United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
P.O. Box 93478
Las Vegas NV 89193-3478
EPA 600/R-93/226
November 1993
Preissue copy
Research and Development
&EPA Determination of
Hemoglobin Ad ducts
Following Acrylamide
Exposure
9312-0181ead
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DETERMINATION OF HEMOGLOBIN ADDUCTS FOLLOWING ACRYLAMIDE
EXPOSURE
Lucio G. Costa, Ph.D., Principal Investigator
Carl J. Calleman, Ph.D., Co-Principal Investigator
Department of Environmental Health, SC-34
University of Washington
Seattle, WA 98195
Cooperative Agreement CR-816768-01-0
Project Officer:
Charles H. Nauman, Ph.D., M.P.H.
Exposure Assessment Research Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
US ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193
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NOTICE
The information contained in this document has resulted from research funded by the
United States Environmental Protection Agency through Cooperative Agreement
CR-816768-01-0 to the University of Washington, Seattle, Washington. It has been
subjected to the Agency's peer and administrative review, and it has been approved for
publication as an EPA Project Report. Mention of trade names or commercial products
does not constitute endorsement or recommendation for use.
11
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ABSTRACT
The present project was undertaken to develop new methodologies for biological
monitoring of exposure to the toxicant acrylamide in laboratory animals as well as
humans. Methods were developed to measure the adducts of acrylamide and its epoxide
metabolite glycidamide to cysteine in rat hemoglobin and to valine in human hemoglobin
by means of gas chromatography/mass spectrometry.
Studies in rats indicated that both acrylamide and glycidamide adducts are formed
following acute or chronic exposure to acrylamide, while only the glycidamide adducts are
formed after exposure to glycidamide. Both adducts, in addition to acrylonitrile adducts,
were measured in a group of Chinese workers exposed to acrylamide during its synthesis
and polymerization. Significant signs of neurotoxicity were also found in this population.
Additional studies in rats indicated that acrylamide, but not glycidamide, is the proximate
neurotoxicant while glycidamide may be responsible for the male reproductive and
genotoxic/carcinogenic effects of acrylamide.
These studies suggest that these novel biomarkers to assess exposure to acrylamide are
useful to assess potential health hazards (including possible cancer risks) due to exposure
to acrylamide in occupationally exposed workers as well as in the general population.
This report was submitted in fulfillment of Cooperative Agreement CR-816768-01-0 by
the University of Washington under the sponsorship of the U.S. Environmental Protection
Agency. The report covers a project period from September 1990 to September 1992,
with a no-cost extension until March 1993 to permit analysis of the extensive field data
collected in the People's Republic of China. Work was completed as of March 1993.
Funding for the research was provided by the Office of Research and Development
(ORD), Environmental Monitoring Systems Laboratory, Las Vegas, Exposure Biomarkers
Research Program, under the Toxic Substances Budget Sub-Activity (L104). Partial
support for the field trip to the People's Republic of China was provided by the ORD
Health Effects Research Laboratory (HERL), Research Triangle Park, NC, under the
ORD/HERL program in Research to Improve Health Risk Assessments (REHRA).
111
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CONTENTS
NOTICE ii
ABSTRACT iii
FIGURES vi
TABLES vii
ABBREVIATIONS ix
ACKNOWLEDGMENTS x
INTRODUCTION 1
Acrylamide 1
Determination of hemoglobin adducts for exposure assessment 2
AIMS 2
METHODS 4
Aiml: 4
Synthesis of deuterated analogues 4
Treatment of animals 5
Isolation of globin and analysis of hemoglobin adducts 5
Gas chromatography/mass spectrometry analysis 6
Alkylation of hemoglobin in vitro 6
Aim 2: 6
Synthesis of alkylated valines 6
NMR Spectroscopy and fast atom bombardment mass spectrometry (FAB-MS) 9
Analysis of Hb adducts in globin hydrolysates .. 9
Analysis of hemoglobin adducts with the modified Edman degradation method 9
Gas chromatography-mass spectrometry analysis 9
Alkylation of hemoglobin in vitro 10
Calibration 10
Field study in the People's Republic of China 11
Physical description of the factory 11
Air monitoring 11
Description of study populations 11
Determination of mercapturic acids in urine 13
Blood samples for hemoglobin adducts 13
Measurements of vibration thresholds 14
Electroneuromyography (ENMG) 14
Clinical and laboratory examinations 15
Questionnaire information 15
IV
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Definition of neurotoxicity index 15
Statistical methods 15
Aim 3: 17
Animals and treatments 17
Neurotoxicity testing 17
Reproductive toxicity testing 17
RESULTS AND DISCUSSION 18
Aim 1: 18
Development of a GC/MS method for measuring Hb adducts of AA and GA 18
Acrylamide and glycidamide hemoglobin adducts following administration
to rats 19
General discussion of Aim 1 27
Aim 2: 29
Development of methods for acrylamide and glycidamide Hb adducts
in humans 29
Symptoms and signs in acrylamide exposed workers 30
Vibration thresholds 30
Eiectroneuromyographic measurements 34
The neurotoxicity index as predictor of clinical diagnosis of
peripheral neuropathy 34
Hb adduct levels in humans exposed to acrylamide 34
Calculation of in vivo doses 44
Other biomarkers of exposure 44
Biomarkers of exposure as predictors of neurotoxicity 44
General discussion of Aim 2 .. 52
Hemoglobin adducts 52
Field study 54
Aim 3: 58
Comparative neurotoxicity of acrylamide and glycidamide 58
Behavioral tests 58
Morphological observations 58
Male reproductive toxicity of acrylamide and glycidamide 62
General discussion of Aim 3 • 62
REFERENCES 65
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FIGURES
Figure 1 Reactions of AA and GA with cysteine residues in Hb 3
Figure 2 Reactions of AA, GA and AN with the N-terminal valine of Hb 8
Figure 3 Hb cysteine adducts of AA and GA in rats injected with 20
different single doses of AA
Figure 4 Mass fragmentogram from a rat treated with 10 mg/kg AA 21
Figure 5 Hb - cysteine adducts of GA in rats injected with different 22
single doses of GA
Figure 6 Conversion of AA to GA in rats injected with AA 23
Figure 7 Neurotoxicity Index in workers with and without clinical diagnosis 36
of peripheral neuropathy
Figure 8 Mass fragmentogram of AA and GA valine adducts 39
Figure 9 Correlation between AA and GA Hb adducts in workers 40
exposed to AA
Figure 10 Correlation between neurotoxicity (NIn) and levels of free 46
AA in plasma
Figure 11 Correlation between neurotoxicity (NIn) and urinary .. 47
mercapturic acids levels
Figure 12 Correlation between neurotoxicity (NIn) and AA Val adducts 48
Figure 13 Correlation between neurotoxicity (NIn) and AN Val adducts 49
Figure 14 Correlation between neurotoxicity (NIn) and lifetime in vivo 50
doses of AA
Figure 15 Body weights of rats treated with AA or GA v 59
Figure 16 Rotarod test performance of rats exposed to AA or GA 60
Figure 17 Hindlimb splay test in rats exposed to AA or GA 61
VI
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TABLES
Table la - Air concentration of acrylamide (mean +. SD, mg/m5) 12
Table Ib - Air concentrations of acrylonitrile (mean ± SD, mg/m3) 12
Table 2 - Definition of Neurotoxicity Index 16
Table 3 - Hemoglobin adducts in rats treated with different dose 24
regimens of acrylamide
Table 4 - Pharmacokinetic parameters of acrylamide and 26
glycidamide in the rat
Table 5 - Second-order rate constants for the reaction of acrylamide 31
and glycidamide with amino acid residues in hemoglobin
Table 6 - Percentage of acrylamide-exposed workers and controls 32
with symptoms of adverse health effects
Table 7 - Comparison of vibration thresholds (VU ± SD) between 33
acrylamide workers and referents
Table 8 - Comparison of motor (MCV) and sensory (SCV) nerve 35
conduction between the acrylamide and reference groups
Table 9 - Hemoglobin adducts of acrylamide (AAVal), glycidamide 37
(GAVal) and acrylonitrile (ANVal) in workers
Table 10 - Air concentrations of AA and AAVal Hb adduct levels 41
in workers in different locations
Table 11 - Individual values for parameters predictive of adverse 42
health outcome in workers exposed to acrylamide
Table 12 - Biomarkers according to job classification 45
Table 13 - Correlation coefficients and levels of statistical 51
significance for the relationships between different
predictive parameters and the Neurotoxicity Index
vn
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Table 14 - Effects of acrylamide and glycidamide on male 63
reproductive organs in rats
Table 15 - Effects of acrylamide and glycidamide on male 64
reproductive parameters in rats
Vlll
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ABBREVIATIONS
AA Acrylamide
AACys S-(2-carbamoylethyl)cysteine
AAVal N-(2-carbamoylethyl)valine
ANOVA Analysis of Variance
ANVal N-(2-cyanoethyl)valine
Bq Bequerel
CEVal N-(2-carboxyethyl)valine
CHEVal N-(2-caiboxy-2-hydroxyethyl)valine
EI^o Effect Level, 50%
El Electron impact
EMG Electromyography
ENMG Electroneuromyography
eV Electronvolt
FAB-MS Fast Atom Bombardment Mass Spectrometry
GA Glycidamide
GACys S-(2-carbamoyl-2-hydroxyethyl)cysteine
GAVal N-(2-carbamoyl-2-hydroxyethyl)valine
GC/MS Gas Chromatography/Mass Spectrometry
Hb Hemoglobin
HFB Heptafluorobutyryl
HFBA Heptafluorobutanoic Anhydride
HOEtVal Hydroxylethyl Valine
HPLC High Pressure Liquid Chromatography
LOEL Lowest Observed Effect Level
MCV Motor Nerve Conduction Velocity
MeOH Methanol
NIn Neurotoxicity Index
NMR Nuclear Magnetic Resonance
NOEL No Observed Effect Level
PCI Positive Chemical lonization
PFPITC Pentafluorophenyl isothiocyanate
PFPTH Pentafluorophenylthiohydantoin
SEM Standard Error of the Mean
SCV Sensory Nerve Conduction Velocity
TLC Thin-Layer Chromatography
TLV Threshold Limit Value
TWA Time-Weighted Average
VU Vibration Units
IX
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ACKNOWLEDGEMENTS
This research project was carried out in the Department of Environmental Health at the
University of Washington in Seattle.
In addition to the Principal Investigator and Co-Principal Investigator, a major contributor
was Dr. Emma Bergmark, who based her Ph.D. thesis on this work.
Dr. David Kalman and Russell Dills were instrumental in facilitating the mass
spectrometry work.
Drs. F. He, H. Deng, Y. Wu, S. Zhang and Y. Wang from the Institute of Occupational
Medicine at the Chinese Academy of Preventive Medicine in Beijing (PRC) and Drs. G.
Tian and B. Xia from the Institute of Occupational Health of XinXiang City, Hunan
Province, (PRC) were important collaborators for the field study in China.
Dr. Charles Nauman, Project Officer, EPA, offered useful guidance in the development
and realization of this project.
Chris Sievanen provided secretarial assistance.
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INTRODUCTION
Acrylamide
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.
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 NIOSH, some
10,000 workers in 27 occupations are potentially exposed to acrylamide, including about
one thousand persons involved in the synthesis of polyacrylamides and another thousand
licensed to perform grouting operations. The number of laboratory workers exposed to
acrylamide in the preparation of chromatography gels has been estimated to be as high as
100,000-200,000.
Since the early fifties the neurotoxic effects, involving both the central and the peripheral
nervous system, have been the primary health concern of human exposure to acrylamide;
close to 150 cases of human intoxication have been reported in the last 30 years (He et al.
1989). These reports have been paralleled by extensive studies in experimental animals,
notably aiming at the elucidation of the mechanism of neurotoxic action of acrylamide
(Miller and Spencer, 1985; Tilson, 1981). In more recent years, following the
demonstration that acrylamide induces tumors in mice and rats, attention has been
increasingly focused on the genotoxic and reproductive effects of the compound (Dearfield
et al. 1988). Thus, acrylamide has been shown to be mutagenic in vitro in mouse
lymphoma cells, and to give rise to heritable translocations and dominant lethal mutations in
rodents. Unexpectedly for a compound producing these types of effects, acrylamide has
consistently given a negative response in the Ames/'Salmonella assay both in the absence
and in the presence of an S-9 fraction.
Despite the fact that acrylamide has been shown to induce this wide range of adverse effects
in mammalian species, few studies have investigated the role of metabolism in the toxicity
of this compound and although reports have appeared indicating that acrylamide may
undergo further metabolism, the structures of the intermediates generated have not been
elucidated. From the point of view of quantitative risk assessment of human exposure this
has represented a serious gap of knowledge, since the extrapolation of toxic effects in
animals requires not only the identification of the agent(s) responsible for the induction of
the effects, but, ideally, also information about the relationship between exposure dose and
in vivo dose (defined as the time-integrated concentration of free electrophilic agent in
vivo). In addition, since exposure to acrylamide may occur through several different routes,
the total amount absorbed by a person may be very difficult to assess.
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Determination of hemoglobin adducts for exposure assessment
The structural elucidation and quantitative determination of hemoglobin (Hb) adducts can be
used to obtain this type of information. For example, in response to long-standing
speculation about the possible involvement of a cytochrome P-450-generated agent in the
toxicity of acrylamide, we were recently able to demonstrate the formation in vitro and in
vivo in rats of glycidamide, an epoxide formed by oxidation of the olefinic bond of
acrylamide, through the identification of S-(2-carboxy-2-hydroxyethyl)cysteine (Fig. 1) in
hydrolyzed Hb samples from acrylamide exposed rats (Calleman et al. 1990).
Determination of Hb adducts as a way of assessing the relationship between environmental
concentrations and in vivo dose, as well as for the dosimetry-based risk estimation of toxic
effects, has been undertaken for several types of alkylating agents in humans. The
advantages of such measurements are:
- Hb adducts are stable during the 4-month life span of the human erythrocyte, meaning
that in vivo doses are integrated over a long and well-defined period of time.
- Large quantities of Hb are easily available from exposed animals and humans allowing for
detection of low levels of adducts, e.g. by gas chromatography-mass spectrometry.
- Differences between species and individuals with regard to rates of uptake, metabolic
activation and deactivation of electrophiles, as well as induction status of enzymes, are
reflected in the levels of Hb adducts formed in vivo, thus providing a basis for extrapolation
between species and dosimetry-based risk estimation.
- The relationship between dose in red blood cells and in target organs can be studied in
experimental animals, and extrapolated to humans if due consideration is given to reaction
kinetic and physicochemical parameters of the electrophile.
