United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas, NV 89193-3478
Research and Development
EPA/600/SR-93/226
April 1994
\yEPA Project Summary
Determination of Hemoglobin
Adducts Following Acrylamide
Exposure
L. G. Costa and C. J. Calleman
The present project was undertaken
to develop new methodologies for bio-
logical monitoring of exposure to the
toxicant acrylamide (AA) in laboratory
animals as well as humans. Methods
were developed to measure the adducts
of AA and its epoxide metabolite
glycidamide (GA) to cysteine in rat he-
moglobin (Hb) and to valine in human
Hb by means of gas chromatography/
mass spectrometry (GC/MS).
Studies in rats indicated that both
AA and GA adducts are formed follow-
ing acute or chronic exposure to AA,
while only the GA adducts are formed
after exposure to GA. Both adducts, in
addition to acrylonitrile (AN) adducts,
were measured in a group of Chinese
workers exposed to AA during its syn-
thesis and polymerization. Significant
signs of neurotoxicity were also found
in this population.
Additional studies in rats indicated
that AA, but not GA, is the proximate
neurotoxicant while GA may be respon-
sible for the male reproductive and
genotoxic/carcinogenic effects of AA.
These studies suggest that these
novel biomarkers to assess exposure
to AA are useful to assess potential
health hazards (including possible can-
cer risks) due to exposure to AA in
occupationally exposed workers as well
as in the general population.
Funding for this research was pro-
vided by the Office of Research and
Development's (ORD's) Environmental
Monitoring Systems Laboratory, Las
Vegas, NV (Exposure Biomarkers Re-
search Program), under the Toxic Sub-
stances Budget Sub-Activity (L104).
Partial support for the field trip to the
People's Republic of China was pro-
vided by the ORD's Health Effects Re-
search Laboratory (HERL), Research
Triangle Park, NC, under the ORD/HERL
program in Research to Improve Health
Risk Assessments.
This Project Summary was developed
by EPA's Environmental Monitoring
Systems Laboratory, Las Vegas, NV, to
announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
Introduction
Humans are potentially exposed to
acrylamide (AA) in industrial processes,
grouting operations, synthesis of chroma-
tography gels and leakage of the mono-
mer from polyacrylamide used in the puri-
fication of drinking water. According to a
survey undertaken by NIOSH, some
10,000 workers in 27 occupations are po-
tentially exposed to AA, including about
one thousand persons involved in the syn-
thesis of polyacrylamides and another
thousand licensed to perform grouting op-
erations. The number of laboratory work-
ers exposed to AA in the preparation of
chromatography gels has been estimated
to be as high as 100,000-200,000.
Since the early '50s the neurotoxic ef-
fects, involving both the central and the
peripheral nervous system, have been the
primary health concern of human expo-
sure to AA; close to 150 cases of human
intoxication have been reported in the last
30 years. In more recent years, following
the demonstration that AA induces tumors
Printed on Recycled Paper
-------
in mice and rats, attention has been in-
creasingly focused on the genotoxic and
reproductive effects of the compound.
Despite the fact that AA has been shown
to induce this wide range of adverse ef-
fects in mammalian species, few studies
have investigated the role of metabolism
in the toxicity of this compound, and al-
though reports have appeared indicating
that AA may undergo further metabolism,
the structures of the intermediates gener-
ated have not been elucidated. From the
point of view of quantitative risk assess-
ment of human exposure this has repre-
sented a serious gap of knowledge, since
the extrapolation of toxic effects in ani-
mals requires not only the identification of
the agent(s) responsible for the induction
of the effects, but, ideally, also informa-
tion about the relationship between expo-
sure dose ana m vivo dose (defined as
the time-integrated concentration of free
electrophilic agent in vivo). In addition,
since exposure to AA may occur through
several different routes, the total amount
absorbed by a person may be very diffi-
cult to assess.
The structural elucidation and quantita-
tive determination of hemoglobin (Hb) ad-
ducts can be used to obtain this type of
information. For example, in response to
long-standing speculation about the pos-
sible involvement of a cytochrome P-450-
generated agent in the toxicity of AA, we
were recently able to demonstrate the for-
mation in vitro and in vivo in rats of
glycidamide (GA), an epoxide formed by
oxidation of the olefinic bond of AA,
through the identification of S-(2-carboxy-
2-hydroxyethyl) cysteine in hydrolyzed Hb
samples from AA-exposed rats.
