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EPA 600/4-89/035
October 1989
PROTEIN ADDUCT FORMING CHEMICALS FOR EXPOSURE MONITORING:
CHEMICALS SELECTED FOR FURTHER STUDY
by
Frank C. Schnell
Tom C. Chiang
Lockheed Engineering and Sciences Company
Las Vegas, Nevada 89119
Contract No. 68-03-3249
Work Assignment Manager
Charles H. Nauman
Exposure Assessment Research Division
Environmental Monitoring Systems Laboratory
Las Vegas, Nevada 89193
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193
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NOTICE
The information in this document has been funded wholly or
in part by the United States Environmental Protection Agency
under contract 68-03-3249 to Lockheed Engineering and Sciences
Company, Environmental Monitoring Systems Laboratory, Las Vegas,
Nevada. It has been subjected to the Agency's peer and
administrative review, and it has been approved for publication
as an EPA document. Mention of trade names or commercial
products does not constitute endorsement or recommendation for
use.
11
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ABSTRACT
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 in the
evaluation of health risks and in the development of regulations
that may be needed for protection from chemicals released into
the environment. 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 risk to individuals and populations. Only by
relating biological measurements to environmental monitoring
measurements may the relationships between total exposure,
effective dose and disease be determined.
In a 1987 report entitled "Carcinogen-DNA Adducts:
Introduction, Literature Summary and Recommendations", it was
recognized that hemoglobin and serum albumin adducts may be more
advantageous than DNA adducts as biological markers of exposure,
because the protein adducts are more stable and are accessible
from humans in much larger quantities.
Subsequently, a 1988 U.S.EPA report entitled "Protein
Adduct-Forming Chemicals For Exposure Monitoring: Literature
Summary and Recommendations" 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. Conclusions were drawn and
proposals 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.
The present report is an expanded'treatment of those
chemicals recommended for further study in the 1988 protein
adducts report. The recommended chemicals were ranked by their
potential for exposure monitoring by protein adduct-based
methods. The prioritized list of selected chemicals is here
reproduced in the introduction, where the ranking scheme is also
explained. The individual chemicals are discussed in subsequent
sections 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.
iii
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CONTENTS
ABSTRACT iii
FIGURES V
TABLES vi
ACKNOWLEDGEMENTS vii
1. INTRODUCTION 1
2 . CONCLUSIONS AND RECOMMENDATIONS 13
3 . ETHYLENE OXIDE 25
4 . PROPYLENE OXIDE 41
5. STYRENE OXIDE 47
6. 4-AMINOBIPHENYL 54
7 . BENZIDINE 65
8 . 4.4' -METHYLENEBIS (2-CHLOROANILINE) 72
9 . o-TOLUIDINE 77
10. N-NITROSONORNICOTINE 81
11. BENZO[a]PYRENE 86
12 . 1-NITROPYRENE 93
13 . VINYL CHLORIDE 96
14 . ETHYLENE DICHLORIDE . . 101
15 . ACRYLONITRILE 106
16. ACRYLAMIDE Ill
17 . CHLOROFORM 114
18 . BENZENE 119
19 . FORMALDEHYDE 125
20. 2,4-TOLUENE DIISOCYANATE 131
21. 7,12-DIMETHYLBENZ ANTHRACENE 134
22 . EPICHLORHYDRIN 137
23 . BENZYL CHLORIDE 141
24 . PENTACHLOROPHENOL 144
REFERENCES 147
IV
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FIGURES
Number pace
1. Relationship between environmental exposure,
biologically effective dose, and chemical
carcinogenesis ............................... .... 2
2. Procedure for analysis of total protein (i.e.,
hemoglobin) for adducts using GC-MS .............. 36
3. Edman degradation procedure applied to (A)
unmodified and (B) alkylated N-terminal valine
in Hb ............................................ 38
4. Modified Edman procedure for analyzing N-terminal
valine adducts in hemoglobin by GC-MS ............ 39
5. Proposed scheme for formation in vivo and
hydrolysis in vitro of acid-sensitive
hemoglobin-4-ABP adducts ......................... 57
6. Hemoglobin adduct formation as a linear
function of single intraperitoneal doses
of 4-aminobiphenyl ................... « ........... 59
7. Accumulation and elimination of hemoglobin
adducts with chronic administration of
4-aminobiphenyl .................................. 60
8. Procedure for analyzing arylamine-hemoglobin
adducts by GC-MS ................................. 63
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TABLES
Number Page
1. Prioritized list of protein adduct-forming
compounds of interest to the EPA 10
2. U.S. Manufacturers of Ethylene Oxide 26
3. Exposure and Background Levels of Some Human
Hemoglobin-Ethylene Oxide Adducts 34
4. U.S. Production of Propylene Oxide 42
5. U.S. Production of Styrene 48
6. Exposure and Background Levels of Some
Hemoglobin-Aminobiphenyl Adducts 62
7. U.S. Production of 0-Toluidine 78
8. U.S. Production of Vinyl Chloride 97
9. U.S. Production of Ethylene Dichloride 102
10. U.S. Production of Acrylonitrile 107
11. U.S. Production of Chloroform 115
12. U.S. Production of Benzene 120
13. U.S. Production of Formaldehyde 126
14. U.S. Production of Epichlorohydrin 138
15. U.S. Production of Benzyl Chloride 142
vi
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ACKNOWLEDGMENTS
The authors wish to acknowledge the assistance of the
following persons: John Sanford for his help in updating the
production information in this report; Peggy Oakes and Pam
O'Bremski, for word processing support; Marie Schnell for
editing; and David Schnell for word processing and proofreading.
vii
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1. INTRODUCTION
1.1. BIOLOGICAL VS ENVIRONMENTAL MONITORING
The basic principles and problems of exposure monitoring,
dosimetry and risk assessment have been reviewed by the
following: Perera and Weinstein (1982); Weinstein and Perera
(1982); Lauwerys (1984); Aitio (1984); Van Sittert and De Jong
(1985); Garner (1985); Hattis (1986); Wogan and Tannenbaum
(1987); and Ashby (1988). The emphasis is on monitoring human
exposure to genotoxic chemicals in the environment.
Traditionally, human exposure to an environmental pollutant
has been estimated from direct measurements of its concentration
in one or more environmental compartments, i.e., air, water, soil
and food (Figure 1). Exposure assessments based on ambient
monitoring cannot, however, take into account the effects of: (l)
actual duration of exposure (e.g., intermittent vs. continuous),
(2) multiple sources of exposure, (3) multiple routes of entry
(i.e., respiratory, dermal and gastrointestinal), (4) absorption,
(5) distribution within the body, (6) metabolism, and (7)
excretion (Klaassen, 1980). Consequently, ambient monitoring can
provide only crude estimates of the potential exposure of
populations; for a given individual, the data cannot accurately
reflect either actual exposure or degree of risk.
Only that portion of the total exposure dose actually
absorbed by the organism is relevant to the exposure-related
health risk incurred by the organism. Biological monitoring
represents an effort to estimate that in vivo dose and entails
the measurement of so-called "biomarkers1*, e.g., the
concentration of chemical species, their metabolites, or reaction
products or other biochemical effects of exposure. Biomarkers of
exposure reflect actual absorption via all routes of entry from
single and multiple sources, integrate the consequences of
intermittent as well as continuous exposure, and take into
account subsequent metabolism and distribution in the body
(Figure 1).
Not only can biomarkers of exposure serve as dosimeters of
exposure for a single individual, but, collectively, their
measurement could provide a more accurate and detailed picture of
exposure and risk at the population level. Biological monitoring
in humans should also help bridge the gap between animal
experimentation and human epidemiology, provide an improved basis
for species extrapolation, and define the nature and scope of
interindividual variation (Vahakangas, 1984; Calabrese, 1985).
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COMPARTMENT
SAMPLING
The Environment:
FOOD
SOIL
AIR
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(ABSORPTION)
V
Portals of Entry (skin, lungs, G.I. tract)
ENVIRONMENTAL
MONITORING
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BIOLOGICAL
MONITORING
Figure 1.
Relationship between environmental exposure,
biologically effective dose, and chemical
carcinogenesis.
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The methodologies for biomonitoring human exposure to
chemicals (especially carcinogens) in the environment have been
reviewed by Perera and Weinstein (1982), Wright (1983), Lauwerys
(1984), Lohman et al. (1984), Van Sittert (1984), Van Sittert and
De Jong (1985), Garner (1985), Perera (1987), Wogan and
Tannenbaum (1987) and Santella (1988).
In general, biomonitoring entails making measurements of (1)
the concentration of the parent compound or its metabolites in
biological media such as blood and urine, (2) the concentration
of reaction products between the compound or its metabolites and
macromolecules such as DNA and protein, or (3) biological
endpoints such as chromosomal aberrations, sister chromatid
exchanges (SCE's), unscheduled DNA synthesis, antibody titer and
altered enzyme levels. While these endpoints are not themselves
considered to be adverse health effects, they may indicate the
presence of either the chemical or adverse health effects
resulting from exposure to the chemical.
With some chemicals, levels of urinary metabolites correlate
reasonably well with exposure dose. However, measurements of
urinary metabolites ignore that proportion of the dose that
evades detoxification and exerts its effect at the site of
action, and they generally can only reflect exposures that have
occurred during the previous 48-72 hours. Similar disadvantages
apply to measurements made in other body fluids.
Biological endpoints such as chromosome aberrations and
SCE's are of interest because they are indicative of DNA damage,
and because it is generally considered that DNA damage of some
sort is the initiating event in carcinogenesis. However, the
biological significance of these endpoints remains unclear, and
the methods are generally non-specific and subject to
methodological problems related to sampling time. By contrast,
macromolecular adducts tend to be more specific for the
responsible exposure, and their biological significance is less
obscure.
1.2. METABOLISM AND ADDDCT FORMATION
Excellent reviews of xenobiotic metabolism and chemical
carcinogenesis by Neal (1980), and Weisburger and Williams
(1980), respectively, may be found in the toxicology textbook by
Casarett and Doull (1980). The same topics are addressed in a
text by Hodgson and Guthrie (1980). Jeffrey (1985) has reviewed
DNA adduct formation by chemical carcinogens.
The majority of xenobiotics that enter the body are
lipophilic, hence their ability to penetrate cell membranes and
be transported by lipoproteins in the body fluids. In various
tissues of the animal body, but especially in the liver, several
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enzyme systems exist that render lipophilic xenobiotics more
water soluble and, thus, more readily excreted. This
biotransformation is generally accomplished in two phases. In
Phase I, a polar, reactive group is introduced into the molecule,
thereby increasing the latter's water solubility and, more
important, its suitability as a substrate for Phase II reactions.
In Phase II, reaction between the modified xenobiotic and an
endogenous substrate produces a more water-soluble conjugate
which may then be readily excreted.
The most important Phase I reactions are catalyzed by the
microsomal mixed-function monooxygenases, a non-specific,
multienzyme system having cytochrome P-450 as the terminal
oxidase. As the term "microsomal" indicates, this enzyme system
is located in the endoplasmic reticulum of the cell; it is most
abundant in hepatocytes, i.e., liver cells. Among the reactions
catalyzed by the cytochrome P-450-containing monooxygenases, the
most important ones for protein-adduct formation are epoxidation
and hydroxylation. Epoxidation is the primary activation pathway
for many unsaturated compounds, especially polynuclear aromatic
hydrocarbons. Examples include ethylene, propylene, styrene,
vinyl chloride, acrylonitrile, acrylamide, benzo(a)pyrene (E(a)P)
and 1,2-dimethylbenzanthracene (DMBA). N-hydroxylation is the
primary activation pathway for aromatic amines such as 4-
aminobiphenyl (4-ABP), benzidine, and methylenebis-
(2-chloroaniline) (MBOCA).
Reactive metabolites generated by phase I reactions in the
liver can react directly with macromolecules of the liver and the
blood that perfuses that organ. If the biological half-life of
the ultimate electrophile is long enough, the latter may migrate
to and covalently bind at distant sites of action. Short-lived
reactive intermediates may undergo phase II reactions to form
more stable conjugated species. These can be transported to
extrahepatic target tissues where they may be enzymatically
deconjugated. The "re-activated" ions or radicals thus created
may then bind to DNA and proteins at the remote site of action.