A method for the determination of Hb adducts of the parent compound acrylamide has been
developed by Bailey et al. (1986) based on the liberation of S-(2-carboxyethyl)cysteine (Fig.
1) upon acid hydrolysis of Hb from acrylamide treated rats. Since, however, we are
hypothesizing that glycidamide might be playing a crucial role in the induction of some of
the toxic effects associated with acrylamide exposure, there was a need for the development
of quantitative methods which would be applicable to both animals and humans and that
could be used for the monitoring of both the adduct formed by acrylamide and by its
epoxide metabolite.
AIMS
It was the central aim of this project to develop and apply methods for simultaneously
determining the adducts formed by acrylamide and glycidamide in Hb as a means of
assessing occupational exposure to acrylamide and to use these data in combination with
dosimetric and metabolic studies in experimental animals undergoing toxicological tests to
arrive at a risk estimation of human acrylamide exposure which may serve as a basis for
regulatory action.
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\
0
CH2»CH-C-NH2
Acrylamide
Metabolism
HB CH-CH2-SH
^«^»^
HB CH-CH2-S-CH2-CH2-C-NH2
HCI
I
COOH
\
/
NH,
CH-CH2-S-CH2-CH2-COOH
/°\ 9
CH -CH-C-NH2
Glycidamide
HB CH-CH2-SH
OH 0
HB CH-CH2-S-CH2-CH -C-NH 2
HCI
I
COOH OH
\ '
CH-CH2-S-CH2-CH -COOH
Z
H*
O
S-(2-carboxycthyl)-cysteinc
I
der.
COOCH3
CH-CH2-S-CH2-CH2-COOCH3
NHCOC3F7
S-(2-carboxy-2-hydroxyethyl)-cysteine
I
der.
I
COOCH3 OOCC3F7
CH-CH2-S-CH2-CH-COOCH3
NHCOC3F7
Figure 1. The reactions of AA and GA with cysteine residues in Hb. The products released
during acid hydrolysis are 5-(2-carboxyethyl)-cysteine and 5-(2-carboxy-2-hydroxyethyl)-
cysteine. The structures of the derivatized products are also shown.
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In addition to the practical implications of the proposed methods for biomonitoring,
acrylamide appears to be a suitable compound for the theoretical development of a risk
model based on in vivo dosimetry because of the wide range of toxic effects it is known to
induce. Since both acrylamide and glycidamide are reactive electrophilic compounds, it
was not clear whether the various toxic effects associated with acrylamide exposure are
induced by the parent compound, the epoxide, or the combined action of the two agents.
Our finding that acrylamide was metabolized to glycidamide thus generated a few
hypothetical solutions to theoretical problems particular to the toxicology of acrylamide.
The specific aims of this project were, therefore:
1. To develop a method for measuring the adducts of acrylamide and glycidamide to
hemoglobin in rats and to determine both adducts following administration of acrylamide or
glycidamide.
2. To develop a method for measuring the adducts of acrylamide and glycidamide in
humans, measure adducts in workers occupationally exposed to acrylamide, and correlate
adduct levels with neurotoxicity.
3. To evaluate and compare the neurotoxicity and reproductive toxicity of acrylamide and
glycidamide in the rat.
METHODS
ABMl
Synthesis of deuterated analogues
S-(2-Carbamoylethyl)-3,-3-d2-cysteinewas prepared in analogy with the non-deuterated
compound (AACys), the synthesis of which was described in Calleman et al. (1990).
Following derivatization with MeOH/HCl and HFBA (Calleman et al., 1978) the mass
spectrum obtained upon electron impact ionization gave the fragments m/z = 419 and 205
interpreted as [M]+ and [M - C^CONHD]*, respectively, and the mass spectrum
obtained when scanning for negative ions using methane as the reagent gas gave m/z = 418
[M - H]~. Both spectra were in analogy with the nondeuterated compound. TLC, amino
acid analysis, and GC/MS revealed an impurity consisting of « 5% cystine.
S-(2-Carbamoyl-2-hydroxyethyl)-3,3-d2-cysteinewas prepared as described, in Calleman et
al. (1990) for the nondeuterated analogue (GACys). Following derivatization, the mass
spectrum obtained upon electron impact ionization gave the fragments m/z = 572 [M -
CO2CH3]+, 417 [M - C3F7CONHD]+, and 203 [M - C3F7C02H - C3F7CONHD]+.
Chemical ionization using methane as the reagent gas gave the positive ions m/z = 418 [M
- C3F7CONHD + H]+ and 632 [M + H]+ and the negative ion 611 [M - HFJ. The
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spectra were in analogy with the nondeuterated compound. TLC, amino acid analysis, and
GC/MS revealed cystine as an impurity of « 5%. On amino acid analysis, three peaks
were evident: two peaks, presumably the diastereoisomers (S)- and (R)-S-(2-carbamoyl-
2-hydroxyethyl)-3,3-d2-<#-cysteine (whose presence in approximately equal amounts was
indicated by proton NMR spectroscopy), merging into one major peak corresponding to
90%; and a minor peak, possibly the regioisomer S-(l-carbamoyl-2-hydroxyethyl)-3,3-d2-
cysteine, corresponding to 10%.
The nondeuterated analogue gave the same pattern of peaks but did not contain any cystine.
On GC/MS, the isomers were not resolved on the capillary column that was utilized.
Treatment of animals
Male Sprague-Dawley rats (130-260 g) were obtained from Tyler laboratories and kept on a
12-hr light/dark cycle with free access to food and water. AA (Eastman-Kodak, Rochester,
NY) or GA (Polysciences, Inc., Warrington, PA) were dissolved in 0.9% NaCl and
administered in a volume of 1 ml/kg body wt by ip injection. Control animals were given
vehicle only. Blood was drawn into a heparinized syringe by cardiac puncture under ether
anesthesia.
Groups of three rats were injected with AA (0, 0.5, 1.0, 5.0, 10.0, 50.0, and 100.0 mg/kg
body wt) and blood was drawn 24 hr after the injection. In an additional experiment, a
cumulative dose of 100 mg/kg body wt AA was given in different regimens to groups of
four rats. The groups received a single injection of 100 mg/kg, 10 daily injections of 10
mg/kg, or 30 daily injections of 3.3 mg/kg. Blood was drawn 24 hours after the final
injection.
Isolation of globin and analysis of hemoglobin adducts
Erythrocytes and plasma were separated by centrifugation of blood, samples. The red blood
cells were washed three times with 0.9% NaCl and lysed by addition of 1 vol of water.
Cell membranes and debris were removed by centrifugation at 20,000g for 30 min. The
supernatant was added to 10 vol of 1% HC1 in acetone and the resulting precipitate was
washed twice with acetone and once with ether and then air-dried.
Samples of 30 mg of globin were dissolved in 6 M HC1 to a concentration of 10 mg
globin/ml. The deuterated analogues, serving as internal standards for the quantitation,
were added to the hydrochloric acid to give concentrations of 10 /xmol/g Hb. The samples
were hydrolyzed for 15 hr at 120°C in sealed Pyrex glass ampoules under vacuum (<50 x
10° mm Hg). The hydrolysates were evaporated to dryness, dissolved in 1.5 ml of H2O),
and incubated for 1 hr at 37°C. After adjustment of the pH to 9 with NaOH, the samples
were applied to Dowex 1 x 8 (100-200 mesh, 1 x 15 cm, HCOO -form) anion-exchange
columns. The columns were washed with 80 ml of 5mM formic acid to remove most of
the amino acids upon which the adducts were eluted with 60 ml of 100 mM formic acid.
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The 100 mM eluates were evaporated and derivatized with MeOH/HCl and HFBA.
Derivatized samples were dissolved in 0.5-1.0 ml of ethyl acetate before quantitation by
GC/MS.
Gas chromatography/mass spectrometry analysis
The analyses were carried out using a Hewlett-Packard 5890A gas chromatograph linked to
a Finnigan 4023 mass spectrometer. The operating conditions for the gas chromatograph
were as follows: helium carrier gas at a constant pressure of 15 psi: temperature
programmed from 1 min at 70°C, 15°C/min to 230°C, 5°C/min to 240°C, 20°C/min to
280°C, and holding for 1 min. A 30-m DB-5 (0.25 mm i.d., 0.25-^m phase thickness)
fused silica capillary column was used. The mass spectrometer was operated in the electron
impact mode at an ion source temperature of 250°C and an ionization energy of 70 eV.
Chemical ionization mass spectra for the derivatized synthetical adducts were obtained with
methane as the reagent gas at an ion source pressure of 0.30 Torr and a temperature of
220°C. Both positive and negative ion spectra were obtained at an ionization energy of 70
eV. For the analysis of the rat Hb samples, 1 /*! of the ethyl acetate solutions was injected
directly onto an on-column injector. AACys and its deuterated analogue were monitored at
m/z « 202 and 203, [M - QFTCONH^D)]*, respectively, and GACys and its deuterated
analogue were monitored at m/z = 202 and 203, [M - QF-jCOJH - C3F7CONH2(HD)]+,
respectively. The retention time for AACys was 719 sec and that for GACys was 730 sec.
Quantitation was based on the ratio between peak areas of the analyte and the internal
standard. Correction for isotopic contributions and nonlinear response (at ratios <0.1) was
made with a calibration curve prepared by mixing triplicates of seven rations between zero
and one of known amounts of pure nondeuterated standard and the same amount of
deuterated standards as added in the rat globin samples.
Alkylation of hemoglobin in vitro
The rates of reaction of AA or GA with Hb were determined in blood from nonexposed
rats. Erythrocytes were separated from plasma by centrifugation, washed with saline,
resuspended to the original blood volume using phosphate-buffered saline (PBS), pH 7.4,
and incubated with either AA (1 mM) or GA (2 mM) at 37°C. Aliquots were taken from
the incubation mixture at different times (5, 10, 15, 20, 40, and 60 min) and the reaction
was terminated by adding 5 vol of ice-cold saline followed by centrifugation. The cells
were washed twice with saline and lysed by the addition of water. Globin was isolated and
the adduct levels were analyzed as described above.
ABM 2
Synthesis of alkylated valines
N-(-Carbamoylethyl)-L-valine (AAVal) was prepared by dissolving 1.76 g (15 mmole)
-------
L-valine in 30 ml H20 and 2.6 ml (18.6 mmole) triethylamine and adding 10 g (141 mmole)
AA. The pH of the solution was « 10. After 6 days at room temperature, the product
was precipitated with acetone and recrystallized in H2O/ethanol. The product (yield «
32%) was judged to be pure based on TLC, amino acid analysis, 'H-NMR and FAB-MS.
The Rrvalue on TLC silica plate, eluted with 2-propanol/H2O 7:3, was 0.65. JH-NMR
indicated d 0.95 (6H, dd, val-y), 2.15 (1H, m, val/3), 2.65 (2H, 5, CH2CH2CONHJ,
3.2(2H, m, NHCHjCHj), 3.4 (1H, d, vala). FAB-MS gave the ion m/z = 189 [M+H]+.
AAVal was derivatized with methanol/HCl and HFBA according to Calleman et al. (1978)
and analyzed by GC/MS. The electron impact ionization spectrum gave fragments m/z =
381 [M]*, m/z = 308 [M-CH2COOCH3]+ and m/z = 212 [M-C3F7]+and chemical
ionization with methane as the reagent gas gave the positive ions m/z = 382, 410 and 422
corresponding to [M+l]+, [M+29]+ and [M+41]+. The spectrum was consistent with
Structure A in Fig. 2.
AAVal was also derivatized with PFPITC according to Mowrer et al. (1986). On GC/MS,
the pentafluorophenylthiohydantoin (PFPTH) gave m/z = 395 [M]+ upon electron impact
ionization. Chemical ionization yielded the positive ions m/z = 396, 424 and 436
([M+i;T, [M+29]+ and [M+41]+) and the negative ion m/z = 375 [M-HF]'. See Fig. 2,
structure B.
N-(2-Carbamoyl-2-hydroxyethyl)-L-valine (GAVal) was prepared in the same way as
described for AAVal, but instead of AA, 3.74 g (42.9 mmole) GA was added. The yield
was « 44%. The product was judged to be pure by TLC, amino acid analysis, 1H-NMR
and FAB-MS. The Rrvalue on TLC was 0.77 in the same system as for AAVal. 'H-NMR
indicated 5 0.95(6H, m, val-y), 2.2(1H, val/3), 3.25(2H, m, NHCH2CHOC), 3.5 (1H, dd,
vala), 4.4 (1H, m, CHjCHOHCONHz). FAB-MS gave the ion m/z = 205
The product was derivatized with methanol/HCl and HFBA, and run on GC/MS. Five
GC-peaks were evident in the approximate ratios 2:1:3:0.7:0.7. Upon electron impact
ionization, peak number 2 gave the fragments m/z = 593 [M]*, 242 [M-C3F7]+, 308
[M-CH(OOCC3F7)COOCH3r and 299
Chemical ionization with methane as the reagent gas gave the positive ions m/z = 594,
622, 634 ([M+l]+, fM+29]+ and [M+41]+). The structure of the derivative was
consistent with C in Fig. 2. Peaks number 1, 3, 4 and 5 were interpreted as stereoisomers
of structure E in Fig. 2.
Derivatization with PFPITC gave the PFPTH-derivative, structure D in Fig. 2, which on
GC/MS gave the electron impact spectrum with a fragment at m/z = 411 "[M]* while
chemical ionization gave the positive ions m/z = 412, 440, 452 ([M+l]+, [M-f 29]+ and
[M+41]*) and the negative ion m/z = 391 [M-HF]'.
N-(Carbamoylethyl)-dg-L-valine (d8-AAVal) and N-(2-carbamoyl-2-hydroxyethyl)-dg-L-
-------
A 9
-I
CHj-CH-C-NK,
OycMwiMt
•C-NHj
CHfCH-CM
Acrytafltoflt
-
OH
faapUfon
tlgleba
1 1
1
. jo*. <*pfpnc \
cc
Q.
IU
cc
0.
w
a
<
CO
°S^
COOH OH
CHEVal
^CH-NHCHj-CHj
COOH
CEVcl
°-O'-CIVCH2-OOOCH, o-c" >^^COOCK,
A (CEVlHiFB)
C (CHEVaMtfB)
O-CH
A (CEV«I-KFB)
B (AAVaM>FPTM)
OH
0 (GAV«M>FPTM)
O N-
CHjO
F (ANV«M>FPTH)
Figure 2. The reaction of acrylamide (AA), glycidamide (GA), and acrylonitrile (AN) with
the N-terminal valine of hemoglobin in vivo. Following acid hydrolysis of globin, the
reaction products of AA and AN with valine are released as CEVal, and the reaction
product of GA with valine is released as CHEVal. Compounds A (CEVal-HFB),
C(CHEVal-HFB), and E are formed after derivatization with methanol/HCl and HFBA.
