detected (22). (2) Three unknown non-sulfurcontaining metabolites were found in the urine of acrylamide-treated rats. The chromatographic positions of these metabolites were not altered by incubations with arylsulfatase or &-glucuronidase(22). ( 3 ) In rainbow trout a metabolite containing a primary alcohol function was demonstrated by IR spectrometry (23). (4) Pretreatments of animals with modulators of oxidative metabolism such as phenobarbital and CoClz have demonstrated significant impacts on the neurotoxicity induced by acrylamide (24-27). (5) A total of 18-30% of the cytochrome P-450 activity was lost when acrylamide was incubated with microsomes in the presence of an NADPH-generating system (28). In the absence of an NADPH-generating system no activity was lost, indicating that a reactive metabolite, rather than the parent compound, was responsible for the inhibition of the enzyme. (6) Acrylamide stimulates NADPH consumption by rat liver microsomes (29),and this consumption was blocked by carbon monoxide, an inhibitor of the cytochrome P-450 system. Especially the three latter observations are strong indications of an involvement of the cytochrome P-450 system in the metabolism of acrylamide. It should also be pointed out, however, that Tanii and Hashimoto did not detect any metabolic conversion of acrylamide by mouse liver microsomes (30). Based on analogies with acrolein (31) and acrylonitrile (32),both of which have been shown to be metabolized to epoxides, the most likely candidate as the primary cytochrome P-450 mediated metabolite of acrylamide appeared to be glycidamide (Figure l ) , formed by oxidation of the olefinic bond. Upon reaction with cysteine residues in proteins in vivo, glycidamide is expected to generate either or both of the regioisomers S-(l-carbamoyl-2-hydroxyethyhysteine and S-(2-carbamoyl-2-hydroxyethyl)cysteine. As a result of acid protein hydrolysis the amide group would be cleaved to a carboxylic acid, yielding S-[1-(or 2-)carboxy-2-hydroxyethyl]cysteine.In the present communication, we provide mass spectrometric proof for the formation of S-(2-carboxy-2-hydroxyethyl)cysteinein hemoglobin hydrolysates from animals treated with acrylamide in vivo, strongly implicating glycidamide as a reactive metabolite.

Materials and Methods Chemicals. Cysteine and acrylamide were obtained from Alfa products (Danvers, MA), and dl-glycidamide was obtained from Polysciences,Inc. (Warrington,PA). S(Carboxymethy1)cysteine was from Sigma (St. Louis, MO). S(Carboxyethy1)cysteine was purchased from Fluka (Ronkonkoma, NY), and heptafluorobutanoic anhydride (HFBA) was from Pierce (Rockford,IL). HCl

?

/o\

CH 'CH-C-NH2

C

2 Acrylamide

cl HB CH-CH2-SH

1

R CH - CH 2-S - CH 2-CH 2- C -NH2

C 1 HB

HCl

COOH \ CH-CH2-S-CH2-CH2-COOH / NH2 S-(2-cPrboxyeIhyl)cysleine

?

CH -CH-C-NH2 2 Glycidunide

HB CH-CH2-SH

C I C 1

?H

?