Adverse health effects such as cancer and isocyanate lung disease
can be caused by specific adducts of DNA and protein,
respectively. By monitoring the formation of other, relatively
innocuous macromolecular adducts, potentially hazardous exposures
may be terminated at a pre-symptomatic stage.
1.3. PROTEIN ADDUCTS AS DOSIMETERS
Compared to other biomarkers of exposure, macromolecular
(i.e., DNA and protein) adducts formed in vivo following a
genotoxic exposure tend to be more specific for that genotoxic
chemical exposure, and the methods available for their detection
are more precise and extremely sensitive. In addition, protein
adducts, unlike most other biomarkers, can quantitatively reflect
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exposure that has occurred during the previous weeks or months,
depending on the lifespan of the protein (Ehrenberg, 1984;
Poirier et al., 1987).
As the target molecules of genotoxic xenobiotics or their
electrophilic metabolites, DNA would be the preferred
macromolecule in which to measure the formation of carcinogen-
protein adducts as a biomarker of both exposure and effect.
However, DNA adducts occur at very low levels in vivo, and the
rates of repair and excretion usually limit their usefulness as
biomonitors to 24-48 hours after exposure. Proteins, on the
other hand, tend to form relatively high levels of adducts with
electrophilic compounds in general, including virtually all those
known to bind to DNA. Perhaps more important, protein adducts
are generally not repaired. Rather, they persist and accumulate
for the life of the protein, thereby integrating the relevant
exposure(s), intermittent and/or continuous, over the same period
of time. Also, blood sampling is more convenient and less
invasive than sampling of other tissues such as fat.
Of course, protein adducts generally cannot be used as
biomarkers of genotoxic risk as certain DNA adducts can, because
the adducts formed between carcinogens and blood proteins are not
directly relevant to carcinogenesis. However, in those special
cases where the two share a common ultimate electrophile, a
protein adduct may serve as a surrogate dosimeter for the
corresponding DNA adduct.
Theoretically, any protein will form adducts at nucleophilic
centers with electrophilic chemicals or metabolites, and, thus,
could serve as a biomonitor of exposure to such agents. However,
hemoglobin (Hb) and serum albumin (SA) are particularly suitable
because large quantities are readily available for analysis in a
5-10 ml sample of human blood. Hemoglobin adducts in particular
show great potential as biomarkers of exposure to genotoxic
compounds.
1.4. HEMOGLOBIN ADDUCTS
The biology of the erythrocyte is discussed at length in the
definitive work by Harris and Kellermeyer (1970) and the
biochemistry of hemoglobin has been reviewed by Dickerson and
Geis (1983).
Hemoglobin represents 10-15% of whole blood by weight (5 ml
of whole blood yields 500-750 mg Hb). It is synthesized in the
immature red cell as the latter develops in the bone marrow, and
shares with the erythrocyte a lifespan of approximately 120 days
in man.
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Some sixty compounds, including examples from most of the
important classes of mutagens and carcinogens, have been shown to
form hemoglobin adducts in animals (Calleman, 1986). The number
of fully characterized adducts, while very much smaller, is, and
will most probably remain, greater for Hb than for any other
protein. This is important, since only fully characterized
adducts are of any real use as biomarkers of specific exposures.
The main nucleophilic centers in proteins are (1) the sulfur
atoms of cysteine and methionine; (2) the nitrogen atoms of amine
groups, ring systems, and guanido groups; and (3) the oxygen
atoms of hydroxyl and carboxyl groups. The most intensively
studied protein adducts have been selected alkyl histidines, N-
terminal valines, and cysteine sulfinyl aromatic amines.
Interest in the alkyl histidines has declined in recent years as
improved methodologies have made it possible to study the same
alkyl moieties at alternate sites without the necessity of
analyzing total protein hydrolysates.
Two such methodologies commonly used in the analysis of Hb
adducts are (1) the modified Edman degradation procedure for
adducted N-terminal valines, and (2) GC-MS analysis of acid/base-
labile cysteine sulfinamide adducts using negative chemical
ionization. These two methods are described here in the sections
on ethylene oxide and 4-aminobiphenyl, respectively. In
addition, intact adducts or their cleavage products that are
strongly fluorescent may be measured using fluorescence
spectrophotometry (Jankowiak et al., 1988; Shugart, 1986).
Immunoassays are currently under development and show great
promise for application to both environmental and biological
monitoring (Vanderlaan et al., 1988; Roberts et al., 1986).
In general, the protein adducts studied by these methods
have exhibited a linear dose-response curve over some specified
dose range. Sometimes, as in the case of 4-aminobiphenyl, that
dose range may be quite large. Stable protein adducts also tend
to be accumulated in a dose-related manner and, upon cessation of
the exposure, eliminated at a rate that reflects the turnover of
the adducted protein. Along with the ready availability in blood
of large quantities for analysis, it is the predictable pharmaco-
kinetics of Hb adducts that makes them useful as biomarkers of
exposure to environmental contaminants.
The ability to monitor chemical exposures via Hb adducts is
limited not so much by the sensitivity of available analytical
techniques as it is by the presence in some cases of background
levels that tend to mask the effects of low level exposures.
Confounding exposures (i.e., different chemicals, or different
sources of the same chemical, that can produce identical adducts)
from unknown and/or endogenous sources contribute to these
background levels.
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There are a few adduct-forming chemicals (e.g., aflatoxin
Bx) that do not form detectable levels of Hb adducts in vivo.
Such chemicals may be metabolized in the liver to reactive
species with half-lives too short to allow significant migration
to and reaction with extrahepatic targets. In such cases,
protein adducts may, nevertheless, be measurable in another blood
protein, serum albumin (Sabbioni et al., 1987).
1.5. SERUM ALBUMIN ADDUCTS
The structure and function of serum albumin has been
thoroughly reviewed in a book edited by Rosenoer, Oratz and
Rothschild (1977).
Albumin, the most abundant protein in human plasma (60% by
weight), is synthesized in the liver on the rough endoplasmic
reticulum of hepatocytes (Rothschild et al.,1977), the same liver
cells that generate the reactive metabolites of many xenobiotics.
Newly synthesized albumin is secreted by the hepatocytes
directly into the systemic circulation, from which compartment it
later equilibrates with lymph and interstitial fluid. While 75%
of the intravascular pool of albumin may exchange with the
extravascular pool in a couple of days, complete redistribution
may take more than a week. The plasma compartment contains only
31-42% of the exchangeable albumin pool. Some 30-40% of the
total extravascular albumin is stored in the skin. These facts
would have to be taken into account in any calculation of tissue
dose from levels of serum albumin adducts.
Because it is not protected by a cell membrane as hemoglobin
is, serum albumin becomes modified with age (e.g., asparagine and
glutamine are deaminated) and is subject to random destruction at
sites as yet unidentified. The mean half-life of human serum
albumin (HSA) and, hence, HSA adducts is 14-20 days, which
results in a normal daily turnover of 8.4-10.6% of the
intravascular albumin pool (Waldmann, 1977). Serum albumin may
be lost at a much faster rate in response to gastrointestinal
disease, nephrosis, or severe burns, and in the rare disorder
analbuminemia, serum albumin is lacking altogether.
Thirty-four of the 35 cysteine residues of albumin form 17
disulfide bridges that maintain the protein's tertiary structure
which, due to the large size of the polypeptide chain and the
notorious heterogeneity of serum albumin preparations, has yet to
be determined. (Brown and Shockley, 1982).
Serum albumin functions primarily as a transport protein and
possesses a profusion of binding sites for fatty acids, steroids,
etc. (Brown and Shockley, 1982). Binding of endogenous ligands
such as bilirubin and fatty acids has been shown to inhibit the
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covalent binding of affinity labels at specific sites. In fact,
competitive binding is the basis of the methods that have been
used to map the binding sites of serum albumin. It is reasonable
to expect that a similar process could inhibit formation of
xenobiotic-serum albumin adducts, in which case, adduct levels
would vary not only with exposure, but also with physiological
parameters such as diet and nutritional status. Such potential
sources of individual variation should, therefore, be carefully
explored before any serum albumin adduct is used to monitor
environmental exposure to an environmental contaminant.
Affinity labeling experiments have identified the following
reactive amino acid residues in bovine serum albumin (BSA): Cys-
34, His-145, Lys-220, His-336, and Lys-412 (Brown and Shockley,
1982) . Position 34 in HSA is also occupied by a cysteine residue
which contains the protein's only reactive sulfhydryl group.
This residue binds covalently with free cysteine, glutathione,
and mercury, and is a known site of xenobiotic-serum albumin
adduct formation. The other four positions do not contain the
same amino acids in HSA as they do in BSA, but nearby positions
in HSA do contain the same or similar residues, i.e., His-146,
Gln-221, Arg-218 and -222, His-338, and Lys 413. In addition,
HSA has a highly reactive tyrosine at position 411 that displays
some esterase activity, and a solitary, uniquely reactive
tryptophan at position 214. The specific xenobiotic-serum
albumin adducts described in the literature are formed primarily
at Cys-34, Trp-214 and one or more lysine residues.
Considering that, compared to hemoglobin, serum albumin has
a shorter half-life in the bloodstream, is distributed in two or
more body compartments, exhibits more complicated binding
patterns, and has a less well understood tertiary structure, it
is not surprising that Hb has been preferred over serum albumin
by investigators studying protein adducts as biomarkers of
exposure. However, in those cases where Hb is not an available
option (e.g., aflatoxin B1), serum albumin is the protein of
choice.
1.6. CHEMICAL SELECTION
The individual chemicals discussed in the sections that
follow are, with the exception of aery1amide, those recommended
for further study in a previous report entitled "Protein Adduct-
Forming Chemicals for Exposure monitoring: Literature Summary and
Recommendations" (U.S. EPA, 1989). In that report, the
recommended chemicals were presented in a "Prioritized List of
Protein Adduct-Forming Compounds of Interest to the EPA". That
list, as it appears in a recent update (DRAFT) of U.S.EPA (1989),
is reproduced here as Table 1.
8
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With the exception of acrylamide, all of the chemicals in
Table 1 also appear on a previous EPA list of adduct-forming
chemicals selected for further study (Appendix H of U.S. EPA,
1987) . Thirteen of the chemicals also appear on the first and
second priority lists of 100 SARA compounds (Federal Register,
Apr 17 and Oct 20, 1988). Both of these lists reflect the
importance of a chemical from the Agency's perspective. In
formulating its list of chemicals selected for further study, the
U.S. EPA took into consideration the adequacy of the existing
database, the availability of an exposed human population, and
the level of genotoxic/toxicological activity. DNA adduct
formation was then reviewed for 13 selected chemicals (Uziel et
al., 1988).
The compounds listed in Table 1 are a subset of the larger
number of chemicals discussed in a summary of the protein adduct
literature (U.S. EPA, 1989). 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-character!zed 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).
It was sometimes necessary to compromise on the last two
criteria, because human exposure to known chemical hazards tends
to be reduced by regulation to very low levels soon after the
hazard to human health is established. Exposed populations may
also be unavailable for some chemicals having large research
databases (i.e., those particularly well represented in the
scientific literature), because such chemicals tend to be those
of greatest toxicological interest, i.e., those most likely to be
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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-Tenninal Valine Adducts.
1) Ethylene Oxide
2) Propylene Oxide
3) Styrene
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-Nitropyrene
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
10
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strictly regulated. The absolute ranking of a particular
chemical will therefore depend upon how specific selection
criteria are "weighted" for that compound. The prioritization
scheme used to generate Table 1 was intended to emphasize rather
than obfuscate such ambiguities in the ranking process. That
prioritization scheme is described below.
Because (1) the protein adduct formed, rather than the
chemical itself, is the object of analysis, and (2) the
practicality of that analysis depends largely on the
methodologies available, the chemicals were first put into groups
according to the following characteristics of the adducts that
they form:
(1) Chemical class of adduct, in the case of cysteine
sulfinamides, and N-terminal alkylvalines only. Special
methods are available for the identification of these
particular adducts.