Following derivatization of globin with PFPITC, the compounds formed are B
(AAVal-PFPTH), D (GAVal-PFPTH), and F(ANVal-PFPTH).
-------
valine (dg-GAVal) were prepared as the non-deuterated analogues (AAVal and GAVal), but
with dg-L-valine as starting material. The syntheses were made on a smaller scale, with a
yield of 54% for dg-AAVal and 38% for dg-GAVal. The compounds were pure as judged
by TLC. The mass spectra after derivatization with methanol/HCl and HFBA were in
analogy with the non-deuterated compounds.
NMR spectroscopy and fast atom bombardment mass spectrometry (FAB-MS)
Proton NMR spectra were recorded as described in Calleman et al. (1990) and FAB mass
spectra were acquired as described in Aim 1.
Analysis of Hb adducts in globin hydrolysates
Globin was isolated from whole blood as described previously. Samples of 100 mg of
globin were dissolved in 6 M HC1 to a concentration of 10 mg/ml. The deuterated
reference compounds (dg-AAVal, dg-GAVal, d2-AACys and d2-GACys), serving as internal
standards for the quantitation, were added. The concentrations of standards were 1 /xmol/g
Hb for the in virro-treated Hb and 100 nmol/g Hb for human samples. The samples were
hydrolyzed for 15 h at 120°C under vacuum. The hydrolysates were evaporated to dryness,
dissolved in 2 ml H20 and incubated for 1 hr at 37°C. The pH was adjusted to 9 with
NaOH and the samples were applied to Dowex 1x8 anion exchange columns (1 x 30 cm,
HCOO" -form, 100-200 mesh). The columns were eluted with 150 ml 12 mM formic acid
followed by 60 ml 30 mM formic acid, 65 ml 60 mM formic acid and 100 ml 120 mM
formic acid. The adduct-containing fractions (see Results) were evaporated and derivatized
with methanol/HCl and HFBA. Derivatized samples were dissolved in 100 /xl ethyl acetate
from which 2 /*! were injected for quantitation by GC-MS.
Analysis of hemoglobin adducts with the modified Edman degradation method
Samples of 50 mg of globin were derivatized according to Tornqvist et al. (1988). The
amount of PFPITC reagent was increased from 7 to 10 /*!. Globift alkylated with
d4-ethylene oxide was used as an internal standard, and was added to give a concentration
of 10 nmol/g Hb of N-(d4-2-hydroxyethyl)valine (d2-HOEtVal). The derivatized samples
were dissolved in 50 /*! toluene and 1 /*! was injected for quantitative analysis by GC-MS.
Gas chromatography-mass spectrometry analysis
The analyses were carried out using a Varian 3400 gas chromatograph linked to a Finnigan
4500 mass spectrometer. The operating conditions for the gas chromatograph were as
follows: helium carrier gas at constant pressure of 10 psi; temperature programming 100°C
- 1 min - 20°/min - 240° - 10°/min - 320° - 2 min for PFPTH-derivatives. A 30 m DB-5
(0.32 mm i.d., 1 fim phase thickness) fused silica capillary column Was used.
The mass spectrometer was operated in the electron impact mode at an ion source
-------
temperature of 140°C and an ionization energy of 70 eV. In the chemical ionization mode,
methane was used as the reagent gas at an ion source pressure of 0.40 Torr.
The HFB-derivatives of the valine adducts were analyzed in the positive ion chemical
ionization mode. The derivative of CEVal and its deuterated analogue were monitored at
m/z = 382 and 388 [M+l]+, respectively, and the derivative of CHEVal and its deuterated
analogue were monitored at m/z 622 and 628, [M+29]*, respectively. The retention times
for the derivatives of CEVal and CHEVal were 575 and 580 seconds, respectively.
Quantitation was based on the ratio between peak areas of the analyte and the deuterated
internal standard. The contribution of the deuterated internal standards of the valine
adducts to the ions monitored for the non-deuterated compounds was 0.1%. The
HFB-derivatives of cysteine adducts were analyzed in the electron impact ionization mode,
as described before.
The PFPTH-derivatives were analyzed in the negative ion chemical ionization mode.
AAVal-PFPTH was monitored at m/z = 375 [M-HFJ and d^HOEtVal-PFPTH at m/z =
352 [M-HF]\ In addition to the valine adduct of AA, N-(2-cyanoethyl)valine-PFPTH
(ANVal-PFPTH, Fig. 2 structure F), the valine adduct of AN, was monitored at m/z =
274 [M-103]'. The retention times for the PFPTH-derivatives of AAVal, ANVal and
d4-HOEtVal were 826, 701 and 692 seconds, respectively. The quantitation was based on
the ratio between peak areas of the analytes and d4-HOEtVal.
Alkylation of hemoglobin in vitro
The rates of reaction of AA or GA with N-terminal valine in human Hb were determined in
blood from a non-exposed person, as described earlier. The incubation was made with AA
(380 mM) or GA (100 mM) and aliquots were taken at different times (0-40 min.). The
adduct levels were determined in hydrolyzed globin samples as described above.
Calibration
The analyses of PFPTH-derivatives were calibrated as follows: A calibration curve was
prepared by addition of different amounts of in vitro alkylated (with AA or AN) globins, to
50 mg globin samples from a non-exposed person. The alkylation levels were 5-80
nmol/g Hb. d4-Ethylene oxide alkylated globin was added to give 10 nmol d4-HOEtVal/g
Hb. The calibration curve for AA was slightly concave and for AN linear in the range
studied. Calibration samples were run every day when unknown samples were analyzed in
order to compensate for variations in sensitivity of the mass spectrometer.
The adduct levels of the in vitro alkylated globins were determined after acid hydrolysis and
ion-exchange chromatography by the procedure described above. For the AA-globin,
dg-AAVal was added as internal standard. For the AN-globin, which was alkylated with
I4C-AN (168 Bq/nmol), the radioactivity in the peak corresponding to CEVal was counted
following ion-exchange chromatography.
10
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Field study in the People's Republic of China
Physical description of the factory
f
The acrylamide shop selected for this study was part of a large chemical factory employing
700 individuals in the city of Xinxiang, Hunan Province, China. The production of
acrylamide, which had started in 1982, was operated in three shifts and amounted to 1,000
tons/year. The acrylamide production line operated as follows: acrylonitrile (bought from
an external supplier) was converted to acrylamide (20% solution) by catalytic hydration
with metal catalysts. The latter solution was then concentrated to give a 35% acrylamide
solution, which was purified by cation and anion exchange. This solution is stored in large
vessels and then transferred to smaller barrels: all these processes are performed in the
same room, the "synthesis room." Barrels are then moved to an adjacent room, the
"polymerization room," where acrylamide is mixed with acrylic acid, starch (35% of final
product) and sodium allylsulfonate to generate a polyacrylamide co-polymer. After
grinding and drying, the co-polymer is then bagged in another adjacent building, "the
packaging unit." The use of the copolymer is primarily in the oil industry to fill the
drilling tube and decrease friction and to harden soil and allow extraction of compact
samples.
Air monitoring
The air concentrations of acrylamide were monitored twice during the summer months and
twice in the month of September of 1991 when the study was undertaken. Each time
several determinations were made. Acrylamide was sampled for 10 minutes (flow rate 3
1/min) by station sampling and determined by gas chromatography with a FFAP column and
an electron capture detector following bromination. The detection limit of this method is
0.5 ng of acrylamide, equivalent to an air concentration of 0.03 mg/m3 under the testing
and sampling conditions used. The results of these analyses are shown in Table la. In the
summer the air concentrations ranged from 0.3-8.8 mg/m3 with an extreme value of 153.1
during the discharge of acrylamide (not included in Table la), whereas during the month of
September when specimens for the determination of biomarkers were collected, a relatively
narrow range of air concentrations of 0.11-1.64 was determined. Air levels of acrylonitrile
were also monitored in June 1991 using a charcoal sampler and gas chromatography
analysis (Table Ib).
Description of study populations
A total of 41 workers, 34 males and 7 females, occupationally exposed to acrylamide while
working in the processes of synthetization, condensation, filling, polymerization, and
maintenance, etc. were selected for the study. Depending on the primary location where
11
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Table 1
la. Air concentrations of acrylamide (mean ± SD, mg/m3).
Summer
Autumn
Average
Polymerization room
5.95 ± 2.55 (n = 6)
0.58 ± 0.21 (n = 6)
3.27 ± 3.29 (n = 12)
Synthesis
1.65±0.89(n = 8)
0.61 ± 0.55 (n= 10)
1.07±0.88(n=18)
Ib. Air concentrations of acrylonitrile (mean + SD, mg/m3)
Summer
Polymerization room
8.4±3.7(n = 6)
Synthesis room
44.7±37.9(ir=6)
12
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these workers performed thek work, they were classified into synthesis workers,
polymerization workers, packaging workers and ambulatory workers which included
foremen, repairmen, etc. Two individuals who had only minimal exposure to acrylamide
during the preceding 4- month period and two others who had been employed in the
workshop for less than half a year, and thus were not expected to have had time to develop
neurotoxic symptoms, were excluded from the regression analysis. The ages of the workers
ranged between 18 and 42 years (mean 27 years). The exposure duration was 3 years on
average with the shortest being one month and the longest 9 years. Ten workers had been
exposed to acrylamide for more than 5 years. Thek personal protection measures included
gauze masks, cotton clothes, rubber gloves and boots.
The workers were brought into the hospital ward approximately 1 hr after they had started
the morning shift. Blood was collected immediately upon arrival and the workers remained
in the ward for medical examination and collection of urine for a 24-hr period.
For the Vibratron measurements the reference group consisted of 105 unexposed and
healthy adults (51 males and 54 females ages 20-60 years) including mechanical workers
and office personnel and for the ENMG a historical control group of 80 persons was used.
Persons with diabetes and nervous diseases were excluded from both groups. For the
biomarkers of exposure and signs and symptoms of neurotoxicity, ten unexposed individuals
from the same city as the exposed group were studied.
Determination of mercapturic acids in urine
Urine samples were collected during 24 hrs and the volumes and specific gravities were
determined. 1 ml of these samples was hydrolyzed with 1 ml of 6N HC1 at 85°C for 4 hr.
Under these conditions the N-acetyl group of the mercapturic acid as well as the amide
group was hydrolyzed to give S-(2-carboxyethyl)cysteine. After centrifugation at 3,000g
for 10 min, 0.1 ml of supernatant hydrolysate solution was evaporated to dryness under N2
at 65°C. The residue was dissolved in 1 ml of water. 200-/*1 aliquots of the hydrolysate
were mixed with 100 j*l of the fluoraldehyde reagent. After 1 min, 10 fd of sample was
subjected to HPLC. Urine samples were acidified with 1% HC1 and frozen, and kept at
-20°.
Solvent A was tetrahydrofuran-methanol-0.1 N sodium acetate (5:95:900, pH 7.2). Solvent
B was methanol-water (9:1). The column was kept at ambient temperature, and 10 (A of
standard or sample were injected into the column. Elution was achieved by running a
gradient of 10% solution B over 8 min at a flow rate of 1.5 ml/min, then flashing the
column with 90% solvent B at the end of each injection to eliminate the less polar
compounds in urine.
Blood samples for hemoglobin adducts
The blood was drawn in vacutainer tubes containing EDTA as anticoagulant. Workers
13
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were involved in the production of AA (synthesized by catalytic hydration of AN) and
polyacrylamide, and had been exposed to AA and AN for 0.1 to 8 years. Blood samples
were also collected from 10 control workers in the same city, who had not been exposed to
AA or AN.
Measurements of vibration thresholds
A Vibratron n vibration sensitivity tester was used for the quantitative measurements of
vibration threshold of bilateral index fingers and big toes of each subject in both groups at a
room temperature of 22-25°C. The instrument consisted of two modules with vibrating
posts and a controller. The testing procedure was a two-alternative forced choice
procedure. While the position of vibration and intensity sequence were under control, the
subjects were asked to press their fingers or toes against each rod and to determine which
of the two rods was actually vibrating. The vibration settings of the five errors and the five
lowest correct scores were then determined. The highest and lowest values of the ten
scores were eliminated and the mean of the remaining eight scores determined the vibration
threshold (1 vibration unit (VU) = 0.5 microns peak-to-peak amplitude).
Electroneuromyography (ENMG)
The ENMG was carried out with a Dantec 2000C electromyographic apparatus. The
electric activities of the abductor pollicis brevis, abductor digit! minimi and anterior tibialis
muscles, including insertion activity, spontaneous activity of motor units, and electrical
activity during mild and maximal voluntary contraction, were recorded with concentric
needle electrodes. The mean duration and mean voltage of 20 motor unit potentials and the
percentage of polyphasic potentials in each muscle were determined. The duration of a
motor unit was taken as the time for the oscilloscope tracing to return to the base line after
its original deflection from the beginning of that potential. The voltage of a motor unit was
taken as the peak-to-peak amplitude. The phase of a motor unit potential was defined by
the number of deflections across the base line.
The minimum acceptable skin temperature of the hands and feet during the measurements
of nerve conduction velocity and distal latency was 30°C. Bipolar skin stimulation
electrodes were used in all the measurements of nerve conduction velocity. The voltage
was always 30% supramaximal. For measuring maximal motor nerve conduction velocity
(MCV), the median and ulnar nerves were stimulated at the elbow and the wrist, and the
muscle action potential was recorded from the abductor pollicis brevis muscle and the
abductor digiti minimi muscle with concentric needle electrodes. The peroneal nerve was
stimulated at the knee and ankle, and the muscle action potential was recorded from the
extensor digitorum brevis muscle by surface electrodes.
The sensory nerve conduction velocity (SCV) of the median and ulnar nerve was measured
through the stimulation of the forefinger and the little finger, respectively, with ring
electrodes. The sensory nerve conduction velocity of the sural nerve was recorded with a
14
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surface electrode at midcalf, 14 cm from the stimulating electrode placed just below the
lateral malleolus. The peak-to-peak amplitude of the evoked potentials was also measured.
The normal values of all related ENMG parameters were previously established in our
laboratory using the same EMG apparatus and the same technique in 80 unexposed healthy
adults.
Clinical and laboratory examinations
Physical and neurological examinations were conducted in both groups according to a
protocol previously described (He et al. 1989). The laboratory studies included routine
blood and urine tests, liver function (serum glutamate pyruvate transaminase and the thymol
turbidity test) and serum hepatitis B surface antigen.
