TOXICOLOGICAL SCIENCES 71, 164 –175 (2003)
Copyright © 2003 by the Society of Toxicology
Comparison of the Hemoglobin Adducts Formed by Administration
of N-Methylolacrylamide and Acrylamide to Rats
Timothy R. Fennell,* ,1 Rodney W. Snyder,* ,2 Wojciech L. Krol,* ,3 and Susan C. J. Sumner* ,4
*CIIT Centers for Health Research, P.O. Box 12137, Research Triangle Park, North Carolina 27709
Received August 16, 2002; accepted October 29, 2002
Acrylamide (AM) and N-methylolacrylamide (NMA) are used
in the formulation of grouting materials. AM undergoes metabolism to a reactive epoxide, glycidamide (GA). Both AM and GA
react with hemoglobin to form adducts that can be related to
exposure to AM. The objective of this study was to evaluate the
extent to which NMA could form the same adducts as AM.
N-(2-carbamoylethyl)valine (AAVal derived from AM) and N-(2carbamoyl-2-hydroxyethyl)valine (GAVal derived from GA) were
measured following a single oral dose of AM (50 mg/kg) or NMA
(71 mg/kg) in male F344 rats. AAVal and GAVal were measured
by a modified Edman degradation to produce phenylthiohydantoin derivatives and liquid chromatography/tandem mass spectrometry. In AM-treated rats, AAVal was 21 ⴞ 1.7-pmol/mg globin (mean ⴞ SD, n ⴝ 4), and GAVal was 7.9 ⴞ 0.8 pmol/mg. In
NMA-treated rats, AAVal was 41 ⴞ 4.9 pmol/mg, and GAVal was
1.4 ⴞ 0.1 pmol/mg. Whether AAVal was derived from reaction of
NMA with globin followed by loss of the hydroxymethyl group, or
loss of the hydroxymethyl group to form AM prior to reaction with
globin, is not known. However, the higher ratio of AAVal:GAVal
in NMA-treated rats (29 vs. 2.6 in AM-treated rats) suggests that
reaction of NMA with globin is the predominant route to AAVal in
NMA-treated rats. The detection of GAVal in NMA-treated rats
indicates oxidation of NMA, either directly or following conversion to AM. The lower levels of GAVal on NMA administration
suggest that a much lower level of epoxide was formed than
compared with AM treatment.
Key Words: acrylamide; glycidamide; N-methylolacrylamide;
hemoglobin; adduct.
Acrylamide (AM) is used in the production of polymers,
grouting agents, and specialty monomers (IARC, 1994). Exposure to AM can occur dermally, by inhalation, and by
1
To whom correspondence should be directed at present address: Center for
Bioorganic Chemistry, MCB/HLB 176, RTI International, P.O. Box 12194,
3040 Cornwallis Road, Research Triangle Park, NC 27709-2914. Fax: (919)
541-6499. E-mail: [email protected].
2
Present address: RTI International, P.O. Box 12194, Research Triangle
Park, NC 27709-2914.
3
Present address: GlaxoSmithKline, 5 Moore Drive, Research Triangle
Park, NC 27709.
4
Present address: Paradigm Genetics, Inc., P.O. Box 14528, Research
Triangle Park, NC 27709-2548.
ingestion (Dearfield et al., 1995; IARC, 1994). AM is neurotoxic, genotoxic, and carcinogenic in rodents (Bull et al.,
1984a,b; Dearfield et al., 1988, 1995; Friedman et al., 1995;
IARC, 1994; Johnson et al., 1986; Spencer and Schaumberg,
1974a,b; Tyl et al., 2000). Exposure of humans to AM results
in a peripheral neuropathy (Miller and Spencer, 1985; Spencer
and Schaumberg, 1974b). AM undergoes metabolism by conjugation with glutathione, or by oxidation to glycidamide
(GA), a reactive epoxide (Fig. 1) (Calleman et al., 1990;
Sumner et al., 1992). The genotoxic effects of AM are thought
to be mediated by its metabolism to GA, which is mutagenic
(Adler et al., 2000; Barfknecht et al., 1988; Butterworth et al.,
1992; Generoso et al., 1996; Hashimoto and Tanii, 1985) and
can react with DNA to form adducts (Segerbäck et al., 1995).
DNA adducts from GA have been reported following administration of AM to rodents (Segerbäck et al., 1995). AM is also
reactive, and the direct reactivity of AM with cellular components may also play a role in its toxicity. Hemoglobin adducts
from direct reaction with AM and from reaction with GA have
been described in rodents administered AM, in exposed people,
and in cigarette smokers (Bailey et al., 1986; Bergmark, 1997;
Bergmark et al., 1991, 1993; Calleman et al., 1990, 1994).
Recently the detection of acrylamide in fried foods has raised
considerable concern about human exposure to low levels of
AM in the diet (Tareke et al., 2002).
N-Methylolacrylamide (NMA) is produced by the reaction
of formaldehyde with AM, and is used for the production of
grouting agents (Fig. 1). NMA is carcinogenic in mice, producing tumors of the harderian gland, liver, and lung in both
sexes, and benign granulosa-cell neoplasms of the ovary in
females (Bucher et al., 1990). NMA was not carcinogenic in
rats at the doses tested. NMA is neurotoxic in both rats and
mice (Bucher et al., 1990) and has been estimated to have
approximately 30% of the neurotoxic potency of an equivalent
dose of acrylamide (Edwards, 1975). Like AM, NMA reacts
rapidly with glutathione (Hashimoto and Aldridge, 1970) and
is metabolized by conjugation with glutathione, resulting in
N-acetyl-S-(3-hydroxymethylamino-3-oxopropyl)cysteine as
the major urinary metabolite in rats and mice (Mathews, 2001).