(2) Degree of characterization (including adduct
identification, rate of formation, background levels and
dose-response relationship). Only well-characterized
adducts can be monitored in humans.
(3) Hydrolyzability (i.e., The adduces ability to be
isolated by mild acid/base treatments that leave the
rest of the protein essentially intact). Quantification
of non-hydrolyzable adducts is expensive and time-
consuming, requiring the analysis of total protein
hydrolysates.
Next, these groups of chemicals (rather than the individual
chemicals themselves) were then ranked in descending order
according to the ease with which the respective protein adducts
can be analyzed. Thus, chemicals that form well-characterized,
hydrolyzable N-terminal valines or cysteine sulfinamides were
ranked highest because these adducts may be most readily
quantified by existing, relatively convenient methods.
Similarly, groups of chemicals that form less well-characterized
and/or non-hydrolyzable adducts received lower rankings. The
chemicals in Groups I and II may be considered to share the top
ranking; they were listed separately to emphasize the fact that a
different method of protein-adduct analysis is peculiarly
applicable to each group.
Finally, the individual chemicals within a given group were
loosely ranked according to a) the size and quality of the
published database pertaining to the protein-adducts they form,
and b) the availability of exposed and control populations for a
human monitoring study. Occasionally, these criteria were in
conflict with one another. For example, the established human
carcinogen 4-ABP is the best documented protein adduct-forming
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aromatic amine. However, with the exception of cigarette
smokers, human populations exposed to significant amounts of 4-
ABP are no longer available in the U.S. By contrast, MBOCA is
not currently regulated and occupationally exposed populations
are available. Presently, however, MBOCA is poorly represented
in the protein-adduct literature. The relative ranking of such
chemicals will depend on how the two criteria are weighted.
This prioritization scheme is more informative and flexible
than a more conventional numerical ranking system. The grouping
of a compound says something specific about the chemical nature
of the adduct it forms and the methods appropriate to its
analysis. From the compound's relative position within a group,
one may infer something about population availability and/or
research status. Also, the grouping of compounds as well as
their relative position within a group may be routinely
readjusted as additional data warrant. As more information
becomes available for a particular adduct or new methods are
developed for its analysis, the appropriate new position for the
corresponding chemical should become readily apparent.
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2. CONCLUSIONS and RECOMMENDATIONS
2.1 GENERAL COMMENTS
Protein adducts have been demonstrated, in man as well as in
laboratory animals, to be useful as biomonitors of exposure to
environmental chemicals. The list of chemicals that form protein
adducts is long, and includes virtually all genotoxic carcinogens
as well as many non-carcinogens (Calleman, 1986). Protein
adducts are generally stable and accumulate in a dose-dependent
manner over the lifetime of the protein, thereby integrating the
effects of both intermittent and continuous exposures from all
sources and by all routes. This property is particularly
important at the low levels of environmental contaminants to
which humans are chronically exposed.
Levels of protein adducts also tend to be related to levels
of DNA adducts (Neumann, 1984b, 1986). The exact ratio between
the two will vary, depending on both the chemical and the tissue
under study, and must be determined on a case-by-case basis. It
is important to keep in mind, however, that absolute levels of
protein binding do not consistently correlate with known
carcinogenic potency (Pereira and Chang, 1981). For example,
aflatoxin B, is a potent liver carcinogen, but it binds to Hb in
very low yield (Wogan and Tannenbaum, 1987). Also noncarcinogens
as well as carcinogens can bind to Hb (Garner, 1985).
Hemoglobin and serum albumin adducts are readily available
in large amounts in a 5-10 ml blood sample, and existing
analytical methodologies are sufficiently sensitive to measure
adduct levels resulting from low-level human exposures to adduct-
forming chemicals. The hemoglobin adducts of over 30 chemicals
have been studied in vivo, and several of these have been used to
monitor exposure in human beings.
By contrast, the albumin adducts of only a few compounds
have been studied in vivo, e.g., 4-aminobiphenyl (Skipper, et
al., 1985), and aflatoxin B. (Sabbioni, et al., 1987). Serum
albumin has a shorter half-life than does Hb, and is not as
conveniently isolated. Consequently, albumin adducts tend to
interest researchers only when a chemical does not form useful
levels of hemoglobin adducts (e.g., aflatoxin B,), or when the
albumin adduct may provide additional information that the
hemoglobin adduct alone can not (e.g., the effect of acetylator
status on 4-aminobiphenyl adduct formation). Because albumin is
synthesized in the same cell (i.e., the hepatocyte) that
generates the majority of reactive metabolites, albumin adducts
will continue to warrant special scrutiny wherever the reactive
metabolites of the chemical under study are too short-lived to
migrate far from the tissue of origin, i.e., the liver.
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2.2 GROUP I CHEMICALS: ETHYLENE OXIDE, PROPYLENE OXIDE,
and STYRENE.
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 tissues than any other
chemical studied thus far.
PO, a closely-related homologue of EO, should exhibit similar
pharmacokinetics. The highest exposures to PO are likely to
occur among production workers. The background levels of PO-Hb
adducts are lower than those of EO-Hb adducts, making the former
the more informative biomarkers of exposure over a larger range
of exposure. Osterman-Golkar's group in Sweden has performed
protein-adduct studies on industrial workers exposed to EO and PO
in the work place. Adduct levels in workers with low exposure to
PO were significantly higher than those in controls (Osterman-
Golkar et al., 1984), whereas high levels of exposure to EO were
required to raise adduct levels above background (Farmer et al.,
1986a; Tomgyist et al., 1986a).
Compared with ethylating agents, the simple arylating agent
styrene produces adducts which exhibit lower background levels
and a cleaner GC elution profile. Both factors would enhance the
sensitivity with which styrene adducts could be detected by
chromatographic procedures. Studies of human exposure to styrene
have been proposed by Nordqyist et al. (1985) and Perera (1987).
Investigators at Columbia University are currently conducting a
molecular epidemiological study of occupational exposure to
styrene in boat-building facilities in Maine and Connecticut.
The heaviest exposures occur during hull and deck lamination. In
addition to hemoglobin adducts, this study proposes to
characterize and validate four other biomarkers of exposure:
lymphocyte DNA adducts, micronuclei, sister chromatid exchanges,
and unscheduled DNA synthesis. Blood samples collected from
styrene-exposed workers and currently being analyzed.
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Human exposure to ethylene oxide, propylene oxide and
styrene can best be monitored by measuring their hydroxyethyl-,
hydroxypropyl- and hydroxyphenylethyl-valine adducts,
respectively. Background levels of the valine adducts are lower
than those of the histidine and cysteine adducts, and can be
measured by the modified Edman degradation technique (Torngvist
et al., 1986a; Mower et al., 1986) which eliminates the need to
perform tedious and time-consuming analyses of total protein
hydrolysates or enzymatic digests. Under basic conditions in the
presence of pentafluorophenyl isothiocyanate, the modified
terminal amino acids (valines in hemoglobin) are selectively
cleaved from the protein. The resulting pentafluorophenyl-
thiohydantoin (F.PTH) derivatives may then be extracted and
analyzed by negative chemical ionization mass spectrometry
(NCIMS).
Because styrene,7,8,oxide, the major reactive metabolite of
styrene, is more reactive and has a shorter biological half-life
than ethylene oxide, and because serum albumin is synthesized in
the hepatocyte where the reactive metabolites are generated, it
is possible that more styrene adducts are formed with albumin in
the liver cells than with hemoglobin in the red cell. However,
further research is required in this area, as no reports of
styrene-albumin adducts were found in the literature. If
styrene,7,8,oxide does form adducts at the N-terminal aspartate
of serum albumin, it might be possible to detect such adducts
using the modified Edman procedure. Again, however, more
research is required since 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. The
method of Waith et al. (1987), which employs an antibody to N-(2-
HOEt)Val in the form of the tryptic N-terminal heptapeptide of
the -chain of human hemoglobin, has been fully validated against
GC-MS and used to successfully distinguish between EO-exposed and
non-exposed groups of hospital workers. If an antibody could be
raised against the FSPTH derivative of modified 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 F.PTH 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
(Wallin et al., 1987) .
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2.3 GROUP II CHEMICALS: AROMATIC AMINES
The aromatic amines are 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 6-93 cysteine
of hemoglobin. The ultimate electrophile, a nitroso compound, is
formed in the erythrocyte by co-oxidation of the N-hydroxylamine
metabolite and hemoglobin. 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 are 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. Benzidine and 4-ABP are established
human bladder carcinogens. Benzidine and MBOCA also rank high on
the list of adduct-forming chemicals of interest to the U.S. EPA.
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". Acetylation of aromatic
amines, which, after subsequent hydroxylation, yields the
corresponding hydroxamic acid, appears to represent the
detoxification pathway in man. By contrast, hydroxylation of
aromatic amines to the corresponding hydroxylamine appears to be
critical to the initiation of arylamine-associated bladder
cancer. Since the hemoglobin (i.e., cysteinyl) adduct mentioned
above is formed from the hydroxylamine, higher levels of this
adduct might reasonably be expected in members of the high risk
group (i.e., slow acetylators) as compared to those of the low
risk group (i.e. fast acetylators), under similar conditions of
exposure. If these expectations should be born out in a pilot
monitoring study, then a single protein adduct will have been
proven useful as a biomarker of risk as well as exposure.
In the case of 4-aminobiphenyl, a unique protein adduct
marker also exists for the hydroxamic acid pathway, i.e., the N-
acetyl-4-ABP adduct of the tryptophan residue at position 214 in
serum albumin. (The acetylated amino group of aromatic amines
does not bind to the cysteine of hemoglobin.) It should be
instructive to measure this adduct simultaneously with the
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cysteine sulfinamide of hemoglobin, the expectation being that
levels of the two adducts would vary inversely with one another
depending upon acetylator phenotype. Unfortunately, however, the
serum albumin adduct (in the rat) is formed in amounts two orders
of magnitude lower than the hemoglobin adduct, and, unlike the
latter, it cannot be made to regenerate ABP. These problems
might be overcome by the use of an anti-ABP antibody (Skipper et
al., 1986). However, immunoassay research on ABP adducts is
still in the developmental stage, and it has not yet been
demonstrated that similar albumin adducts are formed with
benzidine and other aromatic amines.
Because benzidine has two amino groups instead of one, it
may be possible to measure the partially acetylated metabolite of
benzidine as a more abundant hemoglobin adduct. In rats (fast
acetylators by human standards), the major benzidine-hemoglobin
adduct is a sulfinic acid amide formed from the reaction of 4-
nitroso-4'-N-acetylaminobiphenyl with cysteine (Albrecht and
Neumann, 1984; Dolle et al., 1980); it constitutes 70% of the
total binding of benzidine to rat hemoglobin. Upon treatment
with 0.1N HC1, the modified hemoglobin releases monoacetyl-
benzidine which can then be measured by GC as the
pentafluoroproprionic acid amide derivative.
If, as seems likely, the same adduct is formed to some
extent in human hemoglobin (especially fast acetylators), then
acid-released monoacetylbenzidine could be used as an indicator
of benzidine exposure and metabolism (of one nitrogen, at least)
via the detoxification pathway in man. If acetylation of both
amino groups occurs in man, the resulting adduct would have to be
measured some other way, perhaps by an albumin adduct, since
diacetylbenzidine will not form hemoglobin adducts.
To the extent that both amino groups of benzidine are
metabolized in man via the activation pathway, the Hb adducts
formed by the resulting dihydroxylamine should yield the same
hydrolysis product that N-hydroxybenzidine-Hb adducts do, i.e.,
the parent amine. More research is required to determine the
precise identity and relative amounts of benzidine adducts in
human hemoglobin. However, if it is confirmed in the laboratory
that benzidine and monoacetylbenzidine are released by acid
treatment of human hemoglobin-benzidine adducts, then it may be
possible to simultaneously measure, in the same sample of blood,
two different biomarkers of exposure to benzidine, and to relate
those measurements to the individual's established acetylator
status, i.e., relative risk for bladder cancer.
As toxicologically interesting as they are, aromatic amines
may prove difficult to study in humans because, outside of
cigarette smokers, exposed populations will be difficult to find.
