United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas, NV 89193-3478
Research and Development
EPA/600/S4-89/035 Feb. 1990
ŁEPA Project Summary
Protein Adduct-Forming
Chemicals for Exposure
Monitoring :Chemicals
Selected for Further Study
F. C. Schnell and T. C. Chiang
The present report is an expanded
treatment of those chemicals
recommended for further study by a
previous U.S. EPA internal report en-
titled "Protein Adduct-Forming
Chemicals For Exposure Monitoring:
Literature Summary and Recom-
mendations." The latter report
summarized the literature regarding
adducts formed by xenoblotlcs with
proteins, particularly hemoglobin and
serum albumin, and examined the
feasibility of their use as dosimeters
of exposure. Recommendations were
made with respect to those
compounds, protein adducts and
detection methods best suited to
monitoring human exposure to toxic
chemicals, particularly those
occurring at Superfund sites and
others of interest to the EPA. Those
recommendations were summarized
in the form of a table entitled
"Prioritized List of Protein Adduct-
forming Compounds of Interest to the
EPA."
The above mentioned, prioritized
list of chemicals recommended for
further study is reproduced in the
introduction of the present report,
where the ranking scheme is also
explained. In subsequent sections of
this report, the individual chemicals
are discussed in the same order in
which they appear on the prioritized
list. The topics covered for each
individual chemical are as follows:
manufacture and use, sources and
levels of exposure, known health
effects, metabolic detoxification
and.,activation, host factors, adduct
characterization, rates of adduct
second order rate constants), dose-
response relationships, background
adduct levels, methods of adduct de-
tection, and research needs.
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
The Environmental Protection Agency
(EPA) is charged with protecting human
health and the environment, and it has
acted by placing restrictions and
regulations on the use of chemicals that
have been shown to be detrimental to
human health or to the environment.
Accurate dose measurements are critical
to the evaluation of chemical-related
health risks and to the subsequent
development of realistic regulations to
ameliorate those risks. Accordingly, the
EPA has developed an initiative designed
to develop, refine and apply appropriate
biomarkers that can be used in
conjunction with environmental
monitoring data to provide a better
estimate of exposure and risk for
individuals and populations. Macro-
molecular adducts formed as a result of
chemical exposure are among the
biomarkers that the Agency, under its
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Biomarker Initiative, is currently
evaluating for use in future human
biomonitoring studies.
A 1987 report entitled "Carcinogen-
DNA Adducts: Introduction, Literature
Summary and Recommendations"
summarized the literature concerning
adducts formed by xenobiotics with DNA
and discussed the feasibility of their use
as monitoring tools in exposure
assessment. Current interest in this area
is based on the knowledge that certain
chemicals, including the majority of
carcinogens and mutagens, are or may
be metabolized to form reactive
electrophilic species. These reactive
electrophilic covalently bind to
nucleophilic sites in cellular
macromolecules such as DNA, RNA and
various proteins. It was recognized that
measurements of protein adducts,
particularly those of hemoglobin and
serum albumin, may provide a more
quantitative measure of exposure over
longer periods of time than is generally
obtainable from measurements of DNA
adducts.
A previous U.S. EPA internal report
entitled "Protein Adduct-Forming
Chemicals For Exposure Monitoring:
Literature Summary and Recommenda-
tions" summarized the literature
regarding adducts formed by xenobiotics
with proteins, particularly hemoglobin and
serum albumin, and examined the
feasibility of their use as dosimeters of
exposure. Recommendations were made
with respect to those compounds, protein
adducts and detection methods best
suited to monitoring human exposure to
toxic chemicals, particularly those
occurring at Superfund sites and others
of interest to the EPA. Those recom-
mendations were summarized in the form
of a table entitled " Prioritized List of
Protein Adduct-forming Compounds of
Interest to the EPA".
The selection criteria were designed to
identify the most suitable protein adducts
for use in monitoring human exposure to
chemicals of interest to the EPA. Those
criteria were as follows:
(1) The chemical should be identified
as being of interest to the U.S. EPA.