HB CH-CH2-S-CH2-CH-C-NH 2

HCI

COOH

?H CH-CHZ-S-CHZ-CH -COOH

\ /

NH2 S.(2-clrboxy2.hydmxyclyl)Eyttcinc

Figure 1. Reactions of acrylamideand glycidamide with cysteine residues of hemoglobin. (1.25 M) in methanol was prepared by dissolving HCl gas, prepared from the dripping of concentrated HzS04 on NaCl, in methanol. Preparation of S-(2-Carbamoylethy1)cysteine.Acrylamide (1.2 mmol) dissolved in 2 mL of HzO was added to cysteine (1.0 mmol) and triethylamine (1.0 mmol) and allowed to react for 1 h at room temperature. The reaction product was precipitated by the addition of 100 mL of acetone, filtered, and dried, giving a 94% yield. S-(2-Carbamoylethyl)cysteinewhich has previously been synthesized by Dixit et al. (33) was judged to be pure by TLC (Pr-OH/H20,7:3, cellulose, R 0.39) developed by ninhydrin and by the fact that, f o l i o k g derivatization with MeOH-HC1 and HFBA (34),it gave only one peak with the same mass spectrum upon electron impact as previously reported for derivatized S-(2-~arboxyethyl)cysteine(35). The mass spectrum obtained when scanning for negative ions with methane as the reagent gas gave a prominent fragment at m/z = 416 interpreted as [M - HI-. 'H NMR 6 2.6 (2 H, t, CH2CONH2),2.8 (2 H, t, SCH,CH,), 3.05 (1H, dd, cyst@, 3.15 (1H, dd, cysta'), and 3.9 (1 H, dd, cysts). 13CNMR 6 29.7 (SCHZCH,), 34.5 (cyst@),37.3 (CH,CONH,), 56.3 (cysta), 175.3 (CO,H), dnd 179.7 (CONHz). Preparation of S-(2-Carbamoyl-2-hydroxyethyl)cysteine. dl-Glycidamide (1.15 mmol) was added to a solution of cysteine (1.3 mmol) and triethylamine (1.3 mmol) in 2 mL of HzOand left to react at room temperature for 1h. Twenty milliliters of acetone was added to the mixture, and the precipitate was filtered and washed with 140 mL of acetone. The compound was judged by TLC (R, = 0.31) to be pure, and the yield of S-(2-carbamoyl-2hydroxyethy1)cysteinewas 83%. Upon derivatization with MeOH HC1 and HFBA two peaks were evident on GC: a major peak at 601 s giving the fragments m / z = 570,416,356, and 202 upon electron impact ionization interpreted as [M - C02CH3]+,[M C3F&ONHZ]+, [M - C3F7C02H - COZCH3]+,and [M - C3F7C02H - C3F7CONH2]+, respectively, and a minor peak corresponding to -5% of the main peak eluting at 569 s giving m/z = 415,356, and 202 which was interpreted as a vinyl derivative formed by elimination of heptafluorobutanoicacid. Chemical ionization using methane as the reagent gas gave the positive ions m / z = 416 [M - C3F7CONH2]+and 630 [M + H]+and the negative ion m/z = 609 [M - HF]- for the major peak. 'H NMR 6 3.05 (4 H, cyst@ + cystp + CH,CHOH), 3.9 (1 H, d, cysta), and 4.4 (1 H, dd, CH,CHOH) (Figure3). '% NMR 6 36.6 (cyst@),39.2 (CHzCHOH), 57.1 ( c Y s ~ 73.6 ) , (CH&HOH), 176.2 (COZH),and 180.8 (C0NH.J. Formation of S-(2-Carbamoyl-2-hydroxyet hy1)cysteine in Vitro. Acrylamide (final concentration 8 mM) was added to 0.2 M Tris buffer (pH = 7.5) containing NADPH (2 mM), MgSO, (10 mM), and EDTA (1.2 mM). Phenobarbital-induced (50 mg/kg/day for 3 days) rat liver microsomes (2.4 mg) were then added to 1mL of buffer. An incubation mixture not supplemented with microsomes served as control. Samples were shaken for 15 min in a 37 "C water bath before addition of 10 mM L-cysteine. Following a 2-h incubation, the samples were acidified by the addition of 2 M HC1 to pH = 1.0 and applied to Dowex 50 (H+, 5.5 X 0.9 cm) columns eluted with 50 mL of H,O followed by 25

Calleman et al.

408 Chem. Res. Toricol., Vol. 3, No. 5, 1990

mL of 1 M HC1. The acidic eluates were evaporated to dryness on a rotary evaporator, and the dried remainders were derivatized for gas chromatographicanalysis by previously described methods

188,

91

(34).


Formation of S-(2-Carboxy-2-hydroxyethyl)cysteine in Hemoglobin in Vivo. Male Sprague-Dawley rats (body wt 150-175 g) were divided into three groups (10 animals in each) and were administered 0, 50, and 75 mg/kg acrylamide intraperitoneally in physiological saline for 5 consecutive days, corresponding to cumulative doses of 0, 250, and 375 mg of acryl-