Questionnaire information
All subjects were interviewed with the aid of a structured questionnaire collecting
information on demographic factors, smoking and drinking habits, height and weight,
occupational history, past illnesses, reproductive history and family history.
Definition of Neurotoxicity Index
A neurotoxicity index (NIn) with a maximal score of 50 points was defined so as to provide
a quantitative parameter expressing the peripheral neuropathy which could be correlated to
the different biomarkers of exposure. The different observations scored in the
Neurotoxicity Index are shown in Table 2. Clarifications for this Table include the
following: (1) workers who had lost their pain or touch sensation got 1 to 3 points
depending on the extent of this loss in fingers, hands, or forearms, (2) up to 2 points each
for increased vibration thresholds in hands or feet as measured with the Vibratron
instrument were given. Vibratron thresholds were compared to the corresponding control
group with regard to age. If their ratios to the corresponding controls were 0-4 for toes or
0-2.5 for index fingers, a score of 1 point was given for each, whereas if the ratios were
4-8 or 2.5-5 for toes or fingers, respectively, 2 points were given for each, (3) for each
observation of an ENMG abnormality 0.5 points were given, with a maximal possible total
of 12 x 0.5 = 6 points.
Statistical methods
The chi-square test was used to analyze the symptoms and signs, and the Student's t-test
was used in the analysis of ENMG parameters. The variance analysis and Q-test
(Neuman-Keuls methods) were used in the comparison of vibration thresholds between the
reference group and the acrylamide group, both with different age categories. Univariate
and multivariate regression analysis was used to estimate correlation coefficients and levels
of statistical significance for the relationship between the Neurotoxicity Index and the
15
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TABLE 2
Definition of Neurotoxicity Index
Points
Numbness of extremities 1
Cramping pain 1
Loss of position sensation 2
Loss of pain sensation 0-3
Loss of touch sensation 0-3
Loss of vibration sensation:
According to tuning fork 1
Vibration threshold in big toe 0-2
Vibration threshold in index finger 0-2
Clumsiness of hand 4
Difficulty grasping 4
Unsteady gait 4
Muscular atrophy 6
Decrease or loss of ankle reflexes 3 or 5
EMG 6
ENMG 0.5 per abnormality (max 6)
Maximum £ 50
16
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biomarkers and ANOVA analysis was used for comparing the means of the different
categories of workers.
AIM 3
Animals and treatments
Male Sprague-Dawley rats weighing 270-3 lOg were divided into five groups and allowed
free access to food and water. Rats were injected ip with AA or GA once a day for 8
days. The doses were 25 and 50 mg/kg for AA and 50 and 100 mg/kg for GA. Control
rats were injected with the vehicle, distilled water (1 ml/kg). All animals were tested and
sacrificed on day 9, 24 hr after the last injection. For the reproductive toxicity
experiments, sexually mature Sprague-Dawley rats (350g) were utilized. Animals were
administered either AA (50 mg/kg/day for 7 days) or GA (50 mg/kg/day for 14 days).
Neurotoxicity testing
Rotarod. The rotarod apparatus consisted of a horizontal plastic rod (6 cm in diameter)
positioned 30 cm above a switch floor. The rod was divided by partitions so that 4 rats
could be tested simultaneously. All rats were trained to stay on the rod (rotating at 20 rpm)
twice daily for 5 consecutive days prior to the beginning of treatment. The cut off-time
was 100 sec. The test was repeated 3 times at 20-min intervals and the average value of
the measurements (in seconds) was utilized.
Hindlimb Splay. This method was similar to that described by Edwards and Parker (1977).
Each rat was held in a horizontal position with the dorsal side up, 32 cm above a flat
surface which was covered with a thin layer of sawdust. The rat was then dropped and the
position of the fourth digit of each hindlimb on landing was marked and the distance
between the toe(s) marks was measured. Values represent the average of 3 consecutive
measurements.
Reproductive toxicity testing
Twenty-four hours after the last injection, animals were sacrificed by cervical dislocation
under ether anesthesia and testis, epididymis and vas deferens were dissected and weighed.
The head, body and tail of epididymis were minced and homogenized and the number of
spermatozoa was determined. Sperm viability was measured as described by Bishop and
Smiles (1957).
17
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RESULTS AND DISCUSSION
AIM1
Development of a GC/MS method for measuring Hb adducts of AA and GA
The reactions of AA and GA with cysteine residues of hemoglobin are shown in Fig. 1.
Globin isolated from rat blood was subjected to acid hydrolysis and the adducts were
released as S-(2-carboxyethyl)-cysteine and S-(2-carboxy-2-hydroxyethyl)-cysteine. The use
of anion-exchange chromatography for purifying these particular adducts from the neutral
and basic amino acids takes advantage of their extra carboxylic acid function. The
hydrolysate was applied to the column and the most negatively charged amino acids were
most strongly attached. With weak formic acid (5 mM) all amino acids with only one
carboxylic acid group were eluted with the front at 5-20 ml, while 100 mM formic acid
eluted the more strongly bound dicarboxylic acids. Since, however, both glutamic acid and
aspartic acid are dicarboxylic acids, the 100 mM eluate contained, in addition to the two
adducts, part of the glutamic acid content and all of the aspartic acid. The elution volumes
were 70-90 ml for glutamic acid, 90 ml for AACys, 95-110 ml for aspartic acid, and
90-110 ml for GACys. After evaporation, the 100 mM eluates were derivatized for
quantitation with GC/MS. Multiple ion monitoring in the electron impact ionization mode
was chosen because with the instrument used it provided a higher sensitivity than the
negative ion chemical ionization mode. The fragments monitored for the internal standard
had one deuterium instead of the two deuteriums in the parent molecule due to loss of 1
deuterium atom in the fragmentation process (Calleman et al. 1990). The difference in m/z
of only 1 between the analyte and the internal standard is not ideal since the contribution
from the deuterated ion to the nondeuterated was 1% and the contribution from the
nondeuterated ion to the deuterated was 10%. However, this was corrected for by use of
the calibration curve.
The use of free deuterated amino acids as internal standards was based on the assumption
that there is no preferential decomposition during acid hydrolysis of an amino acid
depending on whether it is free in solution or exists as a peptide residue. Studies by
Crestfield et al. (1963) on the close structural analogue S-carboxymethyl-cysteine in
ribonuclease showed that 97, 103, 103, and 101% of this amino acid were recovered after
24, 48, 73, and 102 hrs of hydrolysis in 6 M HC1, respectively. The fact that the
concentration of S-carboxymethyl-cysteine did not decrease after 24 hr when it existed free
in solution indicates that this amino acid is equally stable as a free amino acid and as a
peptide residue.
The synthetic standards of AACys and GACys for preparation of the calibration curve were
quantified by amino acid analysis employing the response factor of leucine. The
compounds contained no impurities. The detection limit for the GC/MS was 1.7 pmol of
injected derivatized standard compound. The calibration curve on GC/MS was linear
between the ratios of 0.1 and 1.0 with R2 = 0.999 for both derivatized AACys and GACys.
18
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Analyses in triplicate gave an average coefficient of variation (CV) of 6% for AACys and
8% for GACys. Despite the fact that deuterated internal standards were used, the GC/MS
system exhibited a certain drift in the ratios determined between different occasions on
which it was used. Thus, while one globin sample was processed and analyzed several
times, the CVs for AACys (n=4) and GACys (n=5) were 2 and 4%, respectively, on a
single occasion; the CV for repeated (n=5) injections of an identical sample on a different
occasion was 2% for AACys and 13% for GACys. The CVs within the dose groups of rats
were on average 13 and 15% for AACys and GACys, respectively. Background levels
found in the globin from control rats were 0.15 (0.14-0.17) /xmol/g Hb and 0.015
(0.010-0.019) /xmol/g Hb for the AA and GA adducts, respectively. Administration of 1
mg/kg body wt acrylamide by ip injection to rats resulted in a statistically significant
increase in adduct levels (p<0.01).
Acrylamide and glycidamide hemoglobin adducts following administration to rats
In a first experiment, rats were injected with different doses of AA and the adduct levels
(AACys) in the blood were measured 24 hr after injection (Fig. 3). At this time all AA
presumably had reacted since no parent compound was detectable by Miller et al. (1982) in
any tissue after 1 day. The AA adduct level increased approximately linearly with the
administered amount. The levels of the GA adduct (GACys), on the other hand, produced
a concave curve (Fig. 3b). A typical mass fragmentogram from a rat treated with 10
mg/kg body wt of AA is shown in Fig. 4.
In a second experiment rats were administered increasing doses of GA. In this case the
GACys adducts increased linearly with the administered amount (Fig. 5). On a mol/kg
body wt basis, levels of GACys adducts were 3.2 times lower than the AACys levels
measured following AA administration.
From these experiments, the percentage of the initial concentration of AA metabolized to
GA was calculated (Fig 6). For this purpose, the concentration of GA (in mmol/kg)
corresponding to the GACys adduct level in the AA-injected rats was calculated as if GA
had been injected directly. This concentration was divided by the initial concentration of
injected AA. At the lowest concentration of AA (5 mg/kg body wt) about 51% was
converted to GA, while at the highest concentration (100 mg/kg body wt) only about 13%
was metabolized to GA.
In an additional experiment, a cumulative dose of 100 mg/kg body wt AA was given as a
single dose, distributed over 10 consecutive days as 10 mg/kg/day, or distributed over 30
days as 3.3 mg/kg/day. Twenty-four hours after the final injection, blood was drawn and
the adduct levels of both AACys and GACys were measured. Table 3 shows the measured
adduct levels and, for comparison, the expected levels calculated according to Eq. (1):
19
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z
o>
o
3.
0»
t>
0
•o
<
10-
8-
6-
4-
2-
0 J
20 40 60 80
Acrylamide (mg/kg bw)
20 40 60 80
Acrylamide (mg/kg bw)
100
Figure 3. Hb cysteine adduct levels of AA and GA in rats injected with different single
doses of AA.(n) Cysteine adduct of AA; (*) cysteine adduct of GA. Symbols represent
means +SD of three to five animals, (a) Full scale, (b) Enhanced detail of (a).
20
-------
202 .
283 .
285 .
785
GACys 0.12 nmol/g Hb
738
•A
internal standard for GACys
=F=
716
7J9
AACys 0.79 nmol/g Hb
729
718
internal standard for AACys
729
725
738
735
746
19520.
977928.
376328.
2453588.
745 S
Figure 4 . A typical mass fragmentogram from a rat treated with 10 mg/kg body wt AA.
The y-axis shows the intensities of the ions monitored, m/z = 202, 203, 204, 205, for the
derivatized nondeuterated and deuterated forms of S-(2-carobxy-2-hydroxyethyl)-cysteine
and S-(2-carboxyethyl)-cysteine. The x-axis shows retention time in seconds.
21
-------
o
3L
«>
O
•o
•o
2.5-
«
2.0-
1.5-
1.0-
0.5
0.0
20 40 60 80 100
Glycidamide (mg/kg bw)
Figure 5. Hb cysteine adduet levels of GA in rats injected with different single doses of
GA. Symbols represent means +, SD of three animals.
22
-------
o
o
o>
>
c
o
100-
90-
80-
70-
60-
50-
40-
30-
20-
10-
0
20 40 60 80 100
Acrylamide (mg/kg bw)
Figure 6. Conversion of AA to GA in vivo in rats injected with AA. The percentage is
calculated in relation to the initial concentration of AA in the body. The fraction converted
to GA is estimated from measured adduct levels of GA.
23
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TABLE 3
Hemoglobin adducts in rats treated with different dose regimens of acrylamide
Measured adduct level
Dose rate Expected adduct level0
(mg/kg body wt) (|imol/gHb) (SD, n) (jimol/gHb)
1 Day x 100
10 Days x 10
30 days x 3.3
AACys* 8.62
GACysc 0.364
AACys 5.72
GACys 1.00
AACys 3.58
GACys 0.749
(0.66,5)
(0.049, 5)
(0.99,4)
(0.17,4)
(0.45,4)
(0.062, 4)
AACys 8.62
GACys 0.364
AACys 5.60
GACys 0.96
AACys 4.20
GACys 0.79
a The expected values were estimated from the data shown in Fig. 3 and were corrected
for the replacement of old erythrocytes and the increase in blood volume due to the
growth of the animal (Eq. (1)). The values of a in Eq (1) were 3.3 mg/kg/day, 0.23 and
0.044 iimol/g Hb for AA and GA, respectively, and, at 10 mg/kg/day, 0.645 and 0.1095
jimol/g Hb for AA and GA, respectively. The body weights increased from 132 to 380 g
during the 30-day treatment and from 264 to 296 g during the 10-day treatment. The
measured adduct levels were not significantly different (p > 0.05) from the expected
adduct levels.
b AACys = The Hb cysteine adduct of acrylamide.
c GACys = The Hb cysteine adduct of glycidamide.
24
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= E o(l - t) x ft, (1)
t=l I.
where {[RYMY]},^ is the accumulated level of adducts at the end of exposure, a is the
daily increment of adducts based on the measured levels in the first experiment
(dose-response of AA), t,, is the duration of the experiment in days, and t^is the lifetime of
the erythrocytes estimated to 61 days in the Sprague-Dawley rat (Derelanko, 1987). The
factor b in the equation (body wt at time t^+i/body wt at tj is used for correcting for the
increase in total blood volume due to the growth of the animal which will cause a dilution
of the adducts. This equation for the accumulation of Hb adducts during chronic exposure
is based on the assumption that the Hb adducts are eliminated at the same rate as the
erythrocytes. The measured adduct levels were not significantly different than the expected
adduct levels.
Levels of AA adducts decreased as the dose rate decreased mainly because of the
elimination of old erythrocytes and dilution of adducts due to body growth, but also because
of a larger fraction of AA metabolized to GA. In fact, levels of GA adducts increased
when the same cumulative dose of AA (100 mg/kg) was given as ten 10-mg/kg doses over
a 10-day period, suggesting higher conversion of AA to GA. When the same amount of
AA (100 mg/kg) was given over 30 days, however, although levels of GA adducts were
higher than after the acute 100 mg/kg dosage, they were not as high as at the 10 x 10-
mg/kg dosage regimen because of elimination of old erythrocytes and body growth.
The constants k^, and kt, as well as t1/2 are presented in Table 4. The second-order rate
constants, k^,, for the reaction between cysteine residues in hemoglobin and AA or GA
were determined in vitro. These rate constants were calculated from k^. = [RY]/[Y]/D,
where [RY]/[Y] is the degree of alkylation measured in the experiment, D is the dose
which may be calculated from D = [RX] x t, and [RX] is the concentration of AA or GA
in the incubation mixture (Segerback, 1990).