(2) The chemical should form
measurable amounts of a distinct, well
characterized protein adduct.
(3) The background levels of that
adduct in non-exposed populations
should be as low as possible.
(4) The adduct must be readily
measurable by existing analytical
techniques.
(5) The adduct must be accumulated
and eliminated in a predictable, dose-
related manner.
(6) Both exposed and control
populations must be available for a
monitoring study of the adduct-
forming chemical.
(7) Current levels of human exposure
to the adduct-forming chemical should
be associated with some potential,
adverse health effect(s).
The present report is an expanded
treatment of those chemicals recom-
mended for further study by the above-
mentioned, protein adducts summary
report. The prioritized list of chemicals
recommended for further study (Table 1)
is reproduced in the introduction of the
present report, where the ranking scheme
is also explained.
In subsequent sections of this report,
the individual chemicals are discussed in
the same order in which they appear on
the prioritized list. The topics covered for
each individual chemical are as follows:
manufacture and use, sources and levels
of exposure, known health effects,
metabolic detoxification and activation,
host factors, adduct characterization,
rates of adduct formation (i.e., second
order rate constants), dose-response
relationships, background adduct levels,
methods of adduct detection, and
research needs.
Conclusions and
Recommendations
Group / Chemicals
Of the simple alkylating and arylating
agents, ethylene oxide(EO), propylene
oxide(PO) and styrene are recommended
as having the greatest potential in human
monitoring studies using protein adducts.
EO, PO and styrene are all produced and
used in the U.S. and rank high on the list
of adduct-forming compounds of interest
to the U.S. EPA. Also, one of the simpler
existing methods of protein-adduct
analysis, the modified Edman
degradation procedure, is applicable to
all three of these compounds.
EO has perhaps the largest research
base of any protein adduct-forming
chemical. Although large amounts of EO
are produced and used by industry in the
U.S., the highest exposures result from a
relatively minor use of the chemical, i.e.,
the sterilization of hospital equipment.
Because EO is distributed almost
uniformly throughout the body and reacts
directly with tissue macromolecules, its
protein adducts more nearly reflect the
level of DNA adducts in different tis
than any other chemical studied thus
PO, a closely-related homologi
EO, should exhibit similar pharrr
kinetics. The highest exposures tc
are likely to occur among produ
workers. The background levels of P
adducts are lower than those of E
adducts, making the former the
informative biomarkers of exposure
a larger range of exposure. Resul
studies of occupational exposure t<
and PO suggest that hemoglobin a<
levels may be more sensitive to
exposure to PO than EO.
Compared with ethylating agents
simple arylating agent styrene proc
adducts which exhibit lower backgr
levels and a cleaner GC elution pi
Both factors would enhance
sensitivity with which styrene adc
could be detected by chromatogr;
procedures. Investigators at Colu
University are currently conductii
study of occupational exposur
styrene in boat-building facilities of N
and Connecticut. The heaviest expo;
occur during hull and deck laminatic
addition to hemoglobin adducts,
study proposes to characterize
validate four other biomarken
exposure: lymphocyte DNA add
micronuclei, sister chromatid exchai
and unscheduled DNA synthesis.
Human exposure to ethylene o
propylene oxide and styrene can be:
monitored by measuring t
hydroxyethyl-, hydroxypropyl-
hydroxyphenylethyl-valine addi
respectively. Background levels of
valine adducts are lower than those o
histidine and cysteine adducts, and
be measured by a modified Ed
degradation technique which elimir
the need to perform tedious and 1
consuming analyses of total prc
hydrolysates or enzymatic digests. U
basic conditions in the presenci
pentafluorophenyl isothiocyanate,
modified terminal amino acids (valini
hemoglobin) are selectively cleaved
the protein. The resulting pe
fluorophenyl-thiohydantoin (F5PTH)
vatives may then be extracted
analyzed by negative chemical ioniz
mass spectrometry (NCIMS).