It was the central aim of this project to
develop and apply methods for simulta-
neously determining the adducts formed
by AA and GA in Hb as a means of
assessing occupational exposure to AA
and to use these data in combination with
dosimetric and metabolic studies in ex-
perimental animals undergoing toxicologi-
cal tests to arrive at a risk estimation of
human AA exposure that may serve as a
basis for regulatory action.
In addition to the practical implications
of the proposed methods for biomonitoring,
AA appears to be a suitable compound
for the theoretical development of a risk
model based on in vivo dosimetry be-
cause of the wide range of toxic effects it
is known to induce. Since both AA and
GA are reactive electrophilic compounds,
it was not clear whether the various toxic
effects associated with AA exposure are
induced by the parent compound, the ep-
oxide, or the combined action of the two
agents. Our finding that AA was metabo-
lized to GA, thus generated a few hypo-
thetical solutions to theoretical problems
particular to the toxicology of AA.
Procedure
The specific aims of this project were,
therefore
1. To develop a method for measuring
the adducts of AA and GA to Hb in
rats and to determine both adducts
following administration of AA or GA.
2. To develop a method for measuring
the adducts of AA and GA in hu-
mans, to measure adducts in work-
ers occupationally exposed to AA,
and to correlate adduct levels with
neurotoxicity.
3. To evaluate and compare the neu-
rotoxicity and reproductive toxicity
of AA and GA in the rat.
Results and Discussion
1. Development of a GC/MS method for
measuring Hb adduct of AA and GA.
A method was developed to determine
Hb adducts formed by the neurotoxic agent
AA and its mutagenic epoxide metabolite
GA. The method was based on simulta-
neous measurements of the cysteine ad-
ducts formed by these two agents by
means of gas chromatography/mass spec-
trometry (GC/MS) in hydrolyzed Hb
samples. Rats were injected ip with AA or
GA in doses ranging from 0 to 100 mg/kg
body wt, and the Hb-adduct levels were
determined. The Hb-binding index of AA
to cysteine was found to be 6400 pmol (g
Hb)'Vjumol (kg body wt)-\ higher than for
any other substance studied so far in the
rat, and 1820 pmol (g Hb)-'/u,mol (kg body
wt)-' for GA In rats injected with AA, for-
mation of adducts of the parent compound
was approximately linear with dose (0-
100 mg/kg), whereas adducts of the ep-
oxide metabolite GA generated a concave
curve, presumably reflecting the Michaelis-
Menten kinetics of its formation. On the
basis of the rate constants for cysteine-
adduct formation determined in vitro, the
first-order rates of elimination of AA and
GA from the blood compartment of rats
were estimated to be 0.37 and 0.48 hr •',
respectively, using a linear kinetic model.
It was further estimated that the percent-
age of AA converted to GA in the rat
decreased from 51% following administra-
tion of 5 mg/kg to 13% after a dose of 100
mg/kg. Subchronic treatment of rats with
AA (10 mg/kg/day for 10 days or 3.3 mg/
kg/day for 30 days) confirmed that the
conversion rate of AA to GA, as deter
mined from Hb-adduct formation, is higher
at low-administered doses. These findings
suggest that dose-rate effects may signifi
cantly affect risk estimates of this com
pound and that different low-dose extrapo
lation procedures should be employed for
effects induced by the parent compound
AA and those induced by the metabolite
GA.
2. Development of methods for AA and
GA Hb adducts in humans.
Hb-adduct determinations were used to
monitor occupational exposure to AA and
acrylonitrile (AN). Forty-one workers in a
factory in the People's Republic of China
who were involved in the synthesis of AA
by catalytic hydration of AN and the manu
facturing of polyacrylamides were studied
Ten nonexposed workers in the same city
served as controls. AA and AN exposures
were monitored using the modified Edman
degradation procedure for the determina
tion of their respective Hb adducts to IM
terminal valine. The adduct levels in the
exposed workers were 0.3-34 nmol/g Hb
for AA and 0.02-66 nmol/g Hb for AN, as
determined by GC/MS. The formation of
GA, the epoxide metabolite of AA, in hu
mans was demonstrated by GC/MS analy
sis of its Hb-adduct N-terminal valine fol
lowing acid hydrolysis, ion-exchange chro
matography, and derivatization. The GA
adduct was detected in samples from ex
posed persons with levels of 1.6-32 nmoi'
g Hb. There was a linear relationship be
tween the AA- and GA-adduct levels
(r=0.96) and the ratio of the in vivo dose
of GA and AA was 3:10. These result,
suggest that A A is metabolized to GA ir
humans, as had previously been shown in
the rat. The high AA-adduct levels in the
exposed workers, as compared to those
expected from air concentrations, indicate
that dermal exposure may contribute sig
nificantly to the total uptake of AA. Obser
vations at the factory are consistent with
this indication. The average daily in vivo
doses of AA and GA in the highest ex
posed Chinese workers were comparable
to the in vivo doses in rats injected with :.i
mg/kg AA. Since a regimen of 2 mg/kg'
day is known to cause a significant in
crease of tumors in rats, preventive mea
sures may be necessary for humans ex
posed to high levels of AA in industrial
settings.