The first-order rate constants for the elimination of AA and GA in the blood compartment,
kj,, were estimated from adduct levels of AA or GA, when AA or GA was injected
directly, using Eq. (2):
'
where [RX]0 is the initial concentration of the injected compound. Since AA distributes
rapidly and evenly in the body to all tissues (Miller et al. 1982), its initial concentration
may be estimated on the basis of 1 kg body wt = 1 liter; the same assumption is also made
for GA since it is a highly water-soluble low-molecular-weight molecule. The k^,
25
-------
TABLE 4
Pharmacokinetic parameters of acrylamide and glycidamide in the rat
Compound
Acrylamide
Glycidamide
a Second-order
kcy$fl
(liter/g Hb x hr)
1.8x10-3
0.92 x 10-3
rate constant for the reaction
ke.*
(hr')
0.37
0.48
with cysteine in rat Hb in
tl/2c
(hr)
1.9
1.5
vitro.
b Estimated first-order rate of elimination in the blood (Eq. (2)).
c Half-life in vivo in the blood. Calculated from i\n = In2
26
-------
determined in vitro are used in Eq. (2) since the rates of reaction of AA or GA with
Hb-cysteine in vivo are assumed to be the same as in vitro. The ratio [RY]/[Y] was taken
from in vivo data assuming complete reaction of the parent compound within 24 hrs. The
half-life is calculated from the relationship t1/2 = In 2/lg.
General discussion of Aim 1
The spectrum of different amino acids in Hb which react with AA and GA is currently not
known. There are several different nucleophilic amino acids such as histidine, cysteine,
and N-terminal valine in hemoglobin (EPA, 1990) which may give rise to adducts useful
for monitoring exposure to these electrophiles. The unsaturated bond of AA is, however,
known to have high reactivity toward -SH groups in Michael type additions, which made
cysteine adducts a natural choice for the development of our quantitative technique along
the lines of the work of Bailey et al. (1986). Although the epoxide GA is expected to react
significantly also with nitrogen- and oxygen-containing nucleophilic amino acids, the
simultaneous determination of adducts formed by both agents provided an analytical
procedure with the appeal of simplicity. Thus, a method was developed to determine the
cysteine adducts formed by the two electrophilic agents.
The adduct levels of AA in our study are in good agreement with those determined by
Bailey et al. (1986) in the concentration range 0-5mg/kg. The background level of AACys
in that study, however, was 7 times lower than the background levels determined in our
study (0.15 /xmol/g Hb), which might be explained either by the use of different strains of
rats (Wistar vs Sprague-Dawley) or by methodological differences, such as the use of
different internal standards for quantitation.
The pronounced concavity of the GA adduct curve in rats injected with AA indicates that
this metabolite is formed by a saturable process following Michaelis-Menten kinetics. It
should be noted that because of our interest in providing 'data useful for relating hemoglobin
adduct levels to neurotoxic effects, the dose range of injected AA and GA is rather high,
compared to most previous studies of Hb adducts with other compounds. Thus,
nonlinearities in the curve shapes, rarely observed at low doses, become evident.
As discussed previously (Calleman et al., 1990), the most likely candidate for the enzyme
system responsible for the metabolic conversion of a simple alkene such as AA to GA is
cytochrome P-450, although this hypothesis has not yet been tested. The saturable kinetics
of this process may provide a possible explanation for the fact that AA has consistently
given a negative response in the Ames/Salmonella test also in the presence of S-9 mix.
Indeed, the case of AA could be similar to that of ethylene and propylene which are
negative in the Ames test while their active metabolites, ethylene oxide and propylene
oxide, respectively, are clearly mutagenic. It has been proposed that the negative responses
of the two parent alkenes are due to the fact that their saturable metabolism may limit the
maximal dose of epoxide generated in the test system to a level below what is required for
a statistically significant response (Osterman-Golkar and Ehrenberg, 1982).
27
-------
The experiments performed show that direct administration of GA results in the formation
in vivo of the same adduct, S-(2-carboxy-2-hydroxyethyl)-cysteine, as that previously
reported as evidence for the formation of this epoxide in AA-treated animals (Calleman et
al. 1990). Adduct formation in GA-treated animals appears to be a linear function of the
injected dose.
The origin of the background levels of adducts is not known. It is unclear whether they are
produced as methodological artifacts or if they reflect true adduct levels. Since the adducts
measured do not contain the amide group originally present, the adducts are not entirely
specific to AA (or GA) exposure. For example, exposure to acrylonitrile has been shown
to give S-(2-carboxyethyl)-cysteine in hydrolyzed Hb (Fennell et al., 1989). Intermediates
in glycolysis, such as 3-phosphoenolpyruvate, 3-phosphoglycerate, and 1,3-diphospho-
glycerate could theoretically give rise to S-(2-carboxy-2-hydroxyethyl)-cysteine following
protein hydrolysis. S-(2-carboxy-2-hydroxyethyl)-cysteine (Ohmori et al. 1965) and
S-(2-hydroxy-2-carboxyethyl)-cysteine (Yao et al. 1970) have been found in the urine of
humans not known to be exposed to electrophilic substances. This indicates that the
formation of the background levels of these specific adducts may be a result of endogenous
processes. Background levels in humans and rodents of several other low-molecular-weight
adducts have been found.
The hemoglobin binding index for AA to cysteine (6400 pmol (g HbWmol (kg body wt/1,
is higher than for any other compound studied in the rat and is higher than that of GA
(1820 pmol (g Hb/V^mol (kg body wt)'1), calculated at 50 mg/kg body wt of AA and GA,
respectively. This difference is primarily explained by a lower reactivity of GA toward
Hb-cysteine and a shorter half-life (t,/^ in blood, compared to that of AA, thus giving a
lower yield of adducts. The half-life for AA (1.9 hr) and GA (1.5 hr) reflects the sum of
all reactions leading to the elimination of the compounds from the blood and is estimated on
the assumption of a linear rate of elimination (Eq. (2)) in the concentration range studied.
The half-life of AA is given as an average over the dose range 0.5-100 mg/kg body wt,
although the elimination rate is expected to decrease with increasing administered doses of
AA due to the saturable formation of GA. The half-life of AA in blood determined by
Miller et al. (1982) and by Edwards (1975) was 1.7 and 1.9 hr, in good agreement with our
data. The second-order rate constant for the reaction of AA with Hb was determined by
Hashimoto and Aldridge (1970) to be 17 x Ifr31 x (g Hb x hr)'1 [as calculated from a
reported value of 18 1 x (mol x min)'1], as compared to 1.8 x 10'31 x (g Hb x hr)'1 in our
experiments. The experimental conditions, however, were not identical in the two studies.
In particular, the pH used by these authors was 8.0, higher than that used in our
experiments (7.4); this is expected to increase the rate of reaction with cysteine residues
because of the increased thiolate ion concentration at higher pH.
Possibly the most notable result of the present study is the high extent of metabolic
conversion in vivo of AA to GA, reaching more than 50% at the lowest dose studied. As
shown in Fig. 6 there is a higher percentage of AA converted to GA at lower doses of AA.
Consequently, if a toxic effect induced by AA is due to GA, the relative importance of the
28
-------
risk for this toxic effect will increase at a lower AA exposure. For risk estimation, it will
be, therefore, necessary to investigate which of the toxic effects induced by AA might be
related to the parent compound, to the metabolite, or to a combination of the two. It is
noteworthy that an injection of 50 or 100 mg/kg body wt AA will give the same tissue dose
of GA as an injection of 10 mg/kg body wt GA. Thus, for a toxic effect which is
exclusively caused by GA, we would expect the same effect if we inject 50-100 mg/kg body
wt AA or 10 mg/kg body wt GA. This is based on the assumption that the tissue dose is
directly related to the toxic effect in a dose-response relationship.
When AA was given in fractionated doses (i.e., in the chronic exposure regimen), a higher
proportion of the cumulative tissue dose consisted of GA than after high-dose acute
exposure (Table 3). Consistent with the results from the acute exposure experiments, this
suggests that the risk for toxic effects caused by GA might be of greater importance at
lower dose rates of AA in long-term exposure situations. In this experiment, the measured
adduct levels were in good agreement with those calculated using the adduct levels from
single injections of AA, suggesting that these particular adducts may be eliminated at the
same rate as the erythrocytes. They appear, therefore, to be stable and useful for
determination of tissue doses. In our calculation of expected values of Hb adduct levels,
we have corrected not only for the elimination of old erythrocytes, but also for the dilution
resulting from the increased body weight (Eq. (1)), and hence blood volume, during the
course of the experiment. The apparently nonlinear decrease of Hb adduct levels often
observed in rats following acute, single treatments of electrophilic agents (Neumann, 1981)
may thus be explained by an increased blood volume during the course of the experiment
rather than by a lack of stability of the adducts.
AIM2
Development of methods for acrylamide and glycidamide Hb adducts in humans
Methods were developed for the determination of AA and GA adducts to N-terminal valine,
both following total hydrolysis of the protein and by means of the modified Edman
degradation procedure (Fig. 2).
In the first method, globin isolated from human blood was subjected to acid hydrolysis. In
this procedure the amide groups originally present in AA and GA were converted to
carboxylic acid groups during acid hydrolysis and thus valine adducts were released as
N-(2-carboxyethyl) valine (CEVal) and N-(2-carboxy-2-hydroxyethyl) valine (CHEVal).
The hydrolysates were applied to anion exchange columns and eluted stepwise with
increasing concentrations of formic acid according to a modification of the method utilized
in rats. Most of the normal amino acids, including glutamic acid, were eluted with 150 ml
12 mM formic acid. CECys eluted in the 60 ml 30 mM fraction, aspartic acid eluted in the
65 ml 60 nM fraction. CEVal eluted partly in the 30 mM fraction and partly in the 60 nM
fraction, and these fractions were therefore pooled. The 100 ml 120 mM fraction contained
29
-------
CHECys and CHEVal. The adduct-containing fractions (30 mM + 60 mM and 120 mM)
were evaporated and derivatized with methanol/HCl and HFBA for GC-MS. The
second-order rate constants for the reactions of AA or GA with N-terminal valine, kvj, and
key,, in human Hb determined in vitro are presented in Table 5. In human Hb, AA is
about 3 times more reactive towards cysteine than versus N-terminal valine, whereas GA is
about equally reactive towards these two sites. The rate constant for the reaction of GA
with N-terminal valine in Hb is comparable for human and rat. In contrast, the reactivity
of cysteine in human Hb is 2 orders of magnitude lower than in rat Hb. This is explained
by the presence of a reactive cysteine in position 125 of the b-chain in rat Hb, which is
lacking in this position in human Hb (Hamboek et al., 1981).
Symptoms and signs in acrylamide exposed workers
The percentage of workers displaying different kinds of symptoms and signs are shown in
Table 6. Fatigue (71%), numbness in hands and feet (71%), sweating (68%) of hands and
feet and peeling of skin (59%) were the early and prominent symptoms in the acrylamide
group and were much more frequently seen than in the reference group. The peeling of the
skin in hands usually appeared within 2-30 days after topical contamination by aqueous
acrylamide. Unsteadiness of grasping and walking were present in 6 and 10 acrylamide
workers, respectively. Systemic symptoms such as dizziness, nausea, insomnia and
headache were also found but their prevalences were not significantly different from
controls.
On neurological examination, impairments of pain (54%), touch (46%) and vibration (41%)
sensation were prominent in the distal part of lower limbs and hands of the exposed
workers. Diminution or loss of ankle reflexes were found in 29% of the acrylamide group.
There were 6 and 8 acrylamide workers having difficulties in standing on one leg and
displaying a positive Rhomberg sign, respectively. None of these abnormalities were seen
in the reference group. No cerebellar involvement was found in either group.
The results of a survey on the reproductive history of each subject showed no difference in
fertility, abortion and birth defects in offspring between the acrylamide workers and
referents.
Vibration thresholds
The vibration thresholds of index fingers and big toes in the reference group showed no
difference between the two sides, nor between the two sexes (data not shown). However,
the difference in vibration thresholds was significant among the 3 age-subgroups of
referents, namely, < 31 years, 31-40 years, and > 41 years (p<0.05), showing an
elevation of vibration thresholds along with the increase of age in unexposed healthy adults.
A significant increase of vibration thresholds was found in acrylamide workers of all age
groups (Table 7).
30
-------
TABLE 5
Second-order rate constants for the reaction of acrylamide and glycidamide with amino
acid residues in hemoglobin [liter (g Hb)'1 hr1] x 106
Compound
Acrylamide
Glycidamide
Human
*Val *Cys
4.4 14
11 9.5
Rat
*Val
N.D."
12*
kCys<
1800
920
a Not determined
b GAVal was determined in globin from a rat treated with 100 mg/kg GA and Jh/al was
calculated from: Aval = (GAVal x JtCys)/GACys.
c Data are taken from Table 4.
31
-------
TABLE 6
Percentage of acrylamide-exposed workers and controls with symptoms of
adverse health effects
Numbness of extremities
Fatigue
Sweating of hands and feet
Skin peeling
Menstruation disorders
Loss of pain sensation
Dizziness
Anorexia
Loss of vibration sensation
Nausea
Loss of ankle reflexes
Insomnia
Headache
Unsteady Gait
Loss of knee jerk
Unsteady Rhomberg sign
Loss of triceps reflexes
Loss of biceps reflexes
Acrylamide-exposed
71
71
68
59
4/7
54
44
41
41
39
29
29
27
22
20
20
10
10
Controls
-
-
-
-
-
-
-
•
-
-
-
10
-
.
-
-
-
32
-------
TABLE 7
Comparison of vibration thresholds (VU ± SD) between acrylamide workers and referents
Acrylamide Workers Reference Group
Age (years)
n fingers toes n fingers toes
<31 32 2.33±1.05* 5.13 ±3.80* 42 1.20 ±0.29 2.40 ±0.85
31-40 9# 2.19 + 0.90* 4.94 ±2.74* 37 1.38 ±0.37 2.85 ±0.85
>41 0 - 26 1.49 + 0.36 3.53 ±0.97
* By variance analysis and Q test, p < 0.01.
# One worker aged 42 was included
33
-------
Electroneuromyographic measurements
The mean duration and amplitude of motor units and the percentage of polyphasic potentials
in muscles abductor pollicis brevis, abductor digiti minimi and anterior tibialis of the
acrylamide group were within normal range. No spontaneous denervation potentials were
found during mild contraction of sampled muscles. However, 20 out of 94 sampled
muscles showed mixed patterns of recruitment during maximal contraction; 9 samples were
of discrete pattern. Abnormal recruitment patterns were also seen in 75% of the 12
samples of anterior tibialis muscles of acrylamide workers.