One possible exception to that statement is MBOCA. MBOCA, a
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suspected bladder and liver carcinogen, is still widely used in
the polyurethane industry, where an occupationally exposed
population should be available.
The N-hydroxy metabolite of MBOCA has been shown to form
hemoglobin adducts in the guinea pig (a fast acetylator). Upon
acid treatment in vitro, these hemoglobin adducts yield the
parent compound as a cleavage product. These findings come from
the ongoing research (not yet published) at the Department of
Pharmacology and Toxicology at Michigan State University. To
date, the literature is mute on the subject of MBOCA-protein
adducts. It is, therefore, strongly recommended that the
research findings at Michigan State University be followed
closely in the future for its relevance to hemoglobin-aromatic
diamine adducts in general and MBOCA adducts in particular.
2.4 GROUP III CHEMICALS: NUN, B(a)P and 1-NITROPYRENE
N-nitrosonornicotine (NNN), benzo(a)pyrene (B(a)P), and 1-
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-(methylnitrosamino)-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 -hydroxylation (the major
activation pathway) to methylating agents (Belinsky et al., 1986
and 1987) or, to a lesser extent, to 4-(3-pyridyl)-4-
oxobutyldiazohydroxide which forms unique, bulky adducts in both
Hb (Carmella and Hecht, 1987) and liver DNA (Hecht et al., 1988)
in rats. The latter adducts are detectable as their hydrolysis
product 4-hydroxy-l-(3-pyridyl)-l-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
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adducts are detectable in blood samples of exposed humans, low
adduct levels and high interindividual variation can be
anticipated.
B(a)P, a carcinogenic polycyclic aromatic hydrocarbon (PAH),
is ubiquitous in the environment, even though it is not
commercially produced in the U.S. The benz(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 (Weston et al.,
1989) .
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 (Jankowiak et al., 1988; Can et al., 1989) and by
competitive ELISA (Lee and Santella, 1988). 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 column.
The highest level of B(a)P adducts occurs in liver proteins
(Wallin et al., 1987), of which newly synthesized albumin is one,
and total binding of radiolabeled B(a)P is three orders of
magnitude higher in serum albumin than in Hb (Balhorn et al.,
1985). An investigation of B(a)P-serum albumin adducts is
therefore recommended. Serum albumin adducts have proven very
useful for monitoring exposure to aflatoxin B1/ another compound
which binds poorly to Hb.
1-Nitropyrene, a widespread environmental PAH, is neither
produced nor used commercially in the U.S. Exposure to this
carcinogenic PAH occurs via inhalation of airborne particulates,
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 (Johnson et al., 1988). The acid-labile Hb
adduct, which is released into solution during the precipitation
of globin in acidic acetone, has been tentatively identified as
the cysteinyl sulfinamide of a ring-hydroxylated N-hydroxylamine
metabolite. This adduct 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.
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2.5 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 work place
are probably quite low (i.e., below the 1 ppm, 8 hr 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
(Williams et al., 1985). A protein adduct-based method for
exposure monitoring would be particularly useful in the case of
vinyl chloride, since no reliable method currently exists for
monitoring exposure to VC at concentrations below 5 ppm.
The reactive metabolites of VC, 2-chloroethylene oxide (CEO)
and 2-chloroacetaldehyde (CAA), introduce 2-oxoethyl groups at
the sulfhydryl groups of cysteine, the 1-N and 3-N positions of
histidine, and the amino nitrogen of N-terminal valine (Osterman-
Golkar et al., 1977; Svensson and Osterman-Golkar, 1986; Walles
et al., 1988). The acid-stable adducts S-(2-oxoethyl)cysteine
and N-(2-oxoethyl)histidine are currently of limited use for
monitoring VC-exposure in humans. The N-terminal valine adduct
should have more potential as a biomarker of exposure, but the
modified Edman degradation procedure has, apparently, not yet
been applied to the study of VC-protein adducts, nor have VC-
serum albumin adducts been investigated. It might prove
worthwhile, therefore, to examine serum albumin as well as Hb
using the modified Edman degradation method.
Ethylene dichloride (EDC), the largest volume chlorinated
organic compound currently produced in the U.S., is used
primarily in the manufacture of vinyl chloride. EDC and VC are
metabolized in much the same way; the epoxide metabolites of both
produce 2-oxoethyl adducts which, when hydrolyzed for analysis,
are reduced to 2-hydroxyethyl adducts (Svensson and Osterman-
Golkar, 1986). Thus, the protein adducts of VC, EDC and EO
(which yields 2-HOEt adducts directly) cannot easily be
distinguished from one another in a protein hydrolysate. As with
VC, the modified Edman procedure has not yet been used to assay
levels of the 2-oxoethylvaline adduct. The glutathione (GSH)
conjugate of EDC also forms a unique, bulky DNA adduct, S-[2-
(N7-guanyl)ethyl]GSH (Guengerich et al., 1987), which, measured
as a urinary metabolite, might serve as a more specific biomarker
for EDC exposure. The related compound, ethylene dibromide, has
been shown to bind to 8.A. (139 nmol/g) in treated rats (Ansari
et al., 1988).
Acrylonitrile is an important industrial monomer used
extensively in the manufacture of synthetic fibers, rubbers, and
resins for a variety of consumer goods. It is also used to
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produce aery1amide, an industrial compound used in the
manufacture of polymers for water treatment. Both chemicals are
neurotoxic in mammals and carcinogenic in rodents, and exposures
are expected to be kept to a minimum. Animal data (the only data
available) indicate that both of these 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-carboxyethy)cysteine (the
major adduct) (Geiger et al., 1983; Bailey et al., 1987), 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) (Van Bladeren et al.,
1981; Calleman and Costa, 1989).
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 that are 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 could then 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 (NTP, 1985; Sax, 1987).
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 carcinogen.
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 (Pereira and
Chang, 1982a,b). In vitro the reactive metabolite of chloroform,
phosgene, forms an unusual cyclic 2-oxothiazolidine derivative
with cysteine in Hb (Pereira et al., 1984). This adduct is
hydrolyzed during preparation for GC-MS analysis and measured as
N-hydroxymethyl cysteine. However, there is some controversy
over the identification of N-hydroxymethyl cysteine as the major
product by GC-MS, because the hydroxymethyl amino moiety should
have been too unstable to be isolated as such.
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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.
2.6 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 the work of Rogene
Henderson, Kirk Maples and William Bechtold 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 urea-
formaldehyde 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 (Swenberg et al., 1985b). 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. Formaldehyde-DNA cross-links tend
to be either unstable or rapidly repaired.
2,4-Toluene diisocyanate (TDI) 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
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(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 (Karol, 1986). Instead,
TDI exposure has been monitored with variable success by
measuring the titer of antibodies to endogenous 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 (Watabe et al., 1983).
However, no in vivo studies of DMBA-protein adducts were found in
the literature.
For epichlorhydrin, benzyl chloride and pentachlorophenol,
little information is available apart from the fact that the
radiolabeled compounds do bind to amino acids or protein in
vitro. No in vivo studies have been performed, and no specific
protein adducts have been identified.
2.7 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 species or the Edman
reagent. If the method could be demonstrated to work with SA 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
23
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of training required for its operation. 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 supernatant after 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 protein
molecule, others will be buried in hydrophobic clefts and be less
accessible to the antibody. Nevertheless, it should be possible
to expose most, if not all, hemoglobin adducts by treating the
sample with denaturing agents or proteases prior to analysis.
More research is required to determine whether antibodies to
protein adducts can be developed which can be used to quantify
adduct levels in intact proteins without severely reducing the
sensitivity of the immunoassay.
As soon as rapid, inexpensive and convenient immunoassays
for their detection are devised, protein adducts, particularly
hemoglobin adducts, should quickly become one of the most
valuable biomarkers available for measuring human exposure to
environmental contaminants. In certain special cases (e.g.,
ethylene oxide), it should even be possible to correlate protein
adduct levels with genotoxic risk.
Macromolecular adduct research is advancing rapidly. As
more information accrues on the identity of critical DNA adducts
and the chemical-specific mechanisms of oncogene activation, it
should become possible to select and analyze specific protein
adducts that correlate not only with specific chemical exposures
but also with the risks of developing certain chemically induced
cancers in man. To keep up with these important developments,
and be able to take timely advantage of them, it is strongly
recommended that a computerized database on protein adducts be
established and maintained in parallel with a similar one on DNA
adducts. The recommended database would contain such chemical-
specific information as: manufacture and use, sources and levels
of exposure, known health effects, metabolic pathways of
activation and detoxification, individual variation (including
species differences), ultimate electrophiles, adduct structures
and rates of formation, background levels, dose-response
relationships, analytical methodologies rated by sensitivity,
and costs and ease of performance. Ready access to such
information as it becomes available will prove invaluable in the
near future to those responsible for devising the most effective
means of monitoring environmental threats to public health.
24
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3. GROUP I CHEMICALS:
ETHYLENE OXIDE
3.1 MANUFACTURE AND USE
Ethylene oxide (EO) (CAS # 75-21-8), also known as ethene
oxide, epoxyethane and oxirane, was the 23rd-highest volume
chemical produced in the U.S. in 1985 (Sax, 1987). EO is
currently manufactured by at least 11 different companies at a
dozen different plants in the U.S., two thirds of which are
located in Texas and Louisiana (Table 2). U.S. production of EO
was 5.365 billion Ibs in 1988 (C&EN, 1989). EO is usually
manufactured by the catalytic oxidation of ethylene. The EO
thereby produced is subsequently diluted approximately 10 times
with a carrier gas to reduce its considerable flammability.
Carboxide and Sterilant 12 are nonflammable mixtures of EO in
carbon dioxide (90% by wt.) and in fluorocarbon 12 (88% by wt.),
respectively (HSDB, 1989a).
EO is used primarily as a chemical intermediate in organic
syntheses. Some 90% of all EO is used by industry, mostly in the
manufacture of antifreeze. EO is also important as a fumigant in
agriculture and the health care industry (HSDB, 1989a). It is a
registered fungicide used for treatment by fumigation of the
following:
(1) dental, pharmaceutical, medical & scientific equipment &
supplies including: laboratory instruments; surgical
instruments & prosthetic parts; hypodermic needles and
syringes; heart & lung machines; heat/moisture labile
materials; hospital critical items made of rubber or
plastic; oral inhalation equipment; diagnostic
equipment; thermometers; stainless steel surfaces;
hospital fabrics, sheeting and paper products; grooming
instruments;
(2) foodstuffs and crops such as spices, black walnuts,
copra, packaged cereals, bagged rice, and tobacco;
(3) beehives (empty and diseased) and beekeeping equipment;
(4) books, clothing, upholstered furniture, furs and
valuable packaged documents in vaults;
(5) aircraft premises, buses and railway cars.
Formerly used as a starting material for manufacture of
acrylonitrile, EO may also be used in the organic synthesis of
the chemical products listed below.
25
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TABLE 2. U.S. MANUFACTURERS OF BTHYLENE OXIDE
MANUFACTURERS
SITES OF PRODUCTION
BASF Wyandotte Corp.,
Parsippany, NJ
Hoechst-Celanese Chem. Co.
Dallas, TX
Dow Chem. Co.,
Midland, MI
Caine Chemical
Wilmington, DE
USI Chemicals,
Cincinnati, OH
Scott Specialty Gas,
Plainfield, NJ
Texaco Chemical Co., Div.,
Houston, TX
Union Carbide Corp.,
Ethylene Oxide/Glycol Div.
Danbury, CT
Olin Corp., Olin Chems Div,
Brandenberg, KY,
Shell Oil Co.,
Shell Chem Co., Div.,
Houston, TX
Atomergic Chemetals Corp.,
Plainview, NY
River Road Plant
Geisman, LA
Clear Lake Plant,
Pasadena, TX
Plaguemine, LA
Bayport Plant,
Pasadena, TX.