The accidental release of AM and NMA from a tunnel
construction project in Sweden resulted in the exposure of
164
HEMOGLOBIN ADDUCTS OF ACRYLAMIDE AND NMA
165
42–5) was obtained from TCI America (Portland, OR) as a powder. The
vendor specified a minimum purity of 99%. 1H- and 13C-NMR analysis
appeared consistent with the specified purity and did not indicate the presence
of any free formaldehyde or acrylamide.
N-(2-carbamoylethyl)valine-leu-anilide (AAVal-leu-anilide) was obtained
from Bachem (King of Prussia, PA). Phenylisothiocyanate was obtained from
Aldrich (Milwaukee, WI). Valine and valine- 13C 5 were obtained from Sigma
(St. Louis, MO) and Isotec, Inc. (Miamisburg, OH), respectively.
FIG. 1. Chemical structures of acrylamide, glycidamide, and N-methylolacrylamide.
workers, ground water contamination, the death of fish, and
poisoning of cattle (Hagmar et al., 2001). In the exposed
workers, N-(2-carbamoylethyl)valine, formed by reaction of
AM with the N-terminal valine residue of hemoglobin, was
measured as an indicator of internal dose. A clear association
was described between levels of the hemoglobin adduct and
symptoms of peripheral nervous-system toxicity (Hagmar et
al., 2001). The extent of formation of globin adducts from GA
was not reported in this study. Whether the N-(2-carbamoylethyl)valine arose from reaction of hemoglobin with AM or
from NMA could not be distinguished.
In this study, we investigated the hypothesis that AM and
NMA can give rise to the same adducts in globin, but are
metabolized quantitatively in a different manner. This hypothesis was tested by measuring the formation of hemoglobin
adducts from AM and GA in rats administered AM or NMA by
gavage. The specific adducts measured were N-(2-carbamoylethyl)valine, formed by direct reaction of AM with the Nterminal valine residue, and N-(2-carbamoyl-2-hydroxyethyl)valine, formed by reaction of GA with the N-terminal valine
residue. To enable a parallel measurement of the extent of
metabolism by 13C nuclear magnetic resonance ( 13C-NMR)
spectroscopy reported elsewhere, the AM used was 13C-labeled
(Sumner et al., 1992). AAVal and GAVal formed in hemoglobin were measured to compare the internal dose of AM vs. GA
on administration of NMA or AM. To accomplish the adduct
analyses, a new method was employed for analysis of valine
phenylthiohydantoin derivatives using liquid chromatography
with tandem mass spectrometry (LC-MS/MS). This new
method could distinguish and quantitate adducts derived from
both the unlabeled NMA and the labeled AM administered.
MATERIALS AND METHODS
Chemicals. [1,2,3- 13C]AM (lot number PR-11085) was obtained from
Cambridge Isotope Laboratories, Inc. (Andover, MA) with a 98% chemical
purity and a 99% enrichment. 13C-NMR spectra of the [1,2,3- 13C]AM, acquired
by the vendor prior to study initiation and acquired at CIIT after delivery, were
consistent with the chemical structure: multiplets at 127–131 ppm and a
doublet (Jcc ⫽ 51 Hz) at 171 ppm, consistent with the 13C-labeled carbons for
the vinyl group (C⫽C) and carbonyl group (C⫽O), respectively.
Methylolacrylamide (NMA, N-hydroxymethylacrylamide, CAS No 924 –
Synthesis of glycidamide. Glycidamide was synthesized by H 2O 2 oxidation of acrylonitrile (Payne and Williams, 1961), and stored at –20°C. The 1H
nuclear magnetic resonance ( 1H-NMR) spectrum for GA in CDCl 3 (7.24 ppm)
contained three 1-proton multiplets centered at 3.45 ppm (CH), 2.95 ppm
(CH 2a), and 2.79 ppm (CH 2b), and a broad 2-proton singlet at 6.2 ppm (NH 2).
The 13C-NMR spectrum (in CDCl 3, 77 ppm) contained signals at 48 ppm
(CH 2), 50 ppm (CH), and 173 (CONH 2) that are consistent with the structure
of GA.
Synthesis of N-(2-carbamoylethyl)valine (AAVal). AAVal was synthesized and purified, as described previously, by Bergmark et al. (1993). AM
(10.01 g) and valine (1.76 g) were reacted in 30 ml water and 2.6 ml
triethylamine for 6 days at room temperature. The 1H-NMR spectrum of
AAVal in D 2O (4.9 ppm) had signals at 3.6 ppm (doublet, CH ␣), 3.4 ppm
(multiplet, CH 2-N), 2.8 ppm (triplet, CH 2-CO), 2.3 (multiplet, CH ␤), and 1.1
ppm (two doublets, valine CH 3). Integration of the signals provided a ratio
appropriate for the number of assigned hydrogens. The 13C-NMR spectrum
showed 2 carbonyl signals at 176 and 179 ppm, a signal at 72 ppm (CH ␣), a
signal at 47 ppm (CH 2-N), 2 signals near 33 ppm (CH ␤ and CH 2-CO), and 2
signals near 21 ppm (2 CH 3).