Because styrene, 7, 8, oxide,
major reactive metabolite of styrene,
a shorter biological half-life than ethy
oxide, it is possible that more sty
adducts are formed with albumin ir
liver cells than with hemoglobin in the
cell. However, no reports of styr
albumin adducts were found in
literature. If styrene oxide does
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Table 1. Prioritized List of Protein Adduct-Forming Compounds of Interest to the EPA
Group I • Simple Alkylating and Arylating Agents That Form N -Terminal Valine Adducts.
(1) Ethylene Oxide
(2) Propylene Oxide
(3) Sfyrene
Group II - Aromatic Amines That Form Hydrolyzable Cysteine Adducts.
(1) 4-Aminobiphenyl
(2) Benzidine
(3) MBOCA
(4) o-Toluidine
Group III - Chemicals That Form Hydrolyzable, but Less Well-Characterized Adducts.
(1) N-Nitrosonornicotine
(2) Benzo(a)pyrene_
(3) 1 - Nrtropyrene"
Group IV - Chemicals That Form Characterized, but Non-Hydrolyzable Adducts.
(1) Vinyl Chloride
(2) Ethylene Dichloride
(3) Acrylonitrile
(4) Acrylamide
(5) Chloroform
Group V - Chemicals That Form Poorly Characterized Adducts.
(1) Benzene
(2) Formaldehyde
(3) 2,4-Toluene Diisocyanate
(4) 7,12 - Dimethylbenzanthracene
(5) Epichlorhydrin
(6) Benzyl Chloride
(7) Pentachlorophenol
dducts at the N-terminal aspartate of
serum albumin, they might be detectable
by the modified Edrnan procedure.
However, no efforts to detect N-terminal
aspartic acid adducts in serum albumin
using the modified Edman procedure
have been reported in the literature.
In a highly desirable alternative to GC-
MS, the modified valine derivatives
produced by the modified Edman
degradation procedure might be detected
using immunological techniques. If an
antibody could be raised against the
pentafluorophenyl-thiohydantoin (F5PTH)
derivative of alkylated N-terminal valine, a
rapid, inexpensive method (e.g., a
competitive ELISA) might be developed
to monitor human exposure to ethylene
oxide, propylene oxide and styrene on a
large scale, one which could
simultaneously be validated by GC-MS of
the same analyte (i.e., the F5PTH valine
derivative). To cite a precedent,
antibodies have been developed and
used to detect benzo(a)pyrene tetrols
released by acid treatment of
benzo(a)pyrene-modified hemoglobin.
Group // Chemicals
Benzidine and 4-aminobiphenyl (4-
BP) are established human bladder
carcinogens, and benzidine and MBOCA
rank high on the list of adduct-forming
chemicals of interest to the U.S. EPA.
The aromatic amines are also particularly
attractive chemicals from the standpoint
of exposure monitoring due to a
remarkable situation that obtains with
regard to their hemoglobin adducts. 4-
ABP, benzidine, MBOCA and o-toluidine,
among others, are all metabolized by a
pathway that leads to the formation of
large amounts of an acid-labile
sulfinamide adduct of the 0-93 cysteine
of hemoglobin. The ultimate electrophile,
a nitroso compound, is formed m the
erythrocyte by co-oxidation of the N-
hydroxylamine metabolite and hemo-
globin. Under mildly acidic conditions,
the sulfinamide bond is cleaved,
regenerating the free amine which can
then be extracted, derivatized and
analyzed by GC-NCIMS. The
development of antibodies to the acid-
released material would make possible a
rapid, inexpensive and extremely
sensitive assay for the entire class of
compounds.
None of the aromatic amines
mentioned above is still made in the U.S.,
but benzidine (and possibly 4-ABP) is a
metabolite of the widely used, benzidine-
based dyes, while the other four all occur
in cigarette smoke and have been
measured in studies of smokers vs non-
smokers. Also, measurable occupational
exposure to 4-ABP may still occur in the
form of an unwanted side product of
other amines or dyes. MBOCA, a
suspected bladder and liver carcinogen,
is still widely used in the polyurethane
industry, where an occupationally
exposed population should be available.
The research findings of investigators at
Michigan State University which are
relevant to hemoglobin-aromatic diamine
adducts in general and MBOCA adducts
in particular, are discussed in this report.