Morris, IL
Plumsteadville, PA
Port Naches, TX
(phasing out production
around 1990)
2 Plants:
Seadrift, TX, and
Taft, LA
Doe Run Plant,
Brandenberg, KY
Geismar, LA
Farmingdale, NY
26
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polyether polyols
mono-, di-, tri- and tetraethylene glycols
mono- and diethylene glycol monoalkyl ethers
mono-, di- and triethanolamines
arylethanolamines, e.g., phenylethanolamines
nonionic & cationic ethoxylated surfactants
ethylene carbonate and polyethylene oxide
ethylene chlorohydrin
choline and derivatives
hydroxyethyl starch
hydroxyethyl & ethyl hydroxyethyl cellulose
unsaturated polyester resins
acetal copolymer resins
crown ethers
beta-phenylethyl alcohol
In 1984, EO was used as a chemical intermediate in the
synthesis of monoethylene glycol (59%), higher glycols (15%),
ethoxylates (10%), ethanolamines (6%), glycol ethers (5%), and
miscellaneous other chemicals (5%) (HSDB, 1989a).
EO has also been used as a rocket propellant, a ripening.
agent for fruits, an inactivator of Kreb's ascites tumor cells,
and a maturation accelerator for tobacco leaves.
3.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
The highest exposures to EO (typically 5-10 ppm for 20
minutes at a time) occur during the sterilization of medical
products in hospitals, a use that represents less than 1% of
production. Frequent exposures result from the regular opening
and closing of sterilization chamber doors and the off-gassing of
recently sterilized products. With the exception of smokers,
sterilization workers in health care facilities represent the
single largest group of exposed individuals.
Brugnone et al. (1986) monitored the blood and alveolar air
of ten workers in a hospital sterilizer unit during and at the
end of an 8-hr workshift. The mean concentration of EO was 5.4
mg/cu m in ambient air and 1.2 mg/cu m in alveolar air. Blood
concentrations were found to be 3 and 12 times higher,
respectively.
Occupational exposures to EO have always been relatively low
and infrequent in the chemical industry where, because of the
extreme explosion hazard of the pure chemical, EO is handled with
extreme caution. OSHA, in response to suggestive carcinogenicity
data, recently lowered the threshold limit value (TLV) for EO to
1 ppm, or 2 mg/cu m (8-hour time weighted average). The OSHA
permissible exposure limit (PEL) is 50 ppm (ACGIH, 1986a).
27
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Indirect exposure to EO may result from inhalation of ethene
(also called ethylene), which is metabolized to EO in vivo .
Ethene is ubiquitous in the environment, with automobile exhausts
being the main source in urban air. In addition, over 36.5
billion Ibs of ethene were commercially produced in the U.S. in
1988 (C&EN, 1989).
3.3 KNOWN HEALTH EFFECTS
The high chemical reactivity of EO accounts for many of its
acute effects such as skin, eye and respiratory irritation.
Aqueous solutions of EO (including EO on moist skin) are
irritating and may lead to severe dermatitis with blisters and
burns. Allergic eczematous dermatitis has also been reported.
EO is absorbed by leather and rubber, and traces of the gas in
clothing, endotracheal tubes and vascular catheters may cause
burns, tracheitis and thrombophlebitis, respectively. High
concentrations of EO vapor may cause irritation and necrosis of
the eyes. Splashed in the eyes, liquid EO can cause severe eye
damage. Large amounts of EO evaporating from the skin can
produce frostbite. If inhaled, EO causes nausea, vomiting,
neurological disorders, central nervous system (CNS) depression,
lung irritation, pulmonary edema and even death.
EO has caused cancer in experimental animals, and is
regarded by EPA, NIOSH and OS HA as a potential leukemogen in man
(Austin and Sielken, 1988). However, the animal data supporting
this position are derived largely from a single species (the
Fisher 344 rat) that exhibits a sizeable spontaneous incidence of
mononuclear cell leukemia. Also, the observed tumors were all
late-occurring neoplasms, whereas potent carcinogens are
generally associated with early induction of tumors.
High levels of ethene failed to cause cancer in exposed rats
during a 2-year inhalation study and, although a dose-related
incidence of lung tumors was observed in rats chronically exposed
to unfiltered diesel exhausts (mean ethene content 1.28 ppm),
filtered diesel exhausts (i.e., particulates removed) did not
induce lung tumors (Tornqvist et al., 1988).
The limited evidence for the carcinogenicity of EO in man
has been recently reviewed by Divine and Amanollahi (1986) and by
Austin and Sielken (1988). Certain chromosome aberrations are
found more frequently in workers exposed to EO than in controls,
and statistically significant increases in tumor incidence have
occasionally been observed. The fact that a number of
epidemiological studies have found either no excess or a
statistically insignificant excess of leukemia in workers with
potential exposure to EO may be attributable to the low exposures
involved.
28
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If the carcinogenicity of EO has not been conclusively
demonstrated in humans, its genotoxicity certainly has. While
genotoxicity per se is not, strictly speaking, a health effect,
it is considered here because of its generally accepted relevance
to potential long-term health effects (i.e., cancer).
Ehrenberg et al. (1974) estimated that, at a given exposure
dose of EO, the risk in man at moderate exercise was
approximately 20 mrad-equivalents per ppm hr. Using this
conversion factor, the genetic risk incurred by epoxide operators
in a Swedish EO factory in 1974 was calculated to be 50 times
greater than that incurred by radiological workers exposed to the
maximal permissible dose (0.1 rad/week). Similarly, Calleman et
al. (1978) estimated that an EO exposure of 10 ppm hr/week,
corresponding to an average concentration of 0.25 ppm, gave the
same risk as whole-body exposure to 0.1 rad hr/week. At that
time, only the Soviet Union's standard of 0.5 ppm (maximal
permissible concentration) was considered reasonable with regard
to this calculated risk.
3.4 METABOLISM
3.4.1 Detoxification
Ehrenberg et al. (1974) demonstrated that, in mice exposed
for 1-2 hr to air containing 1-35 ppm EO (0.03-2% of LD50 and 2-
70% of the MAC), virtually all the EO in alveolar ventilation is
absorbed in a linear fashion, rapidly and almost uniformly
distributed to all organs (tissue dose » 0.5 tM), and rapidly
detoxified and excreted (80% eliminated in 48 hr; biological
half-life approx. 9 min). Most EO absorbed into cells rapidly
undergoes hydrolysis to ethylene glycol. The major urinary
metabolite in rats is 2-hydroxyethylmercapturic acid (N-acetyl-s-
2-hydroxyethyl-L-cysteine), the same excretion product that is
produced by exposure to vinyl chloride, and acrylonitrile. Due
to its high rate of hydrolysis and its low octanol/water
partition coefficient, EO is not expected to bioaccumulate.
3.4.2 Activation
EO is a direct-acting agent, i.e., it requires no metabolic
activation prior to the expression of its genotoxicity. However,
in vivo, the activation of ethene to EO by epoxidation is
catalyzed by the mixed function oxidases (MFO).
Under conditions of low level exposure, about 8% of the
ethene inhaled by mice is metabolized to the epoxide via a
saturable enzymatic process with a K,, of 218 ppm (Segerback,
1983). The maximal rate of metabolism of ethene in mice (0.24
mg/kg/hr) corresponds to an exposure to 4 ppm of EO in the air.
29
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3.4.3 Host Factors
The usual biological variation in metabolism can be expected
to apply in the case of ethene exposure (EO itself is a direct-
acting agent). In addition, variations in epoxide hydrolase
activity will affect tissue concentrations of EO. Similarly,
DNA-EO adducts will be subject to variable rates of repair.
3.5 PROTEIN ADDUCTS
3.5.1 Characterization
EO exposure results in the hydroxyethylation of cellular
macromolecules (Bolt et al., 1988). In the mouse, 90% of the EO-
DNA adducts are 7-(2-hydroxy- ethyl)guanine (Ehrenberg et al.,
1974). In decreasing order of abundance, the major hemoglobin
adducts of EO in the mouse are the 2-hydroxylation products of
(1) the sulfhydryl group of cysteine (HOEtCys), (2) the amino
group of N-terminal valine (HOEtVal) and the Nl nitrogen of
histidine and (3) the N3 nitrogen of histidine (Segerback, 1983).
(Note: The Nl and N3 nitrogens are sometimes referred to in the
literature as the pi and tau nitrogens, respectively.)
Exposure to ethene, a metabolic precursor of ethylene oxide,
results in the same spectrum of macromolecular adducts.
Segerback (1983) demonstrated that, in mice exposed to either
ethene or its metabolite ethylene oxide, the ratio between the
degree of hydroxyethylation of DNA in various organs and of
hemoglobin in red blood cells is approximately the same.
Similarly, the relative amounts of hydroxyethylation products of
cysteine, N-terminal valine, and histidine in hemoglobin is the
same for both compounds.
The dosimetry of EO using Hb adducts has recently been
reviewed by Osterman-Golkar (1987). In general, animal studies
have confirmed that the degree of alkylation of DNA by EO can, at
least approximately, be estimated from the degree of alkylation
of amino acid residues in hemoglobin. Thus, the use of
hemoglobin adducts as biomarkers of ethylene oxide exposure in
humans may make it possible to predict levels of DNA adducts in
other tissues.
3.5.2 Rate of Formation
The second-order rate constant, k, for the alkylation by EO
of valine-N in rat Hb is 5 X 10"5 1/g hr (Segerback, 1985;
Tornqvist et al., 1986,1988). For the reaction of EO with
histidine-N-3, estimated values of k range from 1.4 X 10~5
(Calleman et al., 1978) to 2.7 X 10'5 1/g hr (Osterman-Golkar et
al., 1983a).
30
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In their studies on the mouse using radiolabeled EO,
Ehrenberg et al. (1974) determined the rate constants for the in
vitro reaction of EO with DNA and protein to be 0.9 x 10"4 1/g hr
and 4 x 10"4 1/g hr, respectively. Using the latter rate
constant and the observed degree of protein alkylation in vivo
(0.23 nmole hydroxyethyl/g protein/ppm hr, the average slope of
the curves for spleen and testis protein), they calculated the
tissue dose of EO in exposed mice to be 0.58 /*M hr/ppm hr. By
extrapolating from the mouse data, the tissue dose of EO in a
resting man and a man at moderate exercise were estimated to be
0.06 and 0.28 jimole hr/ppm hr/kg, respectively.
3.5.3 Dose-Response
Osterman-Golkar et al.(1983a) demonstrated an essentially
linear relationship between the level of chronic exposure to EO
and the formation of hemoglobin adducts. In rats exposed to 0,
10, 33, and 100 ppm of EO, 6 hr/day, 5 days/wk, for 2 years,
levels of N3-(2-hydroxyethyl)histidine (HOEtHis) were determined
by GC-MS and amino acid analysis to be 1.3-2.8, 14, 34, and 82
nmol/g Hb, respectively. In acute dosing experiments, levels of
N7-(2-hydroxyethyl)guanine in liver and testicular DNA were about
150% and 50%, respectively, of expected values based on the
observed levels of hemoglobin alkylation, and the assumption that
the tissue dose was everywhere the same as that in red blood
cells. These results are consistent with the finding in the
mouse that the relative doses of EO to liver DNA, hemoglobin and
testis DNA are 1.3, 1.0 and 0.6 (Segerback, 1983).
Tornqvist et al.(1988) demonstrated that long-term exposure
of rats and hamsters to gasoline engine exhausts results in the
dose-related formation of alkylvalines in hemoglobin. The
observed adduct levels corresponded to the epoxidation in vivo of
5-10% of the inhaled alkenes. At high levels of exposure
(estimated 2.28 ppm ethene), HOEtVal levels ranged from 520 to
720 pmol/g, depending on sex and species. Catalytic conversion
reduced the level of ethene in the exhaust to less than 0.1 ppm,
at which concentration the corresponding levels of HOEtVal did
not exceed control values (75-105 pmol/g, depending on species
and sex).
Human exposure to EO has been monitored by Calleman et al.
(1978), Van Sittert et al. (1985), and Farmer et al. (1986a)
using the hydroxyethyl adducts of histidine and by Farmer et al.
(1986a), Tornqvist et al. (1986a,b), Waith et al. (1987), and
Passingham et al. (1987) using the valine adduct.