Synthesis of N-(2-carbamoylethyl)valine- 13C 5 (AAVal- 13C 5). Labeled material for use as an internal standard was prepared on a smaller scale, essentially as described above for the unlabeled standard. 13C 5-Valine (62.5 mg) was
reacted with AM (88.19 mg) in 1 ml of water and 87.5 ␮l of triethylamine for
6 days at room temperature. The NMR spectra of AAVal prepared from
13
C 5-Val contained 13C- 1H and 13C- 13C coupling patterns for signals derived
from the valine portion of the molecule, while signals derived from the AM
portion of the molecule did not contain these patterns. The 1H-NMR spectrum
of 13C 5-AAVal had signals at 3.4 ppm (multiplet, CH 2-N) and 2.8 ppm (triplet,
CH 2-CO), the same as those located in spectra for unlabeled AAVal. A doublet
of multiplets (at 3.4 and 3.8 ppm) were centered on 3.6 ppm, the shift for the
CH ␣ proton. A doublet of multiplets (at 2.1 and 2.5 ppm) were centered on 2.3
ppm, the shift for the CH ␤ proton. A doublet of multiplets (at 0.9 and 1.35
ppm) were centered at 1.1 ppm, consistent with the shift for the 2 CH 3 protons.
Integration of the signals provided a ratio appropriate for the number of
assigned hydrogens. Signals derived from the valine-labeled carbons were
located in the 13C-NMR spectrum near 176 ppm (CO, doublet), 72 ppm (CH ␣,
2 doublets), 33 ppm (CH ␤ , 2 doublets), and 21 ppm (CH 3, 2 doublets).
Synthesis of N-(2-carbamoyl-2-hydroxyethyl)valine. N-(2-carbamoyl-2hydroxyethyl)valine (GAVal) was synthesized and purified as described previously by Bergmark et al. (1993). GA (1.25 g) and valine (0.583 g) were
reacted in 5 ml of water and 87 ␮l of triethylamine for 24 h at 45°C. In addition
to the expected signals for Val CH ␣ (3.65 ppm, 2 doublets), CH ␤ (2.35 ppm,
multiplet), and CH 3 (1.1 ppm, 2 doublets), multiplets for the GA-derived
protons were observed at 4.6 ppm (CHOH) and at 3.3–3.6 ppm (CH 2) in the
1
H-NMR spectrum of GAVal (in D 2O). Integration of the spectrum provided
the appropriate integration ratios for the assigned signals. In the 13C-NMR
spectrum, Val-derived 13C signals were present at shifts similar to those
observed for AAVal, as described above (72, 33, and 21 ppm), while the
GA-derived carbon signals were observed at 52 ppm (N-CH 2) and 74 ppm
(CHOH).
Synthesis of N-(2-carbamoyl-2-hydroxyethyl)valine- 13C 5(GAVal- 13C 5).
Synthesis of N-(2-carbamoyl-2-hydroxyethyl)valine- 13C 5 (GAVal- 13C 5) for use
as an internal standard material was conducted as described above, but on a
smaller scale. 13C 5-Valine (57.3 mg) was reacted with GA (0.121 g) in 0.976
ml water and 85.4 ␮l of triethylamine for 6 days at room temperature. Signals
166
FENNELL ET AL.
in the 13C-NMR spectrum of GAVal- 13C 5 were at shifts consistent with those
obtained for unlabeled GAVal, and contained splitting patterns and relative
signal intensity consistent with incorporation of the labeled carbons of 13C 5valine.
Synthesis of AAVal phenylthiohydantoin. To prepare the AAVal phenylthiohydantoin derivative (AAVal PTH), AAVal (12.2 mg) was incubated with
phenylisothiocyanate (30 ␮l) in 1.5 ml of 0.5 M potassium bicarbonate:1propanol (2:1), pH 8.6, for 2 h at 45°C. The reaction product was isolated by
extracting with n-heptane (2 ⫻ 2 ml), drying under N 2, redissolving in toluene,
drying under N 2, redissolving in methanol, and purifying by HPLC using a
Beckman Ultrasphere ODS column (0.45 ⫻ 25 cm) eluted with 35% methanol/
water. The 1H-NMR spectrum of AAVal PTH (CDCl 3) contained signals for
the phenyl ring protons at 7.2–7.6 ppm. Additional signals were observed at
4.3 ppm (doublet, CH ␣), 3.85 and 4.35 ppm (multiplets, CH 2-N), 2.6 and 3.0
ppm (multiplets, CH 2-CO), 2.5 ppm (multiplet, CH ␤), and 0.95 and 1.25 ppm
(2 doublets, valine CH 3). Nonequivalent CH 2 signals suggest hindered rotation
of the AM side chain in AAVal PTH compared with AAVal. The 13C-NMR
spectra of AAVal PTH contained signals for the Val portion of the molecule
at 68 ppm (CH ␣) 30 ppm (CH ␤), and 16 and 18 ppm (2 CH 3). Signals for
AM-derived carbons were at 34 ppm (CH 2-N), 42 ppm (CH 2-CO), and 129 –
130 ppm for the phenyl ring carbons.