amide/kg, respectively. In another experiment, animals were pretreated with 50 mg of phenobarbital/kg ip for 3 days and then treated daily together with ip injections of 75 mg of acrylamide/kg for 5 days; rats injected with phenobarbital only served as controls. Blood samples were collected by cardiac puncture under ether anesthesia 24 h after the last injection. Globin was prepared from blood samples according to the method of Osterman-Golkar et al. (36). Approximately 5 mg of globin from each animal was dissolved in 6 M HCl to an exact concentration of 10 mg of globin/mL. S-(carboxymethy1)cysteine was added to the hydrochloric acid as an internal standard to a concentration of 1 pmol/mL. The samples were hydrolyzed for 18 h at 110 "C in sealed Pyrex glass ampules under vacuum. The hydrolysates were pooled according to exposure groups, evaporated to dryness, dissolved in 1 mL of H20,and applied to Dowex 50 (H+,5.5 X 0.9 cm) columns. The initial 25 mL of 1M HCl eluted from each column was collected and evaporated to dryness. Aliquots of the eluates were then derivatized according to previously described methods to N,O-bis(heptafluorobutyry1)amino acid methyl esters (34).

Gas Chromatographic-Mass Spectrometric Analysis. The derivatized samples were dissolved in 0.1 mL of ethyl acetate, 2 pL of which was injected splitless onto a 30 m X 0.25 mm i.d. DB-5 gas chromatography column (J & W) with a film thickness of 0.25 pm. The carrier gas was helium with a flow rate of 1.1 mL/min. The GC-MS system was a Perkin-Elmer Sigma model gas chromatograph linked to a Finnigan 1020 mass spectrometer. The oven was held for 1 min at 100 "C and then programmed at a rate of 15 "C/min to 275 "C. Full electron impact mass spectra for derivatized samples were obtained at 70 eV for reference compounds and a highly concentrated sample from a hemoglobin hydrolysate (Figure 5B). For the hemoglobin analyses the mass spectrometer was operated in the multiple ion detection (MID) mode where the ions m / z = 158,202, and 204 were monitored. The retention times of derivatized S-(carboxymethyl)cysteine, S-(2-~arboxyethyl)cysteine, and S-(2-carboxy-2-hydroxyethyl)cysteine were 540, 589, and 601 s, respectively. Chemical ionization spectra for the derivatized adducts were obtained under identical chromatographic conditions on a Hewlett-Packard 5890A GC linked to a Finnigan 4023 instrument using methane as the reagent gas at an ion source pressure of 0.30 Torr and temperature of 220 "C. Both positive and negative ion spectra were obtained at an ionization energy of 70 eV. Fast Atom Bombardment (FAB) MS. FAB mass spectra were acquired on a VG 70 SEQ tandem hybrid instrument of EBbQ geometry (VG analytical, Altrincham, U.K.). The instrument was equipped with a standard unheated VG FAB ion source and a standard saddle-field gun (Ion Tech Ltd., Middlesex, U.K.) producing a beam of xenon atoms at 8 keV and 1 mA. The mass spectrometer was adjusted to a resolving power of 1O00,and spectra were obtained at 8 kV and at a scan speed of 10 s/decade. In this study all samples were applied to the FAB target as solutions with known concentrations. Thioglycerol (2HEDS)was used as matrix in the positive ion FAB MS. NMR Spectroscopy. Proton NMR spectra of the purified products were recorded at 300 MHz, with D20 as solvent, with a Varian VXR 300 instrument. Chemical shifts are reported in ppm downfield from internal sodium (trimethylsily1)propionate. 13C NMR spectra were obtained on the same instrument with 1.4-dioxane as internal standard.

Results and Discussion Identification of reactive, electrophilic metabolites from potentially toxic compounds have in some cases been undertaken by determination of the adducts formed by these

I Figure 2. Fast atom bombardment mass spectrum of S42-carbamoyl-2-hydroxyethy1)cysteineobtained from a thioglycerol

matrix. Fragments 73,91, 149,181,217,and 232 are contributed by the matrix.