In comparison with the reference values of ENMG parameters on a group basis, there was
a prolongation of distal motor latencies in median, ulnar and peroneal nerves as well as a
slowing of MCV of peroneal nerve in the acrylamide group. A prolongation of sensory
distal latencies and a decrease of sensory potentials were also found in the median, ulnar
and sural nerves, as well as a slowing of SCV of sural nerve in acrylamide workers (Table
8).
The neurotoxicity index as predictor of clinical diagnosis of peripheral neuropathy
The neurotoxicity index (Table 2) in the workers with a clinical diagnosis of peripheral
neuropathy and those without are shown in Figure 7. As evident from the plots there is
overlap only for two individuals between the neurotoxicity index and the clinical diagnosis,
which seems to indicate that the neurotoxicity index as defined is a useful quantitative
indicator of acrylamide-induced peripheral neuropathy. In the studied group no worker had
a Neurotoxicity Index exceeding 30 points out of the maximal value of 50.
Hb adduct levels in humans exposed to acrylamide
The Hb adduct levels determined in the workers in the factory in Xinxiang are presented in
Table 9. The AA and AN valine adducts were determined using the modified Edman
degradation procedure. The GA valine adducts were determined after total hydrolysis of
the globin. Since this method is very time-consuming, only 6 such samples were analyzed.
Typical mass fragmentograms from the analyses are shown in Fig. 8a and b. All exposed
workers had detectable Hb adduct levels of AA (0.3 - 34 nmol/g Hb) and AN (0.02 - 66
nmol/g Hb). GA adducts were detected in the samples from the 5 exposed persons (1.6 -
32 nmol/g Hb) but not in the control, and there was an increase in GA adducts with
increasing AA adduct level with a linear regression correlation coefficient of r = 0.96 (Fig.
9). In controls, AA adducts were detected in 1 out of 10 samples (0.01 nmol/g Hb), and
AN adducts were detected only in the smokers (in 4 out of 5 smokers, 0.03 - 0.14 nmol/g
Hb). Table 10 shows the average Hb adduct levels of A A in the workers according to their
location in the factory together with the geometric means of the AA air concentrations.
The Hb adducts of AN were also determined on coded samples in another laboratory (T.
Fennell, C.I.I.T.). All results are shown in Table 11. In this parallel analysis ANVal
adducts were detected also in controls, albeit at low levels.
34
-------
TABLE 8
Comparison of Motor (MCV) and Sensory (SCV) Nerve Conduction between the
Acrylamide and Reference groups (mean ± SD).
ENMG Parameters Acrylamide group Reference group
(N= 41) (N= 80)
MCV (m/sec)
Median nerve 56.8 + 7.2 59.6 + 5.0
Ulnar nerve 60.0 + 9.2 62.5 + 5.4
Peroneal nerve 43.8 + 8.5* 48.1 + 3.5
Motor Distal latency (ms)
Median nerve 4.6+1.3* 3.5 + 0.4
Ulnar nerve 3.1 + 1.1* 2.3 + 0.3
Peroneal nerve 6.0 + 1.9* 3.9 + 0.5
SCV (m/sec)
Median nerve 61.6 + 5.8 60.3 + 4.1
Ulnar nerve 61.9 + 6.4 66.9 + 4.3
Sural nerve 46.6 + 6.6* 56.8 + 4.0
Sensory Distal Latency (ms)
Median nerve 2.8 + 0.4* 1.8 + 0.3
Ulnar nerve 2.5 + 0.8* 1.6 + 0.2
Sural nerve 3.0 + 0.8* 2.4 + 0.2
*p <0.02 by Student's t-test
35
-------
35 -r
30 4-
25 +
20 4-
X
"x
5 15 -f
10 -f
5 4-
•
•
•
8
0 No clinical diagnosis Clinical diagnosis "
Figure 7. Neurotoxicity Index in workers with and without clinical diagnosis of peripheral
neuropathy.
36
-------
TABLE 9
Hemoglobin adducts of acrylamide (AAVal), glycidamide (GAVal) and
acrylonitrile (ANVal) in workers
Subject No. Exposure Smoker Hemoglobin adducts (nmol/g Hb)c Job position
Cl
C2
C3
C4
C5
C6
C7
C8
C9
CIO
El
E2
E3
E4
E5
E6
E7
E8
E9
E10
Ell
E12
E13
E14
E15
E16
E17
E18
E19
E20
E21
E22
E23
E24
E25
E26
E27
E28
E29
E30
E31
Control no
no
no
yes
no
yes
yes
yes
no
yes
Exposed
ti
it
it
it
ti
tt
ii
ti
n
n
n "*
it
ii
it
i
i
t
i
t
• it
ii
ii
"
it
it
it
it
n
"
ti
AAVal
nda
nd
nd
0.01
nd
nd
nd
nd
nd
nd
6.9
6.7
1.6
4.8
2.6
24
12
11
0.6
8.5
34
12
5.6
6.7
7.6
5.8
0.3
11
7.6
4.2
11
4.5
3.2
14
3.7
10
8.5
21
5.4
1.3
4.9
GAVal ANVal
nd nd
-* nd
nd
0.14
nd
nd
0.03
0.06
nd
0.09
17
22
9.1
37
4.3
16 8.3
5.9
9.3 0.12
16
29
66
18
6.4
5.9
14
32
24
2.7
46
26
28
1.6 18
15
8.9 7.3 -
7.2
58
31
14
12
24
2.5
Ambulatory
Synthesis
Ambulatory
Polymerization
Polymerization
Ambulatory
Polymerization
Synthesis
Packaging
Synthesis
Synthesis
Synthesis
Ambulatory
Polymerization
Synthesis
Polymerization
Synthesis
Polymerization
Polymerization
Polymerization
Ambulatory
Packaging
Packaging
Polymerization
Packaging
Ambulatory
Synthesis
Synthesis
Ambulatory
No AA for 4 months
Polymerization
37
-------
Table 9 continued
E32
E33
E34
E35
E36
E37
E38
E39
E40
E41
28
7.1
6.7
3.8
12
7.5
21
32 32
0.6
12
36
0.02
5.7
7.9
21
15
20
28
1.4
22
Synthesis
Polymerization
Polymerization
Synthesis
Synthesis
Packaging
Synthesis
Synthesis
Store house
Ambulatory
a nd = Not detected
b Not analyzed
c The Hb adducts of AA and AN were determined with the modified Edman degradation method,
and the adducts of GA were determined in hydrolyzed Hb samples.
38
-------
1S9C
ZJ63338.
ANVal
IW2
I
1166.
352
375
j \
d4-HOEtVal
54,636.
(see 1580 i6oe
1U24 |1i33 11:42 11:51
l\
T
1881
IC4K30.
AAVal
tese tm
13:36 I3:«
U28 SCAN
14:82
€22
(26
M36
S:?5
1153
GAVal
T
dg-GAVal
; v_
V36
1150
9:35
nee
9:*«
1176
5:45
nee
75056.
125056.
1)96
9-. 55
wee SCAN
16; ee ii*
Figure 8. Mass fragmentogram of A A and GA valine adducts.
39
-------
35
30 -
E
C
15
g 10
10 15 20 25 30
AAVal (nmol/g Hb)
35
Figure 9. Correlation between AA and GA Hb adducts in workers exposed to AA.
40
-------
TABLE 10
Air concentrations of AA and AAVal Hb adduct levels in workers in different locations
Location
AAVal*
(nmol/g Hb)
AA air concentrations*
(mg/mj)
AAVal expected from
air concentrations0
(nmol/g Hb)
Controls
Packaging
Polymerization
Ambulatory
Synthesis
0.0 ±0.0 (10)
3.9 ± 2.5 (5)
7.3 ±3.4 (12)
9.6 ±6. (8)
14.7 ±10.6 (14)
1.52(0.19-8.8,12) 0.93
0.73(0.11-3.01,18) 0.44
a Mean ± SD (n)
b Geometric mean (range, n)
c Cumulative in vivo dose over 17 weeks calculated as:
1.52 mg (m3)-1 x (0.2 liter min-' kg bw1) x (60 min hr1) x (8 hr day1) x (6 day week-') x 17
weeks =
1000 liter (m3)-1 x (71 mg mmoH) x 0.5 hr1
= 0.419mMhr
Expected AAVal adduct level calculated as (cf. Equ. 1-2):
0.419 mMhr x 4.4 x 10'6 liter (g Hb)-1 hr1 x 1/2 = 0.93 nmol/g Hb
41
-------
TABLE 11
Individual values for parameters predictive of adverse health outcome in workers exposed to acrylamide
nr
freeAA#
merc##
AAVal### ANVal* handvibr** footvibr**
job
employment* NIn
cl
c2
c3
c4
c5
c6
c7
c8
c9
clO
9
622
23
25
37
4
5
7
14
16
18
19
20
24
26
33
34
1
3
6
13
21
nd
nd.
nd
65
69
62
nd
nd
nd
nd
nd
nd
nd
251
67
nd
nd
nd
nd
nd
nd
nd
nd
nd
112
nd
78
nd
nd
200
85
nd
nd
2
nd
1.6
2.4
2
3
2.4
3
2.4
9
33
47
258
46
27
19
15
19
15
17
35
16
• 78
133
21
27
9
12
12
40
52
0
0
0
0.01
0
0
0
0
0
0
0.6
4.5
3.2
3.7
7.5
4.8
2.6
12
6.7
5.8
11
7.6
4.2
14
10
7.1
6.7
6.9
1.6
24
5.6
11
, 0.09
0.15
0.03
0.33
0.035
0.17
0.25
0.3
0.05
0.41
18.5
22.2
19.5
10.6
16.2
33.2
8.6
8.7
21.6
27.5
7.8
39.6
31.2
10.1
28.9
0.36
9.6
18.2
15.2
11.9
13
21
1.2
1.6
2.1
2
1.6
1.6
1.3
1.8
1.1
1.3
2.6
1.3
2.3
1.6
4.7
3
1.6
2.3
1.7
2.6
1.1
3.1
1.4
1.2
4.3
1.6
1.8
2.8
1.2
1.7
2.6
2.3
3.3
2.8
3
1.5
3.3
2.3
2.1
4.3
4.2
3
4.2
3.1
7.5
2.6
10.2
5.7
2.9
5.2
4.5
4.3
2.4
6.2
3.3
1.4
10.5
2.4
2.5
7
2.2
3.1
6
5.9
control
control
control
control
control
control
control
control
control
control
packaging
packaging
packaging
packaging
packaging
polymerization
polymerization
polymerization
polymerization
polymerization
polymerization
polymerization
polymerization
polymerization
polymerization
polymerization
polymerization
ambulatory
ambulatory
ambulatory
ambulatory
ambulatory
0
0
0
0
0
0
0
0
0
0
1.9
1.75
1.4
2.3
2.5
2.5
0.75
1.9
2.4
7
1.1
0.6
3.2
1.4
8.2
6
8.4
8
3.1
1.5
2.75
3.2
0
0
0
0
0
0
0
0
0
0
4
8
8.5
0
24
9.5
9
12
7.5
7.5
2
20
10
4
22
8
9
8.5
4.5
2
8.5
12.5
(continued)
-------
TABLE 11 (Continued)
Individual values for parameters predictive of adverse health outcome in workers exposed to acrylamide
nr
freeAA# merc## AAVal### ANVal*
haridvibr** footvibr**
job
employment* NIn
29
41
2
8
10
11
12
15
17
27
28
32
35
36
38
31
39
30
40
nd
nd
nd
141
109
130
93
nd
65
nd
155
45
251
140
180
nd
nd
43
nd
42
129
12
42
78
39
47
23
9
94
28
109
36
163
104
28
318
nd
36
5.4
12
6.7
11
8.5
34
12
7.6
0.3
8.5
21
28
3.8
12
21
4.9
32
1.3
0.6 :
13.5
20.1
14.4
0.22
23.9
26.9
17.9
19.7
22.2
25.9
23.4
26.8
10.6
22.4
26
4.6
32.7
23.7
2.1
1.9
4.3
1.6
1.4
2.6
5.6
2.9
2.1
2.5
3.6
4.7
4.2
1.3
2.3
2.7
1.2
1.5
2
1.5
6.5
4.4
3.1
27
5.8
15.2
7.5
3.3
5.7
7.5
11.9
18.9
2.2
5.7
4.4
2.2
1.9
2.2
2.7
ambulatory
ambulatory
synthesis
synthesis
synthesis
. synthesis
synthesis
synthesis
synthesis
synthesis
synthesis
synthesis
synthesis
synthesis
synthesis
++
++
+++
+++
11.5
4.5
3.5
8
3.75
3.1
3.1
1.6
0.8
5.4
7.9
7.7
2.5
7.5
3
0.1
0.25
1.2
3.5
11
32
16.5
1.5
26.5
34
26
15.5
5
25.5
24.5
26.5
2.5
17
28
2
6.5
12
11.5
P ppm in plasma.
¥ mercapturic acids, jimol/L urine.
¥. nmol/g Hb (from Bergmark et al. 1993).
nmol/g Hb (from McNeela et al., in preparation).
Vibration units.
* years.
** Less than 6 months exposure to AA.
*** not exposed to AA during the 4 last months.
nd = not detected.
-------
Calculation of in vivo doses f
Under the assumption that the adducts are stable during the lifetime of the erythrocytes, the
daily increment of adducts in a chronic exposure situation, ([RY]/[Y])d, can be calculated
as:
([RY]/[Y])tt = ([RY]/[Y])d x iJ2 (1)
where ([RY]/[Y])ttis the steady-state level of adducts at the nucleophilic center, Y, and ^
is the life-span of the erythrocytes, approximately 120 days in man (Osterman-Golkar et al.,
1976). The daily in vivo dose, D, can then be calculated from:
D = ([RY]/[Y])4 / ky (2)
where ky is the second-order rate constant for the reaction of the electrophile with the
nucleophilic center Y in Hb (in this case Y = -NH2 of the N-terminal valine).
As an example, in worker No. E39, the daily in vivo dose of AA is given by:
D= 32 nmol/g Hb x 2 = 0.12 mMhr/day (3)
120 days x 4.4 x 10"6 liter (g Hb x hr)'1
and in analogy, the daily in vivo dose of GA is calculated as 0.048 mMhr in the same
person.
Other biomarkers of exposure
The individual data for the concentrations of free acrylamide in plasma (determined by K.