Calleman et al. (1978) analyzed blood samples from 5 German
workers exposed to relatively well-known doses of EO (daily
exposure dose around 500 ppm hr) in sterilization plants. The
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level of HOEtHis in the workers (0.5-13.5 nmol/g Hb) was
significantly higher than that in controls (<0.05 nmol/g).
Van Sittert et al. (1985) were unable to demonstrate a
significant difference in levels of HOEtHis between workers (2.08
nmol/g) at an EO production facility and a group of controls
(1.59 nmol/g) matched for age and smoking habits, probably
because (1) levels of exposure at the plant were too low, i.e.,
usually below the detection level of 0.05 ppm, and (2) the
background levels of HOEtHis in controls were too high.
In a study of 10 employees (seven exposed, three controls)
from an EO gas bottling plant, Farmer et al. (1986a) determined
the levels of both HOEtHis and of N-terminal N-(2-
hydroxyethyl)valine (HOEtVal) in Hb. A good correlation
(correlation coefficient 0.996) was observed between the HOEtHis
and HOEtVal results, especially at higher levels of alkylated
products. In exposed persons, the HOEtHis levels (0.55-8.0
nmol/g) were on average 0.6 nmol/g higher than the HOEtVal levels
(0.02-7.7 nmol/g). However, in controls, background levels of
unknown origin were also higher for HOEtHis (0.53-1.6 nmol/g)
compared to HOEtVal (0.03-0.93 nmol/g).
Waith et al. (1987) studied levels of hydroxyethylation of
N-terminal valine in hemoglobin samples from hospital
sterilization workers using two different methods: (1) GC-MS of
HOEtVal obtained by the modified Edman degradation procedure, and
(2) radioimmunoassay using antibodies raised against HOEtVal-
containing, N-terminal, tryptic heptapeptides from the a-chains
of human Hb. The two methods gave results that were in very good
agreement with one another and with earlier findings. Levels of
HOEtVal in potentially exposed hospital sterilization workers
overlapped with background levels observed in controls, but there
was a significant difference between the two groups (Waith et
al., 1987).
There was a clear tendency in the above-mentioned studies of
occupational EO exposure, for adduct levels in workers and
controls to overlap. By contrast, Tomqvist et al. (1986b)
showed that the levels of HOEtVal (mean 389 nmol/g Hb; range 217-
690 pmol/g) in smokers of -30 cigarettes/day was significantly
different from and did not overlap with adduct levels in controls
(mean 58 nmol/g Hb; range 27-106 nmol/g). The difference between
the two means (331 pmol/g Hb) was compatible with the difference
of 220 pmol/g expected to result from reaction with the EO formed
by metabolic activation of the ethene in mainstream tobacco smoke
(around 0.25 mg ethene/cigarette).
Passingham et al. (1987) conducted a similar study using an
improved version of the modified Edman degradation procedure. In
that study, background levels of HOEtVal averaged 57.4 pmol/g
(range 23.8-105.7 pmol/g, n=13), and the average smoking-related
32
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increase was 114 pmol/g globin/10 cigarettes/day. Using the same
method, Bailey et al. (1988) found a significant correlation
between the average number of cigarettes smoked per day and
HOEtVal levels. Smoking increased the mean background levels
(49.9 pmol/g Hb) observed in non-smokers by 71 pmol/g Hb/10
cigarettes/day.
3.5.4 Background Levels
In most studies of occupational EO exposure, adduct levels
in workers and controls tended to overlap, especially when
HOEtHis was the analyte. (See Dose-Response above.) This overlap
was greatly reduced (Farmer et al., 1986a) or even eliminated
(Tornqvist et al., 1986b) when HOEtVal was the analyte.
Background levels of unknown origin occur with both adducts, but
are much lower in the case of HOEtVal. Some background levels of
Hb-EO adducts measured in humans are listed in Table 3. These
figures suggest that measurement of the N-terminal valine adduct
by the modified Edman degradation procedure (See Methods below.)
would show the greater sensitivity for low level EO exposure
monitoring.
In the studies of both Tornqvist et al. (1986b) and
Passingham et al. (1987), it was emphasized that (1) the source
of background levels was unknown, and (2) even though EO derived
from ethene in cigarette smoke appeared to be the major source of
HOEtVal in the Hb of smokers, other possible sources had to be
considered. The low amounts of diethanolnitrosamine and
diethylnitrosamine present in cigarette smoke (approximately 30
and 5.2 ng/cigarette, respectively) should not make more than a
marginal contribution to the elevated HOEtVal levels observed in
smokers. However, other smoke components may make indirect
contributions of unknown magnitude by affecting endogenous routes
of ethene production, especially lipid peroxidation (Tornqvist,
1987b). The latter possibility is favored by two observations:
(1) Although the propene content of cigarette smoke is
comparable to that of ethene, as is its rate of
epoxidation in vivo, no significant difference in levels
of 2-hydroxypropyl-valine has been found in comparisons
of smokers and non-smokers.
(2) The significant incremental tissue dose of EO in fruit-
store workers is smaller than expected from the estimated
ethene exposure.
It should also be kept in mind that some of the factors used
to calculate expected values (e.g., rate of metabolism and
clearance of ethene and EO) come from the original mouse data
because the corresponding values in humans are not known.
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Table 3. EXPOSURE AMD BACKGROUND LEVELS OF SOME
HUMAN HEMOGLOBIN-ETHYLENE OXIDE ADDUCTS
(Adduct levels are expressed as nmol/g Hb or nmol/g globin)
Adduct*
Exposure Level**
Reference
Nr-(2-HOEt)His
HOEtVal
0.50-13.5
0.55-8.00
<0.02-9.70
0.22-0.69
0.02-7.70
0.11-1.50
0.04-0.50
Calleman et al. (1978)
Farmer et al. (1986a)
Van Sittert et al.(1985)
Torngyist et al. (1986a,b)
Farmer et al. (1986)
Waith et al. (1987)
Bailey et al. (1988)
Adduct
Background Level
Reference
HOEtCys
Nr-(2-HOEt)His
N'r-(2-HOEt)His
HOEtVal
1.5-4.3
0.11-0.29
0.53-1.60
<0.02-4.70
0.17-1.50
Calleman (1986)
Calleman (1986)
Farmer et al. (1986a)
Van Sittert et al.(1985)
Osterman-Golkar (1983b)
0.06-0.30 Calleman (1986)
0.03-0.80 Torngyist et al. (1986a)
0.03-0.11 Torngyist et al. (1986b)
0.03-0.53 Calleman (1986)
0.03-0.93 Farmer et al. (1986)
0.14-0.44 Waith et al. (1987)
0.02-0.11 Bailey et al. (1988)
**
"N1" and "N"" refer, respectively, to the N3 and N1
nitrogens of the imidazole ring of histidine.
Exposure levels of HOEtHis were measured in workers in a
sterilization plant (Calleman 1978), a production
facility (Van Sittert et al., 1985) and an EO gas
bottling plant. Exposure levels of HOEtVal were measured
in EO gas-bottlers (Farmer et al., 1986), hospital
sterilization workers (Waith et al., 1987), and smokers
(Torngyist et al., 1986a,b; Bailey et al., 1988).
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3.5.5 Methods of detection
Typically, the quantitation of N-(2-hydroxyethyl)histidine
adducts involves the analysis of total protein hydrolysates by
gas chromatography (GC), alone or in conjunction with selective
ion monitoring mass spectroscopy (MS). The method, as described
by Bailey et al. (1987), is sensitive down to <0.1 nmol/g globin.
Figure 2 is a schematic depiction of the method as applied to
hemoglobin adducts.
The products of enzymatic digestion or acid hydrolysis of
modified proteins are not directly amenable to GC analysis and
the modified amino acids must be separated from other materials
by ion exchange chromatography or HPLC. The appropriate
fraction, detected and identified by TLC and/or co-elution with a
labeled internal standard (which must be synthesized in advance),
is then derivatized by esterification followed by
perfluoroacylation. After taking the sample to dryness and re-
dissolving it in an appropriate carrier solvent, the modified
amino acid derivatives are finally ready for analysis by GC-MS.
Because the analysis of total protein hydrolysates entails
such extensive sample preparation, analysis of protein adducts by
GC-MS can be an extremely labor-intensive and time consuming
process. These factors, along with the level of training
required for GC-MS operators, make this method unsuitable for
mass screening. However, it remains, for the time being, the
only proven technique available for the analysis of non-
hydrolyzable adducts of internal amino acids such as alkylated
histidines.
HOEtVal has also been determined as a N2,O-bisheptafluoro-
butyryl methyl ester derivative isolated from the appropriate
fractions of a total protein hydrolysate (Calleman, 1986), but
equally good results may be obtained using a method that is 10-
100 times faster on a per-sample basis.
The HOEtVal adduct can be analyzed by a modified Edman
degradation procedure, an abbreviated GC-MS method that
eliminates the need to analyze total protein hydrolysates. This
method, which is specific for stable adducts of the N-terminal
valine of hemoglobin, is also capable of handling larger amounts
of protein without introducing excessive contamination. The
ability to analyze larger samples, along with the lower
background levels of HOEtVal compared to those of HOEtHis,
accounts for the greater sensitivity of this method over the
measurement of HOEtHis by conventional protein analysis
techniques.
In the normal Edman procedure (Figure 3A), which was used
for protein sequencing before the advent of amino acid analyzers,
phenyl isothiocyanate (PITC) is coupled to the N-terminal amino
35
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BLOOD SAMPLE
(supernatant)
HEMOLYSATE
(precipitate)
HEME
(discard)
GLOBIN
TOTAL PROTEIN HYDROLYSATE
PURIFIED AMINO ACID
CONTAINING ADDUCT
Centrifuge, wash and
lyse erythrocytes.
Pellet cell debris.
Wash supernatant.
Add internal standards.
Precipitate globin in
acidic (3% HC1) acetone.
Hydrolyze in 6N HC1 in
vacuo for 24 hr at 110°C.
Take to dryness and re-
dissolve in 1M HC1.
Purify amino acids on
ion-exchange column.
Isolate adducted amino acid
by comparison w/ previously
synthesized standards.
Derivatize by esterifica-
tion & perfluoroacylation.
Extract derivatives, clean
up, dry down & re-dissolve
in appropriate carrier
solvent.
QUANTIFY BY GC-MS
Figure 2. Procedure for analysis of total protein (i.e.,
hemoglobin) for adducts using GC-MS.
36
-------
acid of a protein under basic conditions. After being extracted
in toluene and dried down, the PITC-modified protein is treated
with anhydrous acid, thereby releasing into solution a cyclic,
anilinothiazolinone derivative of the N-terminal amino acid.
Isomerization in dilute HC1 then yields the desired
phenylthiohydantoin (PTH) derivative.
The modified Edman degradation method (Tornqvist, 1987;
Tornqvist et al., 1986; Mower et al., 1986) relies on the fact
that alkylation of the N-terminal amino acid changes its
properties in such a way that the PTH derivative is formed
directly in the alkaline reaction mixture (Figure 3B). Isolation
of adducts of N-terminal amino acids by this rapid method thus
eliminates the tedious work-up of total protein hydrolysates,
thereby reducing assay time and, consequently, costs by a factor
of 10-100 (Tornqvist et.al., 1986). Replacement of the
conventional PITC reagent with pentafluorophenyl isothiocyanate
results in the formation of pentafluorophenylthiohydantoin
(PFPTH) derivatives which, as a clean toluene extract, are
suitable for analysis by GC-MS (Figure 4). Tornqvist et al.
(1988) report that adjusting the sample to a higher pH before
derivatization increases recovery to about 65%.
Using internal standards, on-column injection, and negative
chemical ionization, Tornqvist et al. (1986) were able to detect
(as HOEtVal-PFPTH) 0.01 nmol of N-(2-hydroxyethyl)valine per gram
of globin in human blood samples. This corresponds to 1 picogram
of injected valine. The choice of negative chemical ionization
was important because excessive interference from reaction by-
products occurs when electron-capture detection is used instead.