Synthesis of AAVal PTH- 13C 5. AAVal- 13C 5 (9.1 mg) was incubated with
phenylisothiocyanate (20 ␮l) in 1.5 ml of 0.5 M potassium bicarbonate:1propanol (2:1), pH 8.6, for 2 h at 45°C, and the product was isolated as
described above. The 1H-NMR spectrum of 13C 5 AAVal PTH contained
signals (multiplets near 4.3, 3.8, 3.0, and 2.6 ppm) for the AM portion of the
molecule consistent with those detected for the unlabeled AAVal PTH. Patterns consistent with 13C- 1H coupling were observed for signals from the
Val- 13C 5 portion of the molecule. The valine 13CH ␣ gave rise to multiplets at
4.0 and 4.5 ppm (centered at 4.3 ppm). The valine 13CH ␤ gave rise to multiplets
at 2.3 and 2.7 ppm (centered at 2.5 ppm). The valine methyl groups give rise
to multiplets at 0.7 and 1.2 (centered at 0.95), and 1.0 and 1.45 ppm (centered
at 1.2 ppm). Signals in the 13C NMR spectrum of 13C 5 AAVal PTH were
consistent with the AAVal PTH, containing the 13C- 13C splitting patterns and
relative intensities consistent with the presence of 13C 5-Val.
Synthesis of GAVal PTH. GAVal (12.0 mg) was incubated with phenylisothiocyanate (30 ml) in 1.5 ml of 0.5 M potassium bicarbonate:1-propanol
(2:1), pH 8.6, for 2 h at 45 °C. The reaction product was isolated by extraction
with n-heptane (2 ⫻ 2 ml), drying under N 2, redissolving in toluene, drying
under N 2, redissolving in methanol, and then isolating on HPLC using a
Beckman Ultrasphere ODS column (0.45 ⫻ 25 cm) eluted with acetonitrile/
water at 30% acetonitrile for 10 min, 40% acetonitrile for 3.5 min, and 100%
acetonitrile for 1.5 min. The 1H-NMR spectrum of GAVal PTH (in CDCl 3)
contained a sharp signal at 4.6 ppm (CHOH) and two broader signals at 4.0 and
4.5 ppm (CH 2) derived from the GA-protons. Signals for the Val-derived
protons were located near 0.9 and 1.3 ppm (CH 3, 2 doublets) 2.5 ppm (CH ␤,
multiplet), and 4.3 ppm (CH ␣). Ring protons were present between 7.2 and 7.6
ppm. The occurrence of nonequivalent CH 3 protons and GA-CH 2 protons
suggest structural hindrance of this molecule. The 13C0-NMR spectrum of
GAVal PTH had signals at 72 ppm (CHOH) and 49 ppm (CH 2-N), derived
from the GA portion of the structure, consistent with signals observed for
GAVal. The Val portion of the molecule had signals at 68 ppm (CH ␣), 30 ppm
(CH ␤), and 15 and 18 ppm (2 CH 3).
Synthesis of GAVal PTH- 13C 5. GAVal- 13C 5 (10.5 mg) was incubated with
phenylisothiocyanate (20 ml) in 1.5 ml of 0.5 M potassium bicarbonate:1propanol (2:1), pH 8.6, for 2 h at 45°C, and the product was isolated as
described above. Signals in the 1H NMR spectrum of GAVal PTH- 13C 5 (in
CDCl 3) contained a sharp signal at 4.6 ppm (CHOH) and two broader signals
near 4.0 and 4.5 ppm (CH 2) derived from the GA-protons. Signals for the
Val-derived protons were centered at 0.9 ppm (CH 3, doublet of multiplets, 0.75
and 1.15) and 1.3 ppm (CH 3, doublet of multiplets, 1.05 and 1.45), 2.5 ppm
(CH ␤, doublet of multiplets, 2.3 and 2.7 ppm ), and 4.3 ppm (CH ␣, doublet of
multiplets, 4.5 and 4.0 ppm). Ring protons were present between 7.2 and 7.6
ppm. The 13C NMR spectrum of GAVal PTH– 13C 5 (in CDCl 3) has intense
multiplets for the Val- 13C 5 portion of the molecule at 69 ppm (CH ␣), 30 ppm
(CH ␤), and 15 and 18 ppm (2 CH 3).
Animals. Male Fischer F344 rats were purchased from Charles River
Laboratories (Raleigh, NC). At the time of dosing, the rats were 9 –10 weeks
old. They were supplied food (NIH 07 diet, Zeigler brothers, Gardner, PA) and
reverse osmosis water ad libitum and maintained on a 12-h light-dark cycle
(0700 –1900 h for light phase) at a temperature of 64 –79°F and relative
humidity of 30 –70%.
Dosing and sample collection. Male Fischer 344 rats (4 per group) were
administered [1,2,3- 13C]AM by gavage at a nominal dose of 50 mg/kg body
weight. NMA was administered by gavage to four male F344 rats at a nominal
dose of 71 mg/kg body weight, targeted to provide an equimolar dose administered for AM and NMA. The AM and NMA dose solutions were prepared in
distilled water and delivered at 1 ml/kg body weight. A control group of four
additional F344 rats were administered distilled water. The dose administered
was calculated by weighing the dosing syringe before and after dosing. The
actual doses administered were 59.5 ⫾ 8.0 mg AM/kg (0.80 ⫾ 0.11 mmol/kg)
and 73.1 ⫾ 3.9 mg NMA/kg (0.72 ⫾ 0.03 mmol/kg). The control rats and rats
treated with [1,2,3- 13C]AM were placed in glass metabolism cages after
administration of the labeled material, and urine was collected over dry ice for
0 –24 h after dosing. The urine samples were stored at – 80°C. The rats
administered NMA were placed in polycarbonate cages after dosing. At 24 h
after administration, rats were euthanized by exposure to CO 2, and blood was
collected by cardiac puncture in a heparinized syringe. The blood was separated into red blood cells and plasma, and the red blood cell fraction was
washed three times with 0.9 % (w/v) saline and stored at –20°C for adduct
analysis.