metabolites with nucleophilic amino acids in hemoglobin in the red blood cells. Thus, the in vivo metabolism of ethylene and propylene to the mutagenic agents ethylene oxide (37) and propylene oxide (38) was demonstrated through the formation of the corresponding 2-hydroxyalkylated adducts formed with nucleophilic amino acids in hemoglobin of animals exposed to the radiolabeled alkenes. Similarly, Axworthy et al. (39) demonstrated the metabolism of acetaminophen to p-benzoquinone through the demonstration of the adduct formed by this quinone to cysteine residues of hemoglobin. Hashimoto and Aldridge (9) found that 12% of the absorbed dose of acrylamide binds to the hemoglobin in the red blood cells of treated rats and that upon acid hydrolysis hemoglobin treated in vitro with acrylamide liberated S-(2-carboxyethyl)cysteine,presumably generated from S-(2-carbamoylethy1)cysteineresidues in the intact protein (Figure 1). On the basis of this finding, Bailey e t al. (35) developed a gas chromatography-mass spectrometry (GC-MS) method for monitoring acrylamide exposure in rats by determining S-(2-~arboxyethyl)cysteine in hemoglobin hydrolysates, and the same group has later indicated the feasibility of monitoring also human exposure (40). In the present study the reaction products formed between cysteine and acrylamide or glycidamide were synthesized with high yields. By use of FAB mass spectrometry these adducts displayed prominent fragments at the expected m / z values 193 and 209 (see Figure 2) for the [M + H]+ ions of the two adducts, respectively, demonstrating that at this step the amide groups are intact. For reference, FAB mass spectra were also obtained from the compounds S-(2-carboxyethyl)cysteine and S-(2-carboxy-2-hydroxyethyl)cysteine, giving prominent fragments a t m/z = 194 and 210, respectivey. Weaker fragments were observed for [M + Na]+ in the FAB mass spectra of the four compounds subjected to analysis, as well as a number of ions derived from the thioglycerol matrix. In order to elucidate which of the possible regioisomers, S-(2-carbamoyl-2-hydroxyethy1)- or S-(1-carbamoyl-2hydroxyethyl)cysteine, was formed in the reaction between glycidamide and cysteine, an NMR spectrum was obtained from a 300-MHz spectrometer (Figure 3). Since the integral of the most downfield signal (not counting the signal a t 4.8 ppm from contaminating HzO) corresponded to one proton, it was concluded that the carbon linked to the most electronegative element, i.e., oxygen, must be linked to a methme proton. The structure shown in Figure 1produced by the reaction of cysteine with the sterically most accessible carbon of glycidamide, C-3, satisfies this requirement. Assignments of 13C NMR signals to the different carbons were based on comparisons with values calculated from the 13C NMR data base (T. Fennell, personal communication). As a result of the derivatization with methanolic HCl,

Chem. Res. Toxicol., Vol. 3, No. 5, 1990 409

Acrylamide Metabolism t o Glycidamide

7

-

T-7T T 7 T 4. .T

r

-I

6

5

rTllT'T7 T - m w - y v - l - r T m 1

4

0 DPY


Figure 3. 300-MHz nuclear magnetic resonance spectrum of S-(2-carbamoyl-2-hydroxyethyl)cysteine in D20.

the amides are converted to methyl esters. Thus, the same derivatives are produced regardless of whether the amides or the corresponding carboxylic acids released from proteins that have undergone acid hydrolysis (see Figure 1) are analyzed. In the second step of the derivatization procedure, the alcohol and amino functions are acylated with heptafluorobutyric anhydride, producing volatile derivatives amenable to gas chromatography-mass spectrometry. N-(Heptafluorobutyryl)-S-(2-carbomethoxyethy1)cysteine methyl ester produced by derivatizing the acrylamide-cysteine adduct gave the same mass spectrum upon electron impact as previously reported (35). The derivatized glycidamide-cysteine adduct, N,O-bis(heptafluorobutyry1)-S-(2-carbomethoxy-2-hydroxyethy1)cysteine methyl ester, yielded a characteristic fragment produced by the loss of a methyl ester radical (mlz = 570) but no detectable molecular ion. In addition, it yielded fragments a t m / z = 416 and 202,presumably produced by two consecutive McLafferty rearrangements leading to the symmetrical, conjugated odd-electron ion [C02CH,)CH= CHSCH=CH(CO,CH,J *+, which was used for mass fragmentography monitoring of the hemoglobin samples. Chemical ionization gave fragments interpreted as [M + H]+ and [M - HF]- when scanning for positive and negative ions, respectively. In addition to the main adduct formed by the reaction of glycidamide with cysteine, a minor adduct ( 5 % ) presumed to be S-(2-carboxyvinyl)cysteine formed by vinylization of the main reaction product during derivatization was evident as a separate peak on GC-MS ( m / z = 415). The derivatized diastereomers of (S)- and (R)-S-(2-carbamoyl-2-hydroxyethyl)-L-cysteine merged into one GC peak on the DB-5 column used for the Hb samples. S-(Carboxymethy1)cysteine was added to the hemoglobin samples prior to hydrolysis to serve as an internal standard correcting for losses during the workup procedure and for the retention times on GC. It was assumed that the two thiwthers produced by reactions of acrylamide and glycidamide with cysteine in hemoglobin would undergo oxidation to the same extent as the internal standard during the acid hydrolysis. Although quantitation of the relatively high levels of S-(2-~arboxyethyl)cysteine using S-(carboxymethy1)cysteineas an internal standard for mass fragmentography gave values consistent with those previously reported (3% the nonlinearity of the response for low levels of the glycidamide adduct prevented accurate quantitative determination of this adduct by this technique.