Any future study of human exposure
to any carcinogenic aromatic amine
should make a point of identifying the
acetylator phenotype of the individual
subjects because so-called "slow
acetylators" are known to have a higher
risk of bladder cancer than so-called "fast
acetylators." This report includes a
discussion of the possibilities of
simultaneously monitoring levels of two
different protein adducts of an aromatic
amine, i.e., one acetylated and one non-
acetylated, in an effort to clarify the
relationship between acetylator status
and effective exposure to aromatic
amines.
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Group /// Chemicals
N-nitrosonornicotine (NNN), benzo (a)
pyrene (B (a) P), and l-nitropyrene (1-
NOP), like the aforementioned aromatic
amines, form Hb adducts the cleavage
products of which may be released into
solution by treatment with dilute acid or
base. However, these hydrolyzable Hb
adducts, unlike those of aromatic amines,
are formed in only small amounts and are
not as well characterized (e.g., the parent
adduct of the hydrolysis product may not
be known.)
The major tobacco alkaloid nicotine is
the precursor to the tobacco-specific
nitrosamines-NNN and 4-(methylnitros-
amino)-l-(3-pyridyl) -l-butanone (NNK).
The latter are among the most important
carcinogens in cigarette smoke and are
the major carcinogens present in so-
called "smokeless" tobacco. Both
compounds may be activated via a.
hydroxylation (the major activation
pathway) to methylating agents or, to a
lesser extent, to 4-(3-pyridyl)-4-
oxobutyldiazohydroxide which forms
unique, bulky adducts in both Hb and
liver DNA in rats. The latter adducts are
detectable as their hydrolysis product 4-
hydroxy-l-(3-pyridyl)-1 -butanone (HPB).
The likely relevance of the HPB-
yielding adduct to carcinogenic risk, and
the absence of confounding sources,-
make the tobacco-specific nitrosamines
NNN and NNK potentially useful model
compounds for human monitoring
studies. To date, however, the scientific
literature contains no report of NNN/NNK-
Hb adducts having been detected in
humans. Because methyl adducts cannot
be easily attributed to any specific
exposure, efforts to detect NNN/NNK
adducts in humans should focus on the
HPB-yielding adduct. The work of
Stephen Hecht of the Naylor Dana
Institute should be followed closely in this
regard. If HPB-yielding adducts are
detectable in blood samples of exposed
humans, low adduct levels and high inter-
individual variation can be anticipated.
Benzo(a)pyrene, a carcinogenic
polycyclic aromatic hydrocarbon (PAH),
is ubiquitous in the environment, even
though it is not commercially produced in
the U.S. The benzo(a)pyrene diol epoxide
(BPDE) metabolite thought to be the
ultimate carcinogen binds to Hb in vivo.
Treatment of the BPDE-modified Hb with
dilute acid releases a minor, acid-labile
adduct into solution as the corresponding
BPDE tetrol. The acid-released tetrols
may then be analyzed using HPLC-
fluorescence spectroscopy.
However, the vast majority of B(a)P-
globin adducts are relatively stable to
acid, and some effort should be made to
characterize these adducts, some of
which may possibly be better biomarkers
of B(a)P exposure than the tetrol-
releasing adducts. Intact B(a)P-Hb
adducts have been analyzed by laser-
induced fluorimetry and by competitive
ELISA. In both cases, Hb was
enzymatically digested prior to analysis
to expose the intact adducts and samples
were enriched in B(a)P-modified peptides
by elution from an immunoaffinity col-
umn.
The highest level of B(a)P adducts
occurs in liver proteins, of which newly
synthesized albumin is one, and total
binding of radiolabeled B(a)P is
reportedly much higher in serum albumin
than in Hb. An investigation of B(a)P-
serum albumin adducts is therefore
recommended. Serum albumin adducts
have proven very useful for monitoring
exposure to aflatoxin B,,another
compound that binds poorly to Hb.