Analysis of AAVal and GAVal in hemoglobin. AAVal and GAVal,
formed by reaction of AM and GA respectively with the N-terminal valine
residue in hemoglobin, were measured by an LC-MS/MS method. Globin was
isolated from washed red cells (Mowrer et al., 1986). Globin samples (approximately 20 mg) were derivatized with phenylisothiocyanate (5 ␮l) in formamide (1.5 ml) with 1 N NaOH (5 ␮l) to form adduct phenylthiohydantoin
derivatives in a manner analogous to the modified Edman degradation (Bergmark, 1997; Perez et al., 1999; Törnqvist et al., 1986). AAVal PTH- 13C 5 (26.6
pmol) and GAVal PTH- 13C 5 (36.0 pmol) were added as internal standards, and
the samples were extracted using a Waters Oasis HLB 3 ml (60 mg) extraction
cartridge (Milford, MA). The derivatized adducts were eluted with methanol,
dried, and reconstituted in 100 ml of MeOH:H 2O (50:50, containing 0.1%
formic acid). Analysis was conducted using a PE Series 200 HPLC system
interfaced to a PE Sciex API 3000 LC-MS with a Turboionspray interface.
Chromatography was conducted on a Phenomenex Luna PhenylHexyl Column
(50 ⫻ 2 ⫻ 3 mm) eluted with 0.1% acetic acid in water and methanol at a flow
rate of 350 ␮l/min, with a gradient of 45–55% methanol in 2.1 min. The elution
of adducts was monitored by multiple-reaction monitoring (MRM) in the
negative-ion mode for the following ions:
AAVal-PTH: m/z 304 3 233 (M-H – 3 M-H – – CH 2-CH 2-CONH 2)
GAVal-PTH: m/z 320 3 233 (M-H – 3 M-H – – CH 2-CHOH-CONH 2)
AAVal-PTH- 13C 5: m/z 309 3 238 (M-H – 3 M-H – – CH 2-CH 2-CONH 2)
GAVal-PTH- 13C 5: m/z 325 3 238 (M-H – 3 M-H – – CH 2-CHOH-CONH 2).
Quantitation of AAVal was conducted using the ratio of analyte to internal
standard, with a calibration curve generated using AAVal-leu-anilide. Quantitation of GAVal was conducted using the ratio of analyte to internal standard.
For samples from rats administered a single dose of [1,2,3- 13C]AM, the
13
CAAVal and 13CGAVal adducts formed can be distinguished in the negative
ion mode since the 13C-containing adduct side chain is lost from the adduct
PTH in the collision cell. An additional set of ions is monitored to quantitate
the adducts formed:
13
C 3-AAVal-PTH: m/z 307 3 233 (M-H – 3 M-H – – 13CH 2- 13CH 213
CONH 2)
13
C 3-GAVal-PTH: m/z 323 3 233 (M-H – 3 M-H – – 13CH 2- 13CHOH13
CONH 2).
Statistical analysis. Statistical analysis was conducted using Instat 2.01
(Graphpad Software, San Diego, CA ). One-way analysis of variance
HEMOGLOBIN ADDUCTS OF ACRYLAMIDE AND NMA
167
FIG. 2. Daughter ion spectra of
AAValPTH (m/z 304, top) and AAVal
PTH- 13C 5 (m/z 309, bottom) in the negative ion mode.
(ANOVA) with the Tukey–Kramer post test was used to compare control,
AM-treated, and NMA-treated groups.
RESULTS
Hemoglobin Adducts of AM and GA
For mass spectral analysis of AAVal and GAVal, the AAVal- and GAVal-PTH derivatives were analyzed in the negative ion mode with a turboionspray interface. In the negative
ion mode, the major ion formed was the parent ion (M-H –), and
the major daughter ions resulted from loss of the AM or GA
side chain (Figs. 2 and 3). This provided the capability to
distinguish between the adduct ions derived from AM, from
[1,2,3– 13C]AM, and from the internal standard labeled with
valine- 13C 5, since the loss of the AM and GA side chains
results in three distinct reactions that can be monitored to
detect each form of the adduct. For example, in the negative
ion mode, AAVal PTH is monitored by m/z 304 3 233
(M-H – 3 M-H – – CH 2-CH 2-CONH 2), while the internal standard AAVal-PTH- 13 C 5 is monitored by m/z 309 3 238
(M-H – 3 M-H – – CH 2 -CH 2 -CONH 2 ), and 13 C 3 -AAVal-PTH
derived from administration of [1,2,3– 13 C]AM is monitored
by m/z 307 3 233 (M-H – -3-M-H – - 13 CH 2 - 13 CH 2 - 13 CONH 2 ).
Conditions for the HPLC analysis of AAVal PTH and GAVal PTH were developed to enable rapid analysis of samples.
Thus chromatography was carried out using a short column
(Phenomenex Luna PhenylHexyl Column, 50 ⫻ 2 ⫻ 3 mm),
enabling rapid separation and elution of the adducts in 3 min,
and recycling of the column for the next injection within 10
min. To enable rapid sample processing and avoid extensive
solvent extractions involved in the original method for the
modified Edman degradation (Törnqvist et al., 1986), a solid-
168
FENNELL ET AL.