6717U.

s10

640

600

SA0

Figure 4. Typical mass fragmentogram of derivatized amino acids in hemoglobin from rats injected with 50 mg/kg acrylamide for 5 days. The arrows indicate the retention times of (A) the internal standard S-(carboxymethyl)cysteine, (B) S-(2-carboxy-2hydroxyethyl)cysteine,and (C) S-(2-carboxyethy1)cysteine.The mass fragmentograms from top to bottom show the intensities of the ions m / z = 158, 202, and 204 indicative of these three compounds, respectively. The 3c axis is retention time in seconds. A

I

.

,

520

+ .. .- - . 560

L

:

-

i

600

.

,

.

,

.

-

1

610

Figure 5. Mass fragmentogram of derivatized amino acids in hemoglobin from control rats. The arrows are the same as in Figure 4.

The occurrence of S-(2-carboxy-2-hydroxyethyl)cysteine was demonstrated by multiple ion detection in hemoglobin hydrolysates from rats treated with 75 and 50 mg of acrylamidelkg bw/day for 5 consecutive days (Figure 4) but was absent in nontreated animals (Figure 5). It was also demonstrated in a microsomal suspension with acrylamide in which cysteine was used as a "trapping agent", but was absent when no microsomes were present. To confirm that the peak a t the retention time of the

410 Chem. Res. Toxicol., Vol. 3, No. 5, 1990

Calleman et al.


B

1y61.

Figure 6. Full mass spectrum (A) at the retention time of the derivatized glycidamidecysteine adduct in a highly concentrated hemoglobin hydrolysate from acrylamide-treated rats and (B) of the synthetic glycidamidecysteineadduct. Because of the scarcity of material from the hemoglobin sample its mass spectra did not display the low-intensity fragments above m / z = 300. The mass spectra for the synthetic standard has been cut at this m / z value to facilitate comparison.

derivatized glycidamide-cysteine adduct was indeed derived from the suspected amino acid, a full mass spectrum was obtained from a highly concentrated samples of a derivatized hemoglobin hydrolysate (Figure 6A). Figure 6B shows the mass spectrum of synthetic S-(Zcarboxy-2hydroxyethy1)cysteine for comparison. Similarly, acrylamide-treated animals (75 mg/kg for 5 days) induced with phenobarbital displayed the glycidamide adduct, whereas in animals given only phenobarbital this adduct was absent. It is noteworthy that S-(2-~arboxyethyl)cysteine was present in treated animals and, as a low background level, also in untreated animals, as previously reported (35),while S-(2-carboxy-2-hydroxyethy1)cysteinewas detected only in acrylamide-treated rats (Figures 4 and 5). The identification of S-(2-carboxy-2-hydroxyethyl)cysteine in hydrolyzed hemoglobin samples from animals treated with acrylamide in vivo is a strong indication that glycidamide, the putative epoxide metabolite of acrylamide, is the reactive species generated. Direct proof of the formation of epoxides in vivo is difficult to obtain, and for the most part their existence has been inferred from the chemical nature of their reaction products with different nucleophiles in the tissues (41). In the case of acrylamide metabolism, the formation of glycidamide clearly represents the simplest and most straightforward explanation for the 2-hydroxylated amino acid observed. This metabolic conversion of acrylamide to glycidamide may also explain why Bailey et al. (35) observed the un-