Crofton, USEPA, HERL, RTF), S-(2-carboxyethyl) cysteine in urine and Hb adducts of
acrylamide and acrylonitrile are shown in Table 11 together with the job classifications of
the different workers. For a majority of workers the levels of free acrylamide in the
plasma could not be measured above the level of detectability. For the other biomarkers of
exposure, statistically significant differences were observed between the exposed and
control groups, which shows that they are in principle useful as biomarkers for workers
heavily exposed to acrylamide and/or acrylonitrile. The group means for the different
categories of workers are shown in Table 12.
Biomarkers of exposure as predictors of neurotoxicity
The relationships between free acrylamide in the plasma, mercapturic acids in the urine,
hemoglobin adducts of acrylamide or acrylonitrile and the lifetime in vivo doses of
acrylamide with the neurotoxicity index are shown in Figures 10-14. The correlation
coefficients and statistical significances of these relationships are shown in Table 13,
assuming linear relationships between the independent and dependent variables. The mean
44
-------
Table 12
Biomarkers according to job classification
controls
Free AA* mere ac.** AAVal#
ANVal#
handVU## footVU##
65 ±4
packaging 159
polymeriz 95
ambulatory 143
synthesis 131 ± 59
1.9 ±1.1 0.0 ±0.0 0.18 ±0.13 1.6 ±0.3 3.0 ±0.9
78.6 ±102 3.9 ±2.5 17.4 ± 4.4 2.5 ±1.3 5.5 ± 3.2
32.7 ±36.4 7.7 ±3.4 18.9 ± 12.8 2.1 ± 1.0 4.3 ± 2.5
36.6 ±44.9 9.5 ±7.3 16.1±3.6 2.4±1.0 5.0±1.8
60.3 ± 45.8 13.4 ± 9.8 20.2 ± 7.7 2.9 ± 1.3 7.2 ± 5.1
Life-time AA§ NIn
0.0 ± 0.0 0.0 ± 0.0
8.1 ±6.6 8.9 ±9.1
27.0 ±23.9 10.0 ±5.8
37.6 ±21.9 11.3 ±9.8
68.3 ± 64.2 19.2 ± 10.6
* Means based only on the values above detectability, ppm
** Mercapturic acids, pmol S-(2 carboxyethyl)cysteine/L hydrolyzed urine
#nmol/ g Hb
## Vibration units
§ Life-time in vivo doses of acrylamide; mMhr
-------
X
c
X
o
c
z
200
Concentration of free acrylamlde In plasma (ppm)
300
Figure 10. Correlation between neurotoxicity index and levels of free AA in plasma.
46
-------
0 100 200 300
Concentration of S-(2-carboxyethyl)cystelne In urine (umol/24 hrs)
Figure 11. Correlation between Neurotoxicity Index and urinary mercapturic acid levels.
47
-------
X
«
T3
X
o
g
«
0 10 20 30 40
Acrylamldd edducts to N-termlnal vallne In hemoglobin (nmol/g Kb)
Figure 12. Correlation between neurotoxicity index and AA Val adducts.
48
-------
0 10 20 30 40
Acrylonltrllo adducts to N-termlnal vellne In Hb (nmol(g Hb)-1)
Figure 13. Correlation between neurotoxicity index and AN Val adducts.
49
-------
40 i
100 200
Accumulated In vivo doses of acrylamlde (mMhr)
300
Figure 14. Correlation between neurotoxicity index and lifetime in vivo doses of AA.
50
-------
TABLE 13
Correlation coefficients and levels of statistical significance for the relationships between
different predictive parameters and the Neurotoxicity Index (NIn).
X variable
Y variable
Corr. Coeff.
P-value
free AA
mere
AAVal
AAVal x time
ANVal
ANVal x time
time
Foot Vibratron
Hand Vibratron
AAVal
NIn
NIn
NIn
NIn
NIn
NIn
NIn
NIn
NIn
ANVal
0.15
0.42
0.67
0.60
0.69
0.60
0.44
0.69
0.81
0.44
0.31
<0.01
<0.001
<0.001
<0.001
<0.001
<0.01
<0.001
<0.001
<0.01
51
-------
values of the different biomarkers, as well as of different indicators of neurotoxicity in the
different job categories, are shown in Table 12.
General discussion of Aim 2
Hemoglobin adducts
Electrophilic compounds react with nucleophilic amino acid residues in Hb, such as the SH
of cysteine or the NH2 of N-terminal valine. The activated double bonds of AA and AN
react with nucleophilic sites in Michael type additions, whereas the epoxide GA reacts
according to a classical nucleophilic substitution mechanism.
When analyzing samples from humans exposed to both AA and AN, the modified Edman
degradation was our method of choice. The reasons for this were two-fold: first, this
method is less time-consuming than the determination of adducts following total hydrolysis
and ion-exchange chromatography; second, the total hydrolysis method does not distinguish
between adducts originating from AA and AN, since both the 2-carbamoylethyl- and the
2-cyanoethyl- groups are converted to the 2-carboxyethyl- group in strong acid.
In our analyses, the amount of the reagent PFPITC was increased compared to the standard
procedure of Tornqvist et al. (1988) because this has been shown to give a better yield of
certain PFPTH-derivatives, and was therefore assumed to increase the yield of
AAVal-PFPTH. d4-HOEtVal was used as an internal standard because of the availability of
a well-characterized standard globin alkylated with d4-ethylene oxide. The overall response
(a combination of yield in the derivatization and extraction procedure, and the response on
GC/MS) was for AAVal-PFPTH somewhat lower (1.5-8 times) and for ANVal-PFPTH
slightly higher (2-6 times) than for d4-HOEtVal-PFPTH. The yield and extraction
efficiency were not determined, but they were assumed to be constant for AAVal-PFPTH
and ANVal-PFPTH versus the internal standard. The difference in response using GC/MS
was compensated for by the use of calibration curve. Although the PFPTH derivative of
GAVal was also formed and was detectable, the overall response was low (1-2 orders of
magnitude lower than that of d4-HOEtVal-PFPTH), and the quantitations were not
reproducible. This might be explained by the fact that the GA adduct is very hydrophilic
so that its PFPTH-derivative may be difficult to extract from the reaction mixture.
The AA and GA valine adducts were generally not detected in unexposed controls and a
possible background could not be established. AN adducts were, however, detected in most
smokers. Based on the lowest levels detected, detection limits for the method appear to be
0.01 nmol/g Hb (signal-to-noise ratio 6:1) and 0.02 nmol/g Hb (signal-to-noise ratio 12:1)
for the AAVal-PFPTH and ANVal-PFPTH derivatives, respectively. These results show
that the determination of AA and AN adducts using the modified Edman degradation
method can be used as a tool to monitor total exposure to AA and/or AN.
As discussed above, adduct formation by GA to N-terminal valine was analyzed using the
52
-------
total hydrolysis method, because of the low yields and hydrophilicity of its
PFPTH-derivative. The detection limit of this method was about 1 nmol/g Hb. This acid
hydrolysis method was used also to determine adduct levels in Hb treated in vitro with AA
or GA for the determination of rate constants with cysteine-SH and valine NH2.
Background levels of the cysteine adducts were high in unexposed controls, probably due to
methodological artifacts, and analysis of these adducts is suitable only for determination of
high alkylation levels such as those in the in vitro experiment.
The valine adducts (CEVal and CHEVal) did not give the expected structures following
derivatization with methanol/HCl and HFBA, apparently due to rearrangement reactions.
The resulting derivatives are interpreted as being formed during the acylation, presumably
by nucleophilic attack of the N-acyl-oxygen on the carbonyl of the "C-terminal ester"
followed by elimination of CH3OH. The derivatives of the dg-analogues therefore contain
only six of the original deuteriums in the dg-valine as was shown by the fragmentation
pattern on GC/MS. Comparison of mass spectra of the deuterated and the non-deuterated
derivatives in the El and the PCI modes proved to be essential in order to elucidate these
structures. The CHEVal derivative gave four additional peaks on GC, whose structures
were identified as stereoisomers of lactons, also formed during the derivatization. The use
of deuterated standards compensated for variations in yield of the different derivatives. The
quantitation of GA adducts was made on the same type of derivative as the one formed for
CEVal.
While performing the analysis of GA valine adducts using total hydrolysis, CEVal was also
determined and found to correspond well (± 8 %) to the sum of the AA and AN valine
adducts determined by the modified Edman procedure in the same individuals. As
mentioned earlier, both AAVal and ANVal form CEVal upon acid hydrolysis of globin.
Based on their roles and positions in the production process we have subdivided the AA
factory workers into four different categories: (1) workers involved in the synthesis of AA
from AN and in the transfer of 35% AA solution into barrels, (2). ambulatory personnel
such as foremen, maintenance workers, etc., with no stationary work place, (3) workers
involved in the copolymerization of AA and acrylic acid, and (4) workers who packaged the
copolymer after mixing it with starch. Among the workers with the lowest levels of AAVal
adducts, one had been free of AA exposure for the past four months (No. 30) and one
worked in the store house (No. 40). The air levels at different locations in the workshop
were monitored on four different occasions prior to blood sampling. In September 1991 the
AA air levels ranged between 0.1 and 1.6 mg/m3, and in the summer months the levels
ranged between 0.3 and 8.8 mg/m3. Although the concentrations measured fall within a
quite narrow range, an occasional level of 153 mg/m3 was recorded during discharge of
AA. The maximal allowable air concentration for AA in the workplace is 0.3 mg/m3 both
in the People's Republic of China and in the United States. A few conclusions may be
drawn from the data presented. First, the average Hb adduct level of AA is related to the
job description and to the location of the different workers in the factory. Second, it
appears that only a small fraction of the total amount of AA absorbed by the workers
53
-------
originated from inhalation of AA. Indeed, assuming a breathing rate of 0.2 1 min'1 kg'1,
estimated for workers doing work involving moderate physical exercise (Calleman et al.,
1978), and that the first-order rate of elimination of AA in humans is the same as in rats
(0.5 hr1), it may be estimated that the observed Hb adduct levels in workers in the
synthesis and polymerization rooms are 10-30 times higher than would be expected from
the measured concentration of AA in the air. Our Hb adduct data are thus consistent with
the notion that dermal exposure may be the predominant route of uptake, as suggested in
several previous studies (EPA, 1988). In addition, the observation of skin peeling of the
hands in several of the workers also suggests that dermal exposure is a significant route of
uptake of AA.
The ratio of the in vivo doses of GA and AA was on average 3:10. In the rat, the
percentage of AA converted to GA is about 60% following a low dose of AA, and the
conversion of AA to GA decreases as the injected amount of AA increases (Bergmark et al.
1991). The metabolism of AA in the rat is therefore saturated at high administered
amounts. It might be assumed that no saturation of metabolism of AA to GA occurs at the
exposure levels found in this study in humans, because of the linear correlation between AA
and GA adducts.
Recent preliminary experiments by D. Segerback (personal communication) showed that in
mice and rats treated with AA, the major DNA adduct is N-7-(2-carbamoyl-2-
hydroxyethyl)-guanine, which is formed by the reaction of GA with DNA. This is
consistent with the hypothesis that GA may be the agent primarily responsible for cancer
initiation following exposure to AA (Calleman et al. 1990). The findings that AA is
metabolized to the genotoxic metabolite GA in humans, and that GA adducts are linearly
related to AA adduct levels, suggest that exposure to AA might be associated with an
increased cancer risk. However, a recent epidemiological study (Collins et al. 1989) has
not found an association between AA exposure and an excess of cancer deaths, although the
exposure levels determined in that study were much lower than in the present one. In this
regard, it is of interest to note that the daily in vivo doses of AA and GA in the most highly
exposed workers correspond to the in vivo doses in rats injected daily with 3 mg/kg AA.
In cancer tests (Johnson et al., 1986), a daily dose of 2 mg/kg increased several types of
tumors in Fisher 344 rats. In addition, exposure to AN is also expected to pose an
increased risk of cancer. Therefore, follow-up studies using Hb adducts as biomarkers for
monitoring reductions in the exposure to AA and AN as well as prospective studies of
cancer incidences in workers heavily exposed to A A and AN are warranted.
field study
As discussed before, the combination of uptake of acrylamide through different routes is a
common characteristic of industrial exposure to acrylamide and speaks in favor of using
biomarkers reflective of the total uptake of the substance through all routes as a basis for
evaluating hazardous conditions in the workplace.
54
-------
The biomarkers of exposure measured in this study reflect, however, different time-periods
of exposure and are thus not expected to be equally useful for predicting an effect which is
believed to be a function of a cumulative dose, or integrated concentration of the neurotoxic
agent.
The concentration of free acrylamide in the plasma could only be detected in a few workers
from each group except for the most extensively exposed group located in the synthesis
room. It must be assumed that the levels of acrylamide detected in three of the controls
reflect an artifact peak eluting at the retention time of the derivatized acrylamide, since no
Hb adducts could be found from acrylamide in this group, and the levels detected (62-69
ppm) are only slightly above the detectability of this method (40 ppm). Given that this
indicator reflects a momentary level and that blood sampling was performed at different
times after the workers had left their work positions, the fact that free acrylamide could not
be found in a high number of workers and that it correlated poorly with the Neurotoxicity
Index, is not surprising. It is, however, noteworthy that the levels of free acrylamide in the
plasma of the most highly exposed group are close to what would be expected from the Hb
adduct values under the assumption of a rate of elimination of acrylamide from human
plasma similar to that estimated in rats (0.5 hr1, Miller et al., 1982; Bergmark et al.,
1991). Thus, the average level of acrylamide Hb adducts (14.7 nmol/g Hb) in this group
corresponded to an average in vivo dose of 6.6 mMhr during the life-span of the
erythrocytes (17 weeks in humans). This corresponds to a steady-state level of 580 ppm of
free acrylamide in the plasma of workers working 8 hrs/day, 6 days/week. While
quantitative inferences from these types of measurements should be made with great
caution, the results seem to indicate that the rate of elimination of acrylamide is of the same
order of magnitude in humans as in rats.
In contrast, the determination of mercapturic acids reflects exposure during approximately 1
to 2 days prior to urine sampling. In our study, the urine was hydrolyzed prior to analysis
to yield the amino acid S-(2-carboxyethyl)-cysteine, the concentration of which reflects not
only acrylamide, but also acrylonitrile conjugates, which are hydrolyzed to the same amino
acid. In addition to this drawback, it should be kept in mind that mercapturic acids are
detoxification products which are not necessarily proportional to the amount of reaction
with target enzymes in nervous tissue. To the extent that interindividual variability in
detoxification efficiency of acrylamide or acrylonitrile comes into play, the amount of
mercapturic acids excreted in the urine may in fact be inversely related to the in vivo doses.
Not surprisingly, the concentration of these mercapturic acids in 24-hr urine is a relatively
poor predictor of the Neurotoxicity Index.