The workers underwent a complete
medical and neurological examination and
provided blood and urine for the determi
nation of several biomarkers of exposure
Among the exposed workers, signs and
-------
symptoms indicating peripheral neuropathy
were found with statistically significant in-
creased frequencies as compared to a
group of controls from the same city.
Based on signs and symptoms and quan-
tifiable indicators of peripheral nervous
dysfunction, such as increased vibration
detection thresholds, neuropathic signs
and electroneuromyography measure-
ments, a Neurotoxicity Index (Nln) spe-
cific for AA-induced peripheral neuropathy
was designed. The Nln, which adequately
predicted the clinical diagnosis of periph-
eral neuropathy, was significantly corre-
lated with the levels of mercapturic acids
(products of AA metabolism) in 24-hr urine,
Hb adducts of AA, accumulated in vivo
dose of AA, employment time, and vibra-
tion sensitivity. The Nln was correlated
also with Hb adducts of AN, which was
explained primarily by a correlation be-
tween AA and AN exposure in this work-
shop. It was however not significantly cor-
related with momentary measures of ex-
posure such as concentrations of AA in
the air or in the plasma of exposed work-
ers.
This study is the first in which adduct
monitoring has been applied to the same
group of individuals where adverse health
effects have been observed. The results
seem to indicate that Hb adducts are use-
ful as predictors of AA-induced peripheral
neuropathy and that measurements of vi-
bration thresholds are useful for identify-
ing early neurotoxic effects in workplaces
with hazardous exposures to AA.
3. Comparative neurotoxicity of AA and
GA.
In rats treated with 0-100 mg/kg of AA,
significant dose-rate effects were observed
on adduct formation by both AA and GA.
The high rate of formation of the metabo-
lite, especially at low doses where ap-
proximately 60% of AA was converted to
GA in vivo, prompted us to investigate its
potential role in the induction of neuro-
toxic and reproductive effects attributed to
AA exposure. In initial neurotoxicological
experiments, the effects of the parent com-
pound (8-14 days, 25 and 50 mg/kg/day)
and the metabolite (8-14 days, 50 and
100 mg/kg/day) were compared. While at
the higher dose both compounds affected
the rats' performance on the rotarod, only
AA had a significant effect in the hindlimb
splay test, which is considered a more
sensitive indicator of peripheral
neuropathy. On the other hand, a stron
ger effect was seen for GA than for AA or
the male reproductive system, especially
on sperm cell viability. These preliminary
results suggest that while the parent com
pound appears to be primarily responsible
for the induction of peripheral neuropathy
other toxic effects associated with AA ex
posure, such as reproductive toxicity, ma>
be attributed to GA.
Conclusions
A GC/MS method was developed for
measuring AA and GA adducts to cys
teine residues of Hb from the rat. Results
of a human study in which AA and GA
were measured as adducted to the N
terminal valine of Hb, suggest that AA is
metabolized to GA in humans as has beer
shown in the rat. Dermal exposure is
thought to be the major route of exposure
in the factory workers who were moni
tored. Indicators of peripheral nervous dys
function were found to be correlated sig
nificantly with Hb adducts of AA. Furthe!
experiments with rats indicate that AA is
the neurotoxicant, while GA may be the
reproductive- and geno-toxin.
•&U.S. GOVERNMENT PRINTING OFFICE: 1994 - 550-067/80229
-------
L. G. Costa and C. J. Calleman are with the University of Washington, Seattle, WA
98195.
Charles H. Nauman is the EPA Project Officer (see below).
The complete report, entitled "Determination of Hemoglobin Adducts Following
Acrylamide Exposure," (Order No. PB94-144235; Cost: $19.50; subject to
change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas, NV 89193-3478
United States
Environmental Protection Agency
Center for Environmental Research Information
Cincinnati, OH 45268
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
Official Business
Penalty for Private Use $300
EPA/600/SR-93/226
------- |