1 -Nitropyrene, a widespread environ-
mental PAH, is neither produced nor
used commercially in the U.S. Exposure
to this carcinogenic PAH occurs via
inhalation of airborne participates, diesel
emissions, coal fly ash, carbon black
photocopier toners and smoke from
nitrate-fortified cigarettes. The formation
of a single major Hb adduct is linearly
related to the oral dose of 1-nitropyrene
in rats. The acid-labile Hb adduct, which
is released into solution during the pre-
cipitation of globin in acidic acetone, has
been tentatively identified as the cy-
steinyl sulfinamide of a ring-hydroxylated
N-hydroxylamine meta-bolite. This ad-
duct may be useful for monitoring human
exposure to 1-nitropyrene. However, no
studies have yet been done in humans,
and the adduct needs to be further
characterized in animals.
Group IV Chemicals
Vinyl chloride (VC), the most important
of the industrial vinyl monomers, is
heavily used in the plastics industry for
the manufacture of polyvinyl chloride and
copolymers. While the potentially
exposed population is probably quite
large, actual levels of exposure to VC in
the workplace are probably quite low (i.e.,
below the 1 ppm, 8 hr time wieghted
average (TWA) standard promulgated by
OSHA in 1983), because VC is an
established human carcinogen. In fact,
hepatic angiosarcoma, a rare form of
neoplasia, occurs almost exclusively
among VC reactor cleaners. A protein
adduct-based method for expos
monitoring would be particularly usef
the case of vinyl chloride, since
reliable method currently exists for r
itoring exposure to VC at concentral
below 5 ppm
The reactive metabolites of VC
chloroethylene oxide (CEO) and
chloroacetaldehyde (CAA), introduci
oxoethyl groups at the sulfhydryl gr(
of cysteine, the 1-N and 3-N positior
histidine, and the amino nitrogen o
terminal valine. The acid-stable add
S-(2-oxoethyl)cysteine and N-(2-oxoe
histidine are currently of limited use
monitoring VC exposure in humans.
N-terminal valine adduct should \
more potential as a biomarkei
exposure,but the modified Edi
degradation procedure has, appare
not yet been applied to the study of
protein adducts, nor have VC-se
albumin adducts been investigate*
might prove worthwhile,therefore
examine serum albumin as well as
using the modified Edman degrade
method.
Ethylene dichloride (EDC), the lar
volume chlorinated organic compc
currently produced in the U.S., is i
primarily in the manufacture of \
chloride. EDC and VC are metabolize
much the same way; the epo:
metabolites of both produce 2-oxo<
adducts which, when hydrolyzed
analysis, are reduced to 2-hydroxy*
adducts. Thus, the protein adducts of
EDC and EO (which yields 2-H
adducts directly) cannot easily
distinguished from one another i
protein hydrolysate. As with VC,
modified Edman procedure has not
been used to assay levels of th<
oxoethylvaline adduct. The glutath
(GSH) conjugate of EDC also forrr
unique, bulky DNA adduct, S-[2-
guanyl)ethyl]GSH, which, measured
urinary metabolite, might serve as a n
specific biomarker for EDC exposure
related compound, ethylene dibrorr
has been shown to bind to se
albumin (139 nmol/g) in treated rats.
Acrylonitrile (ACN) is an impoi
industrial monomer used extensive!
the manufacture of synthetic fib
rubbers and resins for a variety
consumer goods. It is also usec
produce acrylamide (AN), an indu;
compound used in the manufactun
polymers for water treatment. I
chemicals are neurotoxic in mami
and carcinogenic in rodents,
exposures are expected to be kept
minimum. Animal data (the only
available) indicate that both of tl
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direct-acting agents are detoxified
primarily by conjugation with GSH, and
'both are direct-acting agents that may
also form protein adducts via their
epoxide metabolites. When modified Hb
is hydrolyzed for analysis, both the nitrile
group of acrylonitrile and the amide
group of acrylamide are transformed into
carboxyl groups. Thus, the adduct
formed by direct Michael addition of
either compound to cysteine will be
analyzed as S-(2-carboxyethyl)cysteine
(the major adduct), while the adduct
formed by reaction of the epoxide
metabolite of either chemical with
cysteine will be analyzed as S-(2-
carboxy-2-hydroxyethyl) cysteine (a
minor adduct).