Hemoglobin adducts reflect the in vivo dose (integrated concentration) of an electrophilic
substance during the 4-month period prior to blood sampling. As expected, this biomarker
is better correlated with the neurotoxicity index than the other biomarkers of exposure
determined in this study. Two peculiarities deserve attention in this regard: first, the fact
that the Hb adducts formed by acrylonitrile correlate better with the Neurotoxicity Index
than those formed by acrylamide, and second, that the lifetime in vivo doses of either agent
55
-------
as estimated from the Hb adduct levels and time of employment do not correlate as well
with neurotoxicity as do the Hb adducts themselves.
The good correlation between Hb adducts of acrylonitrile and the neurotoxicity index cannot
be explained by a causative relationship since acrylonitrile exposure does not result in the
different signs, symptoms and predictive parameters which were included in this index as
defined here. Humans who have been exclusively exposed to acrylonitrile in occupational
settings may present effects on the central nervous system but not the peripheral neuropathy
characteristic of acrylamide exposure (F. He, personal communication to C. Calleman).
However, although Hb adducts of acrylonitrile correlate better than those of acrylamide in
univariate regression analysis, multivariate analysis gives the relationship: NIn = 0.59
(+0.14) x AAVal + 0.43 (+0.08) x ANVal, where both AAVal and ANVal are correlated
with the NIn at the p < 0.01.
The impairment of vibration sensation has been found to be an early sign of acrylamide
neuropathy and proved to be related to the vulnerability of Pacinian corpuscles to
acrylamide (LeQuesne, 1980). In this study, the prevalence of deficits of vibration
sensation in acrylamide workers examined by a tuning fork (C 128) was 41%, whereas
58.5% of the same acrylamide group detected by a Vibratron n vibration sensitivity tester
showed an increase of vibration thresholds which were higher than the upper limits of
vibration thresholds of the reference group. This suggests that the Vibratron H vibration
sensitivity tester is superior to tuning forks in terms of sensitivity, reliability and
quantitation for the assessment of impairments of vibration sensation. It is therefore
potentially useful in field screening of peripheral nerve dysfunction in acrylamide exposed
workers. In fact, vibration thresholds in hands as determined by the Vibratron instrument
show the best correlation with neurotoxicity of all the potentially predictive parameters.
This good correlation is hardly unexpected given that loss of vibration sensitivity is a more
sensitive indicator of peripheral neuropathy than either loss of pain or touch sensitivity and
the Vibratron instrument may thus be a comparatively inexpensive and useful tool for
identifying workshops with unacceptable exposure conditions.
The ENMG findings in acrylamide workers manifested as a prolongation of distal motor
and sensory latencies and a decrease of distal sensory potentials in peripheral nerves
indicate a damage to the distal part of the peripheral nervous system which coincides with
the pattern of the central-peripheral distal axonopathy found in cats and rats dosed with
acrylamide. The presence of abnormal recruitment patterns without any spontaneous
denervation potentials of the sampled muscles suggests a mild damage to the motor fibers of
the peripheral nervous system. On the other hand, there was a significant decrease of distal
sensory potentials in 17 out of 41 acrylamide workers, the lowest being undetectable,
indicating more involvement of sensory fibers than motor fibers in acrylamide neuropathy
(Sumner and Asbury, 1974).
According to the diagnostic criteria for occupational acrylamide poisoning proposed in our
previous studies (He et al., 1989) and based on the clinical and ENMG data available, 13
56
-------
out of the acrylamide group under study were diagnosed with mild occupational acrylamide
poisoning at a prevalence of 31.7%.
The results of this study provide evidence of neurotoxic effects of acrylamide in exposed
workers, similar to those reported in previous studies (He et al., 1989, LeQuesne, 1980).
The symptoms and signs presented in the acrylamide workers include numbness and
weakness of hands and feet, excessive sweating of hands, impairments of vibration, pain
and touch sensations, as well as diminution and loss of ankle reflexes. These clinical
manifestations, usually preceded by peeling of skin from hands, are more prevalent than in
the reference group and strongly indicate an involvement of the peripheral nervous system.
The results of medical surveillance clearly indicate the necessity of prevention of
acrylamide poisoning in the factory under study.
The neurotoxicity index was designed to be: (1) specific for acrylamide-induced peripheral
neuropathy and to exclude such effects that might be due to acrylonitrile-induced effects on
the central nervous system, (2) objective, i.e. independent of the subjective experience of
the workers, and (3) reflective of the severity of the neurological disturbance. There was
only minimal overlap in the Neurotoxicity Index between those individuals with a clinical
diagnosis of peripheral neuropathy and those without.
Among the workers there was a better correlation between this neurotoxicity index and the
levels of Hb adducts (r = 0.67) than with either the cumulative in vivo dose (r = 0.60) or
the duration of employment (r = 0.44). However, in workers employed for more than 2
years, the correlation between neurotoxicity and the duration of exposure virtually
disappeared (r = -0.06). The interpretation was thus made that the average dose-rate (as
reflected by the Hb adduct levels for the 4-month period prior to blood sampling), rather
than the cumulative in vivo dose, is the better predictive parameter for peripheral
neuropathy in workers exposed for more than 2 years.
A risk analysis was hence performed which included only those workers who had been
exposed to acrylamide for more than 2 years. If the percentages of these workers with
different diagnostic indicators of neurotoxicity were plotted against their Hb adduct levels,
sigmoid curves were evident reaching 100% in the group with the highest level of Hb
adducts. From the plot, NOEL and LOEL and EL^ for clinical diagnosis of peripheral
neuropathy were estimated to 2, 6 and 7.9 nmol AAVal (g Hb)'1, respectively. Assuming a
rate of elimination of acrylamide in man similar to that estimated in the rat, 0.5 hr1, these
Hb adduct levels correspond to intakes of 0.3, 0.8 and 1.1 mg/kg/day, and further
assuming a 100% uptake of airborne acrylamide and a rate of alveolar ventilation of 0.2 1
min'1 (kg bw)'1 in exposed workers, NOEL, LOEL and ELJO for peripheral- neuropathy
correspond to concentrations of 3, 8, and 10 mg/m3, respectively. It may thus be concluded
that in workers exposed for more than 2 years, the currently applied TWA of acrylamide,
0.3 mg/m3, has a safety factor of 10 for adverse neurological effects. It should be also
noted, however, that in the present study the primary route of exposure to AA appeared to
be dermal.
57
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AIM 3
Comparative neurotoxicity of acrylamide and glycidamide
Rats injected with the lowest dose of acrylamide gained weight, while those administered 50
mg/kg acrylamide or glycidamide did not (Fig. 15). Rats in the 100 mg/kg glycidamide
group lost weight during the treatment. Animals in the two high-dose groups started to
appear lethargic and ataxic after 5 injections (cumulative doses of 250 and 500 mg/kg for
acrylamide and glycidamide, respectively). However, the appearance of the animals was
quite different. While acrylamide-treated rats had their hindlimbs extended to both sides in
the hindlimb splay test, glycidamide-treated rats routinely extended both legs to the same
side.
Behavioral tests
The results of the rotarod testing are shown in Fig. 16. At the low doses, both acrylamide
and glycidamide had no significant effect on the ability of rats to stay on the rod. Similar
significant effects were, however, observed starting on day 6 in animals receiving either
acrylamide (50 mg/kg) or glycidamide (100 mg/kg). On the other hand, in the hindlimb
splay test a significant effect was seen only in animals treated with acrylamide (Fig. 17).
At the 50 mg/kg dose, acrylamide increased the spread of the hindlimbs on landing after
two doses, while at the 25 mg/kg dose a significant effect was seen after 8 injections.
Neither dose level of glycidamide had any effect in this test. In the open field tests, no
significant differences were found between control and treated rats at any time point, with
regard to deambulation. However, animals in the 50 mg/kg acrylamide group showed a
decrease in rearing on day 4. On day 8 the values for rearing (mean score jf SEM) were:
control, 26.9 ± 9.4; glycidamide 50 mg/kg, 21.6 ± 9.6; glycidamide 100 mg/kg, 13.0 ±
2.6; acrylamide 25 mg/kg, 19.5 + 5.1; acrylamide 50 mg/kg, 6.1 ± 2.9 (<0.05; n =
10/group).
Morphological observations
One of the most striking features at necropsy was urinary retention and distended bladders
in rats dosed subacutely with acrylamide, as previously reported. The effect of acrylamide
was clearly dose-dependent. Glycidamide appeared to cause some urinary retention but to a
much lesser degree. In animals treated for 8 days with 50 mg/kg acrylamide or 100 mg/kg
glycidamide, no apparent morphological alterations were seen at the light microscopic level
in sections of the sciatic nerve. Therefore, additional groups of rats were injected with the
same doses for 11 or 12 days to reach cumulative doses of 600 mg/kg (acrylamide) or 1200
mg/kg (glycidamide). Characteristic changes of acrylamide neurotoxicity were seen in the
acrylamide group, with degeneration and axonal swelling, but not in the glycidamide-treated
animals.
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320
250
4 6
Days of treatment
Figure 15. Body weights of rats treated with AA or GA.
59
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o
0
•o
o
c
o
0
E
0
o
CO
--•O--
Control
AA25
AA50
GA50
GA100
4 6
Days of treatment
10
Figure 16. Rotarod test performance of rats exposed to AA or GA.
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4 6
Days of treatment
••D--
-A-
Control
AA25
AA50
GA50
GA100
10
Figure 17. Hindlimb splay test in rats exposed to A A or GA.
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Male reproductive toxicity of acrylamide and glycidamide
The effects of AA and its epoxide metabolite GA on the male reproductive system were
determined after 7 and 14 days of treatment, respectively (50 mg/kg i.p./day). GA was
administered for 14 days and no overt signs of toxicity were seen, except for a significant
decrease in total body weight. Administration of AA was stopped after 7 days because at
this time rats showed severe signs of neurotoxicity, i.e., hindlimb weakness and ataxia.
Body weight following AA treatment was also significantly decreased.
GA significantly decreased epididymis weight, the ratio of epididymis weight/testis weight
and the protein content of testicular tissue (Table 14), while AA exposure had no effect on
these parameters. The vas deferens sperm count was significantly decreased by both AA
and GA; however, only GA significantly affected viability (control, 33.1 +. 7.7% viable;
GA, 3.6 ± 1.4% viable) (Table 15). DSP was affected in the epididymis only by GA, but
no effect in the testis was seen for either treatment. Plasma testosterone levels were also
unaffected by either treatment.
No differences in gross morphology and light microscopy of reproductive organs were seen
with either compound. The seminiferous tubules were unaltered in their epithelium.
Sertoli cells and interstitial cells appeared to be unaffected with either treatment.
General discussion of Aim 3
The main conclusion which can be drawn from the present results is that GA is involved in
the reproductive toxicity of AA, but not in its neurotoxicity. Administration of AA, to a
cumulative dose of 400 mg/kg, had a significant effect on the performance of rats in the
rotarod test and in the hindlimb splay test, which is considered more specific for peripheral
neuropathy (Edwards and Parker, 1977). Indeed, A A had a significant effect in this latter
test even at the lower dosage regimen (25 mg/kg/da, for a cumulative dose of 200 mg/kg).
GA, at the cumulative dose of 400 mg/kg, had no effect on either test, while at the
cumulative dose of 800 mg/kg a significant effect was seen in the rotarod test. Animals in
this group were very weak and lost weight compared to the other groups; the positive effect
observed in the rotarod may, therefore, be due to general systemic toxicity rather than to a
neurotoxic effect on peripheral nerves. The lack of effect in the hindlimb splay test seems
to confirm this hypothesis. Furthermore, morphological observation at the light
microscope level did not identify any damage to the tibial and sciatic nerve of GA 100 -
treated rats.
Where male rats were given A A in a dosage regimen of 50 mg/kg/day for a cumulative
dose of 350 mg/kg, mild signs of reproductive toxicity were present, as shown by a
decrease of vas deferens sperm count. Longer treatments with AA were prevented by its
severe neurotoxicity. On the other hand, when GA was given at the same dosage regimen
for a cumulative dose of 700 mg/kg, signs of toxicity were minimal and no neurotoxicity
was observed, although animals displayed significant toxic effects in the reproductive
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TABLE 14
Effects of acrylamide and glycidamide on male reproductive organs in rats
Body wt (g)
Testis wt (g)
Epididymis wt (g)
Testis wt/body wt (%)
Epididy wt/testis wt (%)
Testis protein (mg/g testis)
Testosterone (ng/ml)
Control8
372 ±3.7
3.7 + 0.1
1.2 ±0.04
1.0 ±0.02
33.5+1.4
96.6 ±5.2
0.6 + 0.2
Acrylamide
330 + 80«>
3.4 ±0.1
1.1 ±0.03
1.0 ±0.03
33.1 ±1.1
105.8 ±4.8
0.8 ±0.2
Glycidamide
344 ± 5.5b
3.6 ±0.1
1.0±0.04b
1.1 ±0.04
, 29.1 ±0.9"
77.4 + 4. lb
1.1 ±0.4
* Results are mean ± S.E. of at least 4 animals. No significant difference between controls at 7
versus 14 days.
b Significantly different from control, p <0.05.
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TABLE 15
Effects of acrylamide and glycidamide on male reproductive parameters in rats
Control3 Acrylamide Glycidamide
Vas deferens sperm
count (cell no. x 106)
Sperm cell viability
(% viability)
Testicular DSP
(cell no. x 106)
Epididymal DSP
Testosterone (ng/ml)
14.3 ±2.6
33.1±7.7
67.6 ±18.5
365 ± 39.7
0.6 + 0.2
7.8±0.6b
32.4 ± 6.8
57.6 ±10.2
303 + 25.8
0.8 + 0.2
6.2±1.2b
3.6±1.4b
66.2 ±11.0
68.8 ± 10.3b
1.1 ±0.4
a Results are mean ± S.E. of at least 4 animals. No significant difference between controls at 7
versus 14 days.
b Significantly different from control, p < 0.05.
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system. The finding that GA causes reproductive toxicity agrees with the hypothesis of
Sega et al. (1990) who postulated a role for this epoxide metabolite in the effects of AA on
male gonads.
In summary, these preliminary observations suggest that peripheral neurotoxicity of AA
may be ascribed to the parent compound, while reproductive toxicity may be due to the
formation of the epoxide metabolite GA. Both compounds react readily with proteins and
form adducts to hemoglobin (Calleman et al. 1990) and appear to have similar access to
target tissues, while they may differ significantly in their reactivity with DNA. The cellular
and molecular mechanisms involved in their toxicity in the nervous and reproductive
systems need to be elucidated.
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