Passingham et al. (1987) have further modified and improved
the method. The potential chromatographic adsorption of low
concentrations of HOEtVal-PFPTH was eliminated by reacting the
latter with N,O-bis(trimethyl-silyl)trifluoroacetamide to form
the trimethylsilyl ether derivative. In addition, on-column
injection was replaced with a falling needle type solid injection
device, in order to protect the capillary column from
contamination by non-volatile by-products. By allowing a much
larger fraction of the sample extract to be analyzed by GC-MS,
these alterations have provided a 20-fold increase in
sensitivity. The new detection limit of approximately 20 pmol
HOEtVal/g globin is sufficient for monitoring human exposure to
EO from occupational sources (Farmer, 1988) .
Waith et al. (1987) have developed a radioimmunoassay (RIA)
for N-(2-hydroxyethyl)valine as the N-terminal heptapeptide of
Hb. In an RIA, unlabeled antigen is mixed with predetermined
amounts of labeled antigen and constant amounts of antibody, and
a standard curve is plotted of the ratio of antibody-bound/free
radioactivity as a function of concentration (Note: 100% response
occurs in the absence of unlabeled antigen). The concentration
37
-------
Y OH" Y
* HjjN-CH-CONH-X - CgHg-NH-CS -NH -CH-CONH-X
pluMiyl inuthio- valyl residue plionylthiocarbamoyl (PTC) dvrivacivc
rvnn.iri*
\
\r
2-anilino-4-isopropyl-5- s
I hiaxo I inunv 5-i«opropyl-3-ph«nyl-2-chio»iyd«ntoin
* Y OH" " Y
c H -NsC = S * HN-CH-CONH-X - •• CgHg-NH-CS — N— CH-CONH-X
alkylated valyl residue
»
S
alkylated PTH derivative
Figure 3. Edman degradation procedure applied to (A) an
unmodified N-terminal valine of globin, and (B) an
alkylated N-terminal valine of globin. "X" indicates
the rest of the globin molecule (adapted from Mower
et al., 1989).
B
38
-------
BLOOD SAMPLE
HEMOLYSATE
(supernatant)
-6LOBIN
T
HEME
N-TERMINAL
ALKYLVALINES
Centrifuge RBC's (500g).
Wash in saline, and lyse
in distilled water.
Centrifuge (3000g) to
remove cellular debris,
and wash supernatant.
Add internal standards.
Precipitate globin in
acidic ethyl acetate.
Centrifuge and wash precip.
Treat with F5PITC in a basic
reaction medium.
Extract with n-hexane.
Dry down & redissolve
in toluene.
QUANTIFY BY GC-MS
Figure 4. Modified Edman procedure for analyzing N-terminal
valine adducts in hemoglobin by GC-MS.
39
-------
of unlabeled antigen in a sample may then be determined from the
calculated ratio of bound/free radioactivity in that sample.
The antibody used in the assay of Wraith et al. was raised
against EO-modified N-terminal heptapeptides (N-Val-Leu-Ser-Pro-
Ala-Asp-Lsy-C) which had been released from a-chains of human Hb
by the action of trypsin, coupled to horse serum albumin and
injected into rabbits. The labeled antigen was the
hydroxyethylated heptapeptide radioiodated at the amino group of
the C-terminal lysine. The antibody would bind to the
hydroxylated N-terminal valine only after the latter was exposed
to tryptic hydrolysis. Presumably, the modified N-terminal
valine was insufficiently exposed at the surface of the native
protein to make optimal contact with the antibodies directed
against it. However, when the antibody was tested against
peptides from trypsin-hydrolysed Hb, an overall sensitivity of
0.14 pmol HOEt peptide/ g Hb was achieved. The antibody bound
equally well to the equivalent HOEt peptide from rat Hb but did
not bind to the analogous propylene oxide-modified peptide.
The RIA was fully validated against GC-MS and applied in a study
of hospital workers potentially exposed to EO. Waith et al.
found significant differences between the potentially exposed
group (approximately 0.11-1.50 nmol/g globin) and controls
(approximately 0.14-0.44 nmol/g). The observed background levels
of HOEtVal were in agreement with earlier findings (Calleman,
1986).
3.6 RESEARCH NEEDS
The protein adduct-related literature is more complete on
ethylene oxide than on any other chemical. Hb-EO adducts have
already been employed in a number of studies to monitor human
exposure to this compound. However, two areas in particular
require further research and/or development. First, it is
important to determine the sources of the background levels of
Hb-EO adducts observed in humans. Second, it is desirable to
develop a method of analysis more suitable to mass screening than
those currently available. Such a method might somehow combine
the modified Edman degradation procedure with immunochemical
techniques. If antibodies to the PFPTH derivatives of N-terminal
alkylvalines were available, a fast and inexpensive immunoassay
might be developed which could replace GC-MS analysis of the
products of the modified Edman degradation technique. The
analysis of N-terminal valine adducts would then be a relatively
simple and cost-effective proposition. A survey of the
literature suggests that the modified Edman degradation technique
has yet to be tried on serum albumin. If adducts are formed at
the N-terminal amino acid of serum albumin (i.e., glutamic acid
in the rat and aspartic acid in humans), their detection should
be possible using the modified Edman degradation technique.
40
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4. GROUP I CHEMICALS:
PROPYLENE OZIDE
4.1 MANUFACTURE AND USE
Propylene oxide (PO) (CAS # 75-56-9), also called propene
oxide, 1,2-epoxypropane, and methyl oxirane, has been produced in
the U.S. since 1925, and was the 41st-highest volume chemical
produced in 1985. Currently produced by at least 5 different
companies, most of which are located in Texas (Table 4), PO is
used extensively in the manufacture of polyurethane and propylene
glycol. PO is the reactive epoxide of another industrial
compound, propene, which is produced in large amounts in the U.S.
(ranked 12th in 1985) and used in the synthesis of several other
3-carbon compounds and in the production of polymers (Sax, 1987).
Propylene oxide is currently manufactured in the U.S. by two
different processes, each of which accounts for roughly one half
of total production (IARC monographs, 1985). In the so-called
chlorohydrin process, propylene is treated with hypochlorous acid
(chlorine & water) to produce propylene chlorohydrin, an
intermediate which, in turn, is treated with calcium hydroxide or
sodium hydroxide to produce propylene oxide. In the peroxidation
process, an oxidant such as an organic hydroperoxide (tert-butyl
hydroperoxide or ethylbenzene hydroperoxide) or peracetic acid is
used to convert propylene directly to propylene oxide.
PO is approved by the FDA as a direct and indirect food
additive for use as an etherifying agent in the production of
modified food starch (at use levels of 25% max or less), a
package fumigant for certain fruit products, and a fumigant for
bulk quantities of several food products, provided residues of
propylene oxide & propylene glycol do not exceed specified limits
(IARC monographs, 1985). PO may react with moisture and
inorganic chlorides in foodstuffs to form toxic glycols and
chlorhydrins, respectively (Furia, 1972, p.157). PO was listed
by the National Pesticide Information Retrieval System in 1987 as
a bacteriostat, a fungicide, an insecticide, and a miticide.
A major use of PO is as a chemical intermediate for the
production of polyurethane polyols, mono- and di-propylene
glycol, glycol ethers and nonpolyurethane polyols,
isopropanolamines and propoxylated surfactants, propylene
carbonate and hydroxylated cellulose. In 1984, usage patterns
were as follows: polyurethane-flexible foams, 40%; rigid foams,
7%; non-cellular, 10%; coatings/adhesives, 7%; polyols for
specialty surfactants, 4%; propylene glycol, 22%; detergents, 4%;
other, 6% (HSDB, 1989b).
41
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TABLE 4. U.S. PRODUCTION OF PROPYLENE OXIDE
MANUFACTURERS
SITES OF PRODUCTION
Aberco Inc,
Bath, PA
Aldrich Chemical Co. Inc,
Milwaukee, WI
ARCO Chemical Co,
Philadelphia, PA
Dow Chemical Co,
Midland, MI
Spectrum Chemical Mfg.
Gardena, CA
Houston, Texas
(reprocess only)
Milwaukee, Wisconsin
Bayport, Texas
Channelview, Texas
Freeport, Texas
Gardena, California
(small quantities)
42
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4.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
The greatest exposure to PO occurs in workers involved in
the production and use of the chemical, primarily by inhalation
and, to a lesser extent, by dermal absorption. The number of
workers in the U.S. exposed to PO was estimated at 11,417 in 1985
(NIOSH, 1985). Occupational exposure limits are currently
regulated at 20 ppm time weighted average (TWA) or 50 mg/cu m,
with short-term exposures not to exceed three times the TLV-TWA
for no more than a total of 30 min. during a work day (ACGIH,
1986b; HSDB, 1989b), but actual exposures to PO in both
production and manufacturing facilities are generally very low.
Propene or propylene, the metabolic precursor of PO, occurs in
urban air (from automobile exhausts and combustion of
hydrocarbons) and in cigarette smoke. Occupational exposure may
also occur: almost 20 billion Ibs of propylene was commercially
produced in the U.S. in 1988 (C&EN, 1989).
4.3 KNOWN HEALTH EFFECTS
PO has approximately the same chemical reactivity as its
homologue, ethylene oxide, and, accordingly, shares many of the
same symptoms of acute exposure. PO vapors are irritating to the
skin (dermatitis), eyes (corneal burns) and respiratory system
(edema). Prolonged contact with skin may result in delayed
burns. PO is also a mild depressant of CNS activity and may
cause nausea, vomiting, incoordination, ataxia and general
depression (ACGIH, 1986b).
PO also has approximately the same mutagenic effectiveness
as its homologue, ethylene oxide, and PO is carcinogenic in rats
and mice (Osterman-Golkar, 1984). Although PO induces
chromosomal aberrations and SCE's in human lymphocytes, there is
inadequate evidence for carcinogenicity of PO in humans.
Nevertheless, due to its known carcinogenicity in experimental
animals, PO is regarded as a probable carcinogenic risk to
humans. (IARC monographs, 1985).
4.4 METABOLISM
4.4.1 Detoxification
No data on the metabolic detoxification of PO was found in
the literature; however, it is reasonable to expect the
metabolism of PO to be much the same as that of ethylene oxide.
(See Section 2.4.1.) PO would be hydrolyzed in the body to form
propylene glycol, and a probable urinary metabolite is 2-
hydroxypropyl- mercapturic acid (N-acetyl-S2-hydroxypropyl-L-
cysteine).
43
-------
4.4.2 Activation
Like ethylene oxide, PO is a direct-acting agent, i.e., it
requires no metabolic activation before it can bind covalently
with cellular macromolecules. Also like ethylene oxide, PO is
the product of the MFO-catalyzed epoxidation of its corresponding
alkene. Hence, exposure to propene may result in the formation
in vivo of PO and subsequent macromolecular binding (Svensson
and Osterman-Golkar, 1984).
4.4.3 Host Factors
Same as for ethylene oxide.
4.5 PROTEIN AODUCTS
4.5.1 Characterization
PO reacts with DNA at neutral pH to yield two principal
products, N7-(2-hydroxypropyl)guanine and N3-(2-hydroxypropyl)
adenine (IARC monographs, 1976). Exposure to either propene or
propylene oxide may result in the formation in vivo of 2-hydroxy-
propylated products of cysteine, N-terminal valine and histidine
in hemoglobin (Svensson and Osterman-Golkar, 1984).
In a propene-inhalation study with mice (Svenson and
Osterman-Golkar, 1984), DNA adducts measured as N7-(2-
hydroxypropyl)guanine were below the limits of detection, i.e., 1
and 0.2 nmol/g ONA in liver and pooled organs, respectively.
However, these results may reflect the instability of this adduct
during the interval (13 hrs in this study) between termination of
exposure and sacrifice of the animals.
4.5.2 Rate of Formation
Rate constants for the reaction of PO with proteins or amino
acids could not be found in the literature.
4.5.3 Dose-Response
A linear dose response has been observed for the formation
of N3-(2-hydroxypropyl)histidine (N3-HOPrHis) in rats exposed to
PO (Farmer et al., 1982). An average exposure of 1300 ppm PO in
air produced adduct levels of 12.3 nmol N3-HOPrHis/g Hb/ hr.