FIG. 3. Daughter ion spectra of GAValPTH (m/z 320, top) and GAVal PTH13
C 5 (m/z 325, bottom) in the negative
ion mode.
phase extraction method was developed to separate the adduct
derivatives from the solvent, remaining protein, derivatizing
reagent, and its products. Following extraction, the eluted
adduct derivatives were dried and reconstituted in mobile
phase for LC-MS analysis.
For standardization of the quantitative analysis, AAVal-leuanilide was added to samples containing unmodified globin.
This peptide provides a model for the AAVal in globin, and
will cyclize and cleave in a similar manner (Lawrence et al.,
1996; Osterman-Golkar et al., 1994; Törnqvist et al., 1986).
The samples were derivatized with PITC, and after addition of
the internal standard AAVal-PTH- 13C 5, the PTH derivatives
were extracted and analyzed by LC-MS/MS. A standard curve
for AAVal PTH generated with AAVal-leu-anilide indicated a
linear response with a background present in untreated hemoglobin, consistent with the observations of others (Bergmark,
1997; Perez et al., 1999; Tareke et al., 2000). This background
limits evaluation of the sensitivity that may be obtained for the
analysis method. However, for the analysis of samples of
AAVal PTH without added globin, the limit of detection is
estimated to be approximately 0.5 fmol/injection. Injection of
5-␮l aliquots from a 100-␮l sample derived from 20 mg of
globin gives an estimated limit of detection of 0.5 fmol/mg
globin. Greater sensitivity could be achieved by increasing the
volume of the aliquot injected and by increasing the amount of
globin analyzed. The standard curve generated was used for the
quantitative analysis to convert the ratio of analyte to internal
standard to amount of adduct present in each sample. Comparison of the amount of AAVal-leu-anilide added with the
amount of internal standard added indicated that approximately
70% of the added analyte was recovered. A similar calibration
could not be performed for GAVal with a peptide standard,
HEMOGLOBIN ADDUCTS OF ACRYLAMIDE AND NMA
169
FIG. 4. LC-MS analysis of AAVal
in a rat administered [1,2,3– 13C 3] AM
(50 mg/kg by gavage). The top panel
represents the natural abundance AAVal
analyte (m/z 304 3 233). The middle
panel is from 13 C 3 -AAVal-PTH (m/z
307 3 233). The bottom panel is AAVal-PTH- 13 C 5 internal standard (m/z
309 3 238).
because this is not commercially available. Therefore, quantitation
of GAVal was based on the peak area ratio of analyte:internal
standard, and the amount of GAVal-PTH standard added.
Adducts in AM- and NMA-Treated Rats
Samples from rats administered [1,2,3- 13C]AM or unlabeled
NMA were analyzed for the adducts formed by both the
13
C-enriched and natural abundance forms of AA and GA. A
chromatogram for AAVal in globin from a rat administered
[1,2,3- 13C]AM by gavage is shown in Figure 4. Three separate
chromatograms are shown for two forms of the analyte and the
internal standard. A similar set of chromatograms is shown in
Figure 5 for GAVal in the same animal. For quantitation, the
two peaks for the isomers of GAVal-PTH were integrated
together. Typical chromatograms for AAVal in NMA-treated
rats are shown in Figure 6 and for GAVal in Figure 7.
The results of the analysis of GAVal and AAVal are presented in Table 1. In the 50-mg/kg 13C AM-treated rats, little
change was observed in the amount of unlabeled AAVal and
GAVal compared to the control group. However, 13C AAVal
was 21 ⫾ 1.7 pmol/mg globin (mean ⫾ SD, n ⫽ 4), and 13C
GAVal was 7.9 ⫾ 0.8 pmol/mg. In the NMA-treated rats,
AAVal was 41 ⫾ 4.9 pmol/mg globin, and GAVal was 1.4 ⫾
0.1 pmol/mg. Thus AAVal and GAVal can be detected following administration of NMA. The ratio of AAVal to GAVal in
the NMA-treated rats was considerably higher (29) than that
following administration of an equimolar dose of AM to rats
(2.6 in AM-treated rats at 50 mg/kg body weight).
170
FENNELL ET AL.
FIG. 5. LC-MS analysis of GAVal
in a rat administered [1,2,3– 13C 3]AM (50
mg/kg by gavage). The top panel represents the natural abundance GAVal analyte (m/z 320 威 233). The middle panel is
from 13C 3-AAVal-PTH (m/z 323 3
233). The bottom panel is AAVal-PTH13
C 5 internal standard (m/z 325 3 238).
The AAVal detected in globin from NMA-treated rats may
have arisen by one of two mechanisms (Fig. 8). One possibility
is that NMA undergoes loss of the hydroxymethyl group to
form AM, which can then react with globin to form AAVal.
The second possibility is that NMA reacts directly with globin
and then loses the hydroxymethyl group to form AAVal. Both
reactions, involving loss of formaldehyde, could occur on a
chemical basis without the involvement of metabolism. These
mechanisms cannot be directly distinguished from the AAVal
data alone. The same issue exists for GAVal (Fig. 8). This
adduct may be formed following dissociation of NMA to give
AM, oxidation to GA, and reaction with globin. Alternatively,
GAVal could be formed following oxidation of NMA to an
epoxide and reaction with globin to form an adduct, which then
releases the hydroxymethyl group. If loss of the hydroxymethyl group to form AM with the oxidation to GA is the route
for AAVal and GAVal formation, one would expect that the
ratio of AAVal to GAVal would be similar in the AM- and
NMA-treatment groups. The much higher ratio of AAVal:
GAVal in the NMA-treated rats (29 vs. 2.6 in AM-treated rats)
suggests that reaction of NMA with globin is the predominant
route to these adducts in NMA-treated rats rather than conversion to AM. The detection of GAVal in NMA-treated rats
indicates oxidation of NMA, either directly or following conversion to AA. The lower levels of GAVal on NMA administration suggest a much lower level of epoxide formed in these
animals compared with AM treatment.