The consequence for biological
monitoring is that acrylamide and
acrylonitrile exposures cannot be
distinguished from one another using the
hydrolysis products of their cysteine
adducts. However, if both glycidamide
and glycidonitrile form N-terminal valine
adducts, the milder pH changes involved
in the modified Edman degradation
procedure might leave the amide and
nitrile groups of the respective adducts
intact. If this approach were successful,
then the glycidamide-and glycidonitrile-
valine adducts might be separated and
analyzed by GC-MS (provided the
appropriate column were used) or by LC-
MS. If ACN/AN-adducts of N-terminal
valine cannot be detected in Hb of
exposed animals, then ACN/AN-adducts
of N-terminal aspartate should be sought
in serum albumin.
Chloroform is produced and used in
the U.S. principally for use as a reactant
in the manufacture of freon (fluorocarbon-
22) and fluorocarbon plastics, and as an
extractant and industrial solvent in the
dye and drug industries. Production
workers make up the best exposed
population, but again, exposures are
expected to be low. Although chloroform
is not mutagenic in bacteria, it causes
cancer in rodents, and is classified by the
U.S. EPA as a probable human car-
cinogen.
In vivo binding of' radiolabeled
chloroform to rat Hb increases linearly
over a wide range of dose and is
eliminated at a rate consistent with the
lifespan of erythrocytes. In vitro the
reactive metabolite of chloroform,
phosgene, forms an unusual cyclic 2-
oxothiazolidine derivative with cysteine in
Hb. This adduct is hydrolyzed during
preparation for GC-MS analysis and
measured as N-hydroxymethyl cysteine.
tHowever, it should be noted that there is
some controversy over the identification
of N-hydroxymethyl cysteine as the
major product by GC-MS, because the
hydroxymethylamino moiety should have
been too unstable to be isolated as such.
In view of the unreliability of blood
levels and breath levels of chloroform for
monitoring exposure, a protein adduct-
based method would be particularly
useful. However, no studies of the
formation of chloroform adducts in human
Hb have yet appeared in the literature.
More research is needed to identify the
chloroform-protein adducts formed in
vivo.
Group V Chemicals
Benzene is produced in great quantity
(1.72 billion gallons in 1988) in the U.S.
where it is widely used as a chemical
intermediate in the synthetics industry.
Because it is produced by natural as well
as manmade sources, benzene is
ubiquitous in the environment. The
largest number of people are exposed to
benzene by inhaling automobile exhaust
and cigarette smoke. An established
bone marrow toxin, benzene has been
associated with myelogenous leukemia in
man, and the U.S. EPA classifies
benzene as a Class A human carcinogen.
Hence, the Agency's interest in finding
improved methods of monitoring
exposure to this chemical is well-placed.
However, more research on benzene-
protein adducts is needed in all areas,
from adduct identification to method
development, before the question of the
feasibility of using protein-adducts to
monitor human exposure to benzene can
be adequately addressed. In particular,
efforts should be made to identify and
characterize an N -terminal valine adduct
of benzene in Hb of animals and humans.
It is recommended that work on benzene-
Hb adducts at the Lovelace Inhalation
Toxicology Research Institute be followed
closely.
Formaldehyde is of interest to the U.S.
EPA because of (1) its high level of
production (2.81 million tons in 1985) and
widespread use (e.g., in particle board,
plywood and ureaformaldehyde
insulation), (2) the opportunity for
exposure in the general population (e.g.,
in mobile homes and remodeled offices),
and especially (3) the report that high
doses cause nasal cancer in rats.
However, the induction of nasal cancer in
rats by 6-15 ppm formaldehyde--4-5 ppm
is intolerable to most humans-does not
appear to be particularly relevant to the
human situation. In any case, it is not
presently feasible to monitor
formaldehyde exposure using protein
adducts, because no stable
formaldehyde-protein adducts have been
described in the literature. The DNA
protein cross-links that are formed by
formaldehyde are either unstable or
rapidly repaired.