usual phenomenon of a convex curve a t low doses when plotting the amount of S-(2-~arboxyethyl)cysteinein hydrolyzed hemoglobin samples against the amount of acrylamide injected in rats. The finding that glycidamide is formed by oxidative metabolism of acrylamide prompts an evaluation of its potential role in the toxicity of acrylamide. In preliminary experiments, we have found that glycidamide is less potent than acrylamide (on a mg/ kg basis) in producing neurotoxicity, as judged by the performance of rats on a rotarod apparatus (24)following repeated ip injections (Calleman et al., unpublished results). This finding would be consistent with the decreased neurotoxicity of acrylamide observed by several authors as a result of microsomal enzyme induction by phenobarbital (24,42;Bergmark et al., unpublished results). Glycidamide might, however, be involved in the genetic and reproductive toxicity of acrylamide. In preliminary experiments we have found that glycidamide caused reproductive toxicity in male rats following a treatment regimen (50 mg/kg/day for 14 days) which did not cause any neurotoxicity (Gregotti et a]., unpublished results). Thus, the unknown main radioactive peak observed by Sega et al. (20) in protamine hydrolysates from rat sperm may in fact be the adduct formed between glycidamide and cysteine. Finally, certain observations in the literature are suggestive of an involvement of glycidamide in the cancerinitiating effects of acrylamide: (a) Glycidamide has been shown to be mutagenic in Salmonella typhimurium (43) and in mouse lymphoma cells (44). (b) The radioactivity associated with liver DNA in vivo in mice given acrylamide (45)corresponded to an adduct level of about 1 nmol/mg of DNA at a dose (46)of 100 mg (kg)-' X 1 kg L-' X 0.014 mmol (mg)-' X 2 h X (In 2)-' = 4 X M h [using the value 2 h for the half-life of acrylamide determined in rats (22) and 1 kg bw = 1 L]. This level is almost 30000 times higher than would be expected from the rate of reaction of acrylamide with DNA determined in vitro (8). (A total level of adducts of 11.6 nmol/mg of DNA was produced at a dose of 1.36 M X 40 days X 24 h day-' = 1.3 X lo3 M h a t 37 O C and pH = 7.0 when calf thymus DNA was reacted with acrylamide.) Thus, unless the radioactivity associated with DNA in vivo was a result of metabolic incorporation or protein contamination, the high level of adducts seems to implicate that adducts were formed by an agent, such as, e.g., glycidamide, which is more reactive versus DNA. ( c ) The percentages of rats with testicular mesotheliomas (18) and mice bearing squamous cell carcinomas (17) in oncogenicity studies with acrylamide appear to be consistent with the initiating agent being formed by a metabolic conversion of acrylamide that follows Michaelis-Mentens kinetics (46). In summary, the identification of S-(2-carboxy-2hydroxyethy1)cysteine in hydrolyzed hemoglobin samples from rats treated with acrylamide is strongly suggestive of the metabolic conversion of acrylamide to glycidamide, a reactive epoxide metabolite. The kinetics of this conversion, as well as the potential role of glycidamide in the neurotoxic, reproductive, and genotoxic effects of acrylamide, is now under investigation.

Acknowledgment. This study was supported in part by NIEHS Grant ES-04696 and by a grant from the Department of Environmental Health, University of Washington. E.B. was partly supported by a fellowship from the Sweden-America Foundation. We thank Drs. Dave Kalman and Rasmy Talaat and Mr. Greg Nothstein for facilitating the mass spectrometry work, Dr. Tim Myers

Acrylamide Metabolism to Glycidamide

for the 13C NMR analysis, and Claudia Thomas for secretarial assistance. Registry No. Acrylamide, 79-06-1; glycidamide, 5694-00-8; S-(2-~arbamoylethyl)cysteine,3958-15-4; S-(2-carbamoyl-2hydroxyethyl)cysteine, 128057-42-1; S-(2-carboxy-2-hydroxyethyl)cysteine, 29529-32-6; cysteine, 52-90-4.

References


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’

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Acrylamide is metabolized to glycidamide in the rat: evidence from

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