A further question that was investigated was whether valine
171
HEMOGLOBIN ADDUCTS OF ACRYLAMIDE AND NMA
FIG. 6. LC-MS analysis of AAVal
in a rat administered NMA (71 mg/kg by
gavage). The top panel represents the
natural abundance AAVal analyte (m/z
304 3 233). The middle panel is from
13
C 3-AAVal-PTH (m/z 307 3 233). The
bottom panel is AAVal-PTH- 13C 5 internal standard (m/z 309 3 238).
adducts containing a hydroxymethyl group could be detected in
rats administered NMA. For this purpose, LC-MS/MS of the
valine PTH extracts was performed as described above for
AAVal and GAVal. However, reaction monitoring for a hydroxymethylated AAVal was carried out by monitoring 334 3
233 and for hydroxymethylated GAVal by monitoring 350 3
233 (data not shown). An increase in peak area of approximately 10-fold was observed in monitoring 334 3 233 in the
NMA-treated rats compared with the control rats and those
administered AM. Little change was seen in monitoring 350 3
233. While these observations suggest that adducts can be
formed by direct reaction of NMA with hemoglobin, they are
by no means conclusive. The preparation of authentic adduct
standards and PTH-derivative standards would enable deter-
mination of retention time and fragmentation characteristics.
This, in turn, would enable the choice of appropriate conditions
for adduct quantitation.
DISCUSSION
Paulsson et al. (2002) recently reported a study in which AM
and NMA were administered to rats and mice for measurement
of AAVal and GAVal by modified Edman degradation, with
pentafluorophenylisothiocyanate to form adduct phenylthiohydantoins. GAVal was further derivatized with acidic acetone to
form an N-(2,2-dimethyl-4-oxazolidonylmethyl)valine derivative. AM was administered to male Sprague-Dawley rats by ip
injection in saline at a dose of 100 mg AA/kg body weight or
172
FENNELL ET AL.
FIG. 7. LC-MS analysis of GAVal
in a rat administered NMA (71 mg/kg by
gavage). The top panel represents the
natural abundance GAVal analyte (m/z
320 3 233). The middle panel is from
13
C 3-AAVal-PTH (m/z 323 3 233). The
bottom panel is AAVal-PTH- 13C 5 internal standard (m/z 325 3 238).
142 mg NMA/kg body weight (an equimolar dose). In addition,
male CBA mice were administered 25, 50, or 100 mg AM/kg,
or 35, 71, or 142 mg NMA/kg body weight by ip injection. In
this study, we confirmed the observation that NMA administration results in the detection of the same adducts formed by
AM administration. However, there are considerable quantitative differences between our observations and those reported
previously. A comparison of the two data sets is presented in
Table 2, where the adduct levels have been normalized for the
administered dose. The data obtained from AAVal following
AM administration are similar in the two studies. The amount
of GAVal obtained per unit dose was higher in this study and
may be consistent with the saturation of oxidation and nonlinearity observed in previous studies (Calleman et al., 1992).
Whereas Paulsson et al. (2002) described a higher amount of
AAVal formation with AM treatment (ratio of AM:NMA treatment of 2.7), we found a much higher formation with NMA
treatment (ratio of AM:NMA treatment of 0.53). While Paulsson et al. (2002) described a similar ratio of GAVal:AAVal in
rats between AM (0.26) and NMA treatment (0.23), we observed considerable differences between AM and NMA, with
ratios of GAVal:AAVal of 0.38 for AM treatment and 0.03 for
NMA treatment. Possible reasons for these differences could
be the route of exposure (ip injection vs. gavage administration) or nature of the NMA preparation (supplied as a solution
or obtained as a solid).
An important issue in predicting and relating possible adverse effects of AM and NMA to hemoglobin adducts is
173
HEMOGLOBIN ADDUCTS OF ACRYLAMIDE AND NMA
TABLE 1
Comparison of AAVal and GAVal in Globin from Rats Following a Single Oral Dose of 50 mg/kg AM, or 71 mg/kg NMA
Adduct level (fmol/mg globin)
Treatment
AAVal
Control
13
C-Acrylamide
NMA
156 ⫾ 9
168 ⫾ 22
0556 ⫾ 4945* ,**
13
C-AAVal
19 ⫾ 2
20847 ⫾ 1674*
22 ⫾ 6
GAVal
117 ⫾ 16
114 ⫾ 9
1351 ⫾ 122* ,**
13
C-GAVal
ND
7876 ⫾ 757*
ND
Note. Groups of 4 rats were administered 0.80 ⫾ 0.11 mmol AM or 0.72 ⫾ 0.03 mmol NMA/kg body weight by gavage. Blood was collected at 24 h after
dosing for analysis of adducts. Values represent mean ⫾ SD, n ⫽ 4. ND ⫽ not detected.
*Significantly different from control, p ⬍ 0.001.