2-4-Toluene diisocyanate is a highly
reactive chemical used in the
manufacture of rigid polyurethane
products. Because it is such a potent
allergen, OSHA has set its TLV at 5 ppb
(parts per billion), 8 hr-TWA, and 20 ppb
for 10 minutes. TDI also causes cancer in
rats and female (but not male) mice when
administered by gavage. Although the
isocyanate functional groups of TDI are
reported to react extensively with -OH, -
SH, or -NH groups on proteins, no
specific TDI-protein adducts appear to
have been described in the literature.
Instead, TDI exposure has been
monitored with variable success by
measuring the titer of antibodies to
endogeneous adducts in sera of exposed
persons. Given the efficiency with which
TDI apparently reacts with protein, it
should not be difficult to chemically
identify one or more TDI-protein adducts.
Of special interest for the purpose of
monitoring would be any adducts formed
at the N-terminal amino acids of either
Hb or serum albumin.
7,12-Dimethylbenzanthracene (DMBA)
is an extremely potent animal carcinogen
that is often used as a positive control in
carcinogenicity assays. DMBA is not
produced commercially, and its only use
is as a research chemical. Consequently,
there is no exposed population for study.
The sulfate ester of a major microsomal
metabolite of DMBA, 7-hydroxy-12-
methyl-benz[a] anthracene (HMBA),
covalently binds to cysteine, lysine and
methionine residues in protein -in vitro
However, no in vivo studies of DMBA-
protein adducts were found in the lit-
erature.
For epichlorhydrin, benzyl chloride
and pentachlorophenol, little information
is available apart from the fact that the
radiolabeled compounds do bind to
arnino acids or protein in vitro. No in vivo
studies have been performed, and no
specific protein adducts have been
identified.
Future Directions
No efforts appear to have been made
to use the modified Edman procedure to
detect N-terminal aspartic acid adducts in
serum albumin. The reactivity of the
amino groups of valine and aspartic acid
should be quite similar. The real question
is whether the carboxyl group of
aspartate will compete with the amino
group for either the adduct-forming
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species or the Edman reagent. If the
method could be demonstrated to work
with serum albumin as well as with Hb,
then the modified Edman procedure
might be useful in monitoring exposure to
certain chemicals that react poorly with
Hb.
Although N-terminal alkylvalines and
cysteine sulfinamides can be detected
and quantified by methods that eliminate
most of the time-consuming steps
associated with protein analysis, thorough
extraction and derivatization of each
blood sample is still required. Also, GC-
MS is not ideally suited to mass
screening due to the expense of the
instrumentation and the level of training
January 5, 1990 12:24 PM required for
itsoperation. The obvious solution to
these problems is the development of
antibodies for the modified valine
residues and the free aromatic amines
generated by the procedures described
above. Such antibodies could be used to
quantify hydrolyzable adducts in the
super-natantafter the adducts had been
released from the protein and
concentrated into a smaller volume.
The use of antibodies that could
recognize non-hydrolyzable adducts in
situ would cut out even more steps in the
analysis. While some such adducts will
occur on the surface of the proti
molecule, others will be buried
hydrophobic clefts and be le
accessible to the antibody. Neverthele
it should be possible to expose most
not all, hemoglobin adducts by treat!
the sample with denaturing agents
proteases prior to analysis. Me
research is required to determi
whether antibodies to protein adducts c
be developed which can be used
quantify adduct levels in intact prote
without severely reducing the sensitiv
of the immunoassay.
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F.C. Schnell and T.C. Chiang are with Lockheed Engineering and Sciences Co.,
Las Vegas, NV 89119
Charles H. Nauman is the EPA Project Officer (see below).
The complete report, entitled "Protein Adduct-Forming Chemicals for Exposure
Monitoring: Chemicals Selected for Further Study," (Order No. PB90
1517621 AS; Cost: $23.00, 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 Center for Environmental Research
Environmental Protection Information
Agency Cincinnati OH 45268
Official Business
Penalty for Private Use $300
EPA/600/S4-89/035
000085833 PS
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