**Significantly different from 13C-AAVal from acrylamide treatment, p ⬍ 0.001.
whether the methylol group is lost before or after formation of
the adduct. Rephrasing, the question is whether NMA is converted to AM in vivo prior to adduct formation (possible
pathways leading from NMA to AAVal and GAVal are illustrated in Figure 8). While Paulsson et al. (2002) were unable to
answer this question, the data from this study may provide an
answer. If NMA is converted to AM in vivo prior to reacting
with hemoglobin, one would expect similar ratios of GAVal:
AAVal to be observed with NMA and AM administration.
However, we observed very different ratios of these two adducts on administration of AM and NMA. Thus it would
appear that NMA reacts with hemoglobin prior to the loss of
formaldehyde. Whether this loss occurs in the animal or during
the process of isolation and analysis cannot be addressed at this
point. In a study of the metabolism and disposition of 14Clabeled NMA in male rats, Mathews (2001) found that 79% of
an oral dose of 150 mg/kg NMA was excreted in the urine.
Unchanged NMA was excreted in the urine (30% of the dose),
and N-acetyl-S-(3-hydroxymethylamino-3-oxopropyl)cysteine
was the major urinary metabolite (about 30% of the dose).
Other metabolites were detected in urine in this study but not
identified (about 10% of the dose). This indicates that at least
60% of the dose administered is derived from NMA, rather
than AM. Characterization of the additional metabolites could
indicate whether all of the NMA is metabolized without conversion to AM.
The considerably higher levels of AAVal in NMA-treated
rats suggest that the internal dose of NMA is higher than that
of AM. To calculate the rate of elimination (k el) of AM and
GA, Bergmark et al. (1991) used the relationship:
k el ⫽
关RX兴 0 k cys
关RY兴/关Y兴
between hemoglobin adduct levels expressed as [RY]/[Y], the
initial concentration of injected compound [RX] 0, and the rate
constant for reaction of AM and GA with cysteine in hemoglobin. Calculation of [RX] 0 involved the assumption that AM
and GA were rapidly and evenly distributed to tissues (Miller
et al., 1982) and that 1 kg body weight equals 1 liter. In this
situation, a knowledge of the rate constants of reaction of AM
and NMA with hemoglobin to form N-terminal valine adducts
would prove useful in determining the comparative internal
dose, and to estimate the relative rates of elimination of AM
and NMA.
An ambiguity remains in using AAVal and GAVal in assessing exposure to mixtures of AM and NMA. If AAVal data
alone were used to estimate exposure to AM but the exposure
was in reality to NMA, the exposure using the data generated
in this study would be overestimated on a molar basis by a
factor of two. The amount of GAVal formed on exposure to
FIG. 8. Possible routes of reaction
of NMA to produce AAVal and GAVal.
174
FENNELL ET AL.
TABLE 2
Comparison of AAVal and GAVal per Unit of Administered Dose of AM or NMA in Rats
Treatment
AAVal
GAVal
GAVal/AAVal
AM a
NMA a
Ratio AA/NMA a
AM b
NMA b
Ratio AA/NMA b
26.4 ⫾ 4.9
56.2 ⫾ 8.1
0.47
26.2 (21.1–31.4)
9.8 (6.9–12.8)
2.7
9.9 ⫾ 1.6
1.9 ⫾ 0.2
5.3
6.8 (5.9–7.7)
2.2 (1.9–2.5)
3.1
0.38 ⫾ 0.07
0.033 ⫾ 0.002
0.26 (0.17–0.59)
0.23 (0.11–0.52)
Note. AA Val and GA Val values are given in nmol/g globin per mmol/kg body weight.
a
This study, values are mean ⫾ SD, n ⫽ 4.
b
From Paulsson et al. (2002), values represent mean (95% confidence intervals).
AM would be considerably higher per mole of AAVal when
compared with NMA.
Further investigations should focus on whether NMA undergoes oxidative metabolism to produce hydroxymethylglycidamide and whether the hemoglobin adducts of NMA and
hydroxymethylglycidamide can be detected. The approach
used in this study with LC-MS/MS is much more amenable to
the analysis of polar adducts than is the more widely used
GC-MS approach. Analysis for the presence of NMA and
hydroxymethylglycidamide adducts may provide a means to
distinguish NMA and AM exposure. However, the stability of
these adducts in vivo, and during isolation and preparation for
analysis will need to be evaluated.
The approach used in this study with LC-MS/MS of valine
hemoglobin adducts has considerable advantages over the
more widely used GC-MS approach. This new method requires
less labor-intensive sample preparation: sample analysis is
more rapid as a result of shorter chromatography runs, and the
method much more amenable to the analysis of polar adducts.
This method has been applied to the quantitation of adducts
from other reactive chemicals, the results of which will be
communicated elsewhere.
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ACKNOWLEDGMENTS
Calleman, C. J., Stern, L. G., Bergmark, E., and Costa, L. G. (1992). Linear
versus nonlinear models for hemoglobin adduct formation by acrylamide
and its metabolite glycidamide: Implications for risk estimation. Cancer
Epidemiol. Biomarkers Prev. 1, 361–368.
This study was supported by SNF SA (primary scientific contact, Marvin A.
Friedman). The authors would like to acknowledge the support of the CIIT
Animal Care Staff. Dr. Barbara Kuyper is acknowledged for editorial review.
Calleman, C. J., Wu, Y., He, F., Tian, G., Bergmark, E., Zhang, S., Deng, H.,
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