In mice exposed to 20,000 ppm of [C14]propene 4 hr/day for 8
consecutive days, 2-hydroxypropylated products of cysteine, N-
terminal valine and histidine were identified in the hemoglobin
44
-------
hydrolysates (Svensson and Osterman-Golkar, 1984). The degree of
alkylation of N3-His was 70 nmol/g Hb in treated animals as
compared to 12 nmol/g Hb in controls. The latter figure was
considered to be the upper limit for N3-HOPrHis in controls due
to the presence of background noise interference at the position
for the expected peak. In a separate experiment, N2-(2-HOPr)val
and S-(2-HOPr)cys eluted simultaneously in a single peak.
Alkylated valine accounted for less than 20% of the radioactivity
in this peak. Some 70% of the radioactivity associated with the
hemoglobin of treated mice was attributable to metabolic
incorporation.
Osterman-Golkar et al.(1984) used levels of N3-HOPrHis in
hemoglobin (as determined by GC-MS) to monitor occupational
exposure to PO in 8 workers at a production facility for
hydroxypropylated starch. Good agreement was obtained between
the estimated exposure and the degree of alkylation of
hemoglobin. Levels of HOPrHis (nmol/g Hb) in human blood were
0.1-0.38 (mean 0.145) in 13 controls, 0.85-1.2 in 3 workers with
low-intermediate exposure and 4.5-13 in 4 workers with high
exposure (10 ppm PO for 25-75% of the work time).
4.5.4 Background Levels
According to Farmer et al.(1984), animals and humans show
little or no background of N3-HOPrHis. Background levels of
HOPrHis in human blood tend to be an order of magnitude lower
than background levels of 2-HOEtHis (Osterman-Golkar, 1983). In
one study, they averaged 0.145 nmol/g Hb and ranged from 0.1 to
0.38 nmol/g Hb (Osterman-Golkar et al., 1984).
4.5.5 Methods of Detection
Farmer et al. (1982) describe a gas chromatography-mass
spectrometric method for the determination in Hb of S-(2-hydroxy-
propyl)cysteine following exposure to propylene oxide. Because
epoxidation is not stereospecific, analysis of 2-hydroxypropylat-
ed Hb by GC-MS (or HPLC) yields two peaks for N3-HOPrHis,
reflecting the presence of two diastereomers (Svensson and
Osterman-Golkar, 1984; Mower et al., 1986).
Svensson and Osterman-Golkar (1984) determined N3-HOPrHis in
mouse Hb by means of HPLC. The protein was hydrolyzed in 6 M HC1
for 15 hr. The N3-HOPrHis residues were isolated by ion-exchange
chromatography and derivatized with fluorescamine. After heating
in acid, the fluorescamine derivatives of N3-alkylated histidines
are intensely fluorescent, while the fluorescence of other
fluorescamine-labeled compounds disappears. N3-HOPrHis was
quantified by monitoring fluorescence with a filter fluorimeter
(excitation at 360 run; fluorescence at 418-700 nm) .
45
-------
Farmer et al. (1982) and Osterman-Golkar (1983) measured
N-alkylated histidine by GC-MS analysis of total protein
hydrolysates. Due to the lower background levels of HOPrHis, the
potential resolving power of GC-MS analysis should be ten-fold
higher with HOPrHis relative to HOEtHis. However, GC-MS samples
prepared by the acid hydrolysis of precipitated globin, as
described by Calleman et al. (1978) for the analysis of N3-
HOEtHis, contain substances that interfere with the analysis of
HOPrHis residues by GC-MS. Osterman-Golkar et al. (1984), were
able to avoid such interferences by using enzymatic digestion on
hemolysates that had been previously dialyzed overnight. They
later found that the contaminants were also eliminated if globin
were precipitated from an acidic water-propanol solution by the
addition of ethyl acetate.
Osterman-Golkar et al. (1984) isolated N3-HOPrHis residues
from the protein hydrolysate by ion-exchange chromatography.
After pentadeuterated N3-HOPrHis was added as an internal
standard, the samples were esterified and acylated with
heptafluorobutanoic acid and analyzed by multiple-ion detection
mass spectrometry. N3-HOPrHis was quantified by comparing the
determined peak height ratios (i.e., unlabeled to deuterated N3-
HOPrHis) from duplicate GC-MS analyses with those on a
calibration curve. This method could accurately determine 2 ng
of labeled amino acid/protein sample; smaller amounts could not
be measured accurately due to the presence of background noise
(Osterman-Golkar et al., 1984).
For the purpose of monitoring PO-exposure in large human
populations, it would be more practical to measure the N2-(2-
hydroxypropyl)valine adduct (HOPrVal) by GC-MS using the modified
Edman degradation procedure. (See Section 2.5.5.) The GC-MS
response of the pentafluorophenlythiohydantoin derivatives of
HOPrVal and HOEtVal are similar and the same detection limit can
also be expected (Mowrer et al., 1986).
4.6 RESEARCH MEEDS
PO-exposure monitoring studies should be performed on both
animals and humans in which Hb is analyzed for N2-(2-hydroxy-
propyl)valine by the modified Edman degradation method. In the
course of such studies, rate constants of formation and
background levels of HOPrVal could be determined.
46
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5. GROUP I CHEMICALS:
STYRENE
5.1 MANUFACTURE AND USE
Styrene (CAS # 100-42-5), also called vinylbenzene,
ethenylbenzene, phenylethylene, and styrol, is produced by the
catalytic dehydrogenation of ethylbenzene formed from ethylene
and benzene in the presence of aluminum chloride. The 20th-
highest volume chemical produced in the U.S. in 1985 (Table 5),
styrene is one of the most important monomers used in the
plastics and synthetic rubber industry. When exposed to heat,
light or peroxide catalysts, it polymerizes to form the clear
plastic polystyrene (Hawley, 1987).
In 1982, the pattern of usage of styrene was as follows:
monomer or copolymer for polystyrenes, 67%; acrylonitrile-
butadiene/styrene resins, 9%; styrene-butadiene elastomers, 7%;
styrene-butadiene copolymer latexes, 6%; styrene-acrylonitrile
resins,1%; cross-linking agent in polyester resin manufacture,
5%; other uses, 5% (HSDB, 1989c). Styrene is a chemical
intermediate in the production of styrenated phenols/ oils and
styrene oxide (CAS # 96-09-3). It is used in the manufacture of
protective coatings (Styrene-butadiene latex; alkyds) and paints.
Glass fiber-reinforced, unsaturated polyester resins are used in
construction materials and boats. Styrene is an excellent
electrical and thermal insulator (Merk, 1983). Styrene is an
FDA-approved food additive permitted for addition to food for
human consumption both directly (as a flavoring agent in ice
cream and candy) and indirectly (as a component of adhesives and
rubber articles intended for use in contact with food) [21 Code
of Federal Regulations 175.105 and 177.1810 (4/1/84)].
5.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
A National Occupational Hazard Survey has estimated that at
least 30,000 full-time workers in the U.S. are potentially
exposed to styrene with over 300,000 total workers (including
part-time) potentially exposed. The threshold limit value (TLV)
of styrene has been set at 50 PPM time weighted average (TWA) or
215 mg/cu m., while the short term exposure limit is 100 ppm or
425 mg/cu m. (HSDB, 1989c). Although the full-shift, TWA styrene
exposures associated with styrene monomer and copolymer
production are generally less than 10 ppm, average styrene
exposures in reinforced plastics/ composites plants can range
from 40-100 ppm, with individual TWA and short-term exposures as
high as 150-300 ppm and 1000-1500 ppm, respectively (Santodonato
et al. 1985).
47
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TABLE 5. U.S. PRODUCTION OF 8TYRENB
MANUFACTURERS
SITES OF PRODUCTION
AMOCO Chemicals Corp, Hq
Chicago, IL 60601
Atlantic Richfield Co
ARCO Chen Co, Div
COS-MAR, INC
Dow Chem USA
Midland, MI
Rexene Products
Odessa, TX 79760
Chevron Corporation
Chevron Chemical Company
Shell Chem Co,
Houston, TX
Standard Oil Co (Indiana) ,
AMOCO Chems Corp, subsid,
Texas City, TX
United States Steel Corp,
USS Chems, Div, Houston, TX
Texas City, Texas
Channelview, Texas
Carville, Louisiana
Midland, Michigan
Odessa, Texas
St. James, Louisiana
YEAR
PRODUCTION
SOURCE
(all figures in millions of Ibs)
1985
1986
1987
1988
7,622
7,888
8,014
8,588
C&EN, 1989
C&EN, 1989
C&EN, 1989
C&EN, 1989
48
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E
The heaviest exposures to styrene in the boat industry
result from the extensive use by deck and hull lamination workers
of thermoset polyester resins, which may be 40% styrene by
weight. During manual spraying operations, 10-15% of this
volatile chemical may evaporate into the air (Zeighami, 1987).
Accordingly, inhalation is the primary route of exposure, with as
much as 60-70% of the amount inspired being absorbed (Nordqvist
et al., 1985). Conventional methods for monitoring occupational
exposure to styrene include analysis of styrene in blood and
expired air and of mandelic acid and phenylglyoxylic acid in
urine (Doull et al., 1980).
Low-level exposure of the general population to styrene is
possible by oral ingestion of contaminated food which has been
packaged in polystyrene, by oral ingestion of contaminated
finished drinking water, by inhalation of air contaminated by
industrial sources, auto exhaust, or incineration emission, and
by inhalation of smoke from cigarettes. Styrene is present in
urban air in the low parts per billion. The average domestic
filter-blend cigarette contains 18.0 pq styrene (HSDB, 1989c).
Exposure to styrene may also occur during the use of
miscellaneous products containing styrene such as floor waxes and
polishes, paints, adhesives, putty, metal cleaners, autobody
fillers, and varnishes (NIOSH, 1983). Unsaturated polyester
resin products used in fiberglass boat construction and repair,
auto body fillers and casting plastics may contain styrene at
concentrations of 30 to 50%. (HSDB, 1989c).
Styrene is not expected to bioaccumulate or bioconcentrate
in organisms and food chains to any appreciable extent (U.S. EPA,
1985). However, styrene has been found in subcutaneous fat
samples from workers as long as 3 days after occupational
exposure to more than 4.2 mg/cu m (Ippm) in air (IARC, 1979).
The estimated half-life of styrene in subcutaneous adipose tissue
is 2-4 days (Engstrom et al., 1978). The limited ability of
styrene to partition into fat can be expected to have an impact
on the dose-response relationship, especially as regards
linearity. (See Section 5.5.3. below.)
5.3 KNOWN HEALTH EFFECTS
The principal hazards of acute occupational exposure to
styrene are central nervous system depression and irritation of
the skin, eyes, and upper respiratory tract. Direct contact of
the skin with liquid styrene causes defatting and dermatitis.
Exposure to high concentrations of styrene vapors may produce
irritation of the eyes, nose and throat followed by symptoms of
narcosis and muscular contractions due to respiratory center
paralysis. Characteristic signs of so-called "styrene sickness"
include headache, fatigue, weakness, depression and unsteadiness.
49
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Styrene was not carcinogenic to rodents when administered by
gavage (NCI, 1979), nor were teratogenic effects evident in rats
or rabbits exposed to styrene by inhalation or gavage (Murrat et
al., 1978). However, styrene oxide (SO), the major metabolite of
styrene, has been shown to be both mutagenic and carcinogenic in
laboratory animals (Ponomarkov et al., 1984).
Chromosomal aberrations have been associated with
occupational exposure to styrene. An elevated incidence of
hematopoietic and lymphatic cancer has been reported for workers
in the styrene-butadiene rubber industry (McMichael et al,
(1976). Decreased frequency of births and increased frequency of
spontaneous abortions in female workers have also been reported
(U.S. EPA, 1985).
5.4 METABOLISM
5.4.1 Detoxification
Animal studies using radiolabeled material demonstrate that
styrene is rapidly metabolized and excreted. Some 85-90% of the
injected radioactivity is excreted in the first 24 hours,
primarily in the urine (Plotnick et al., 1979).
In mammals, styrene is metabolized primarily in the liver
and to a lesser extent in extrahepatic tissues, including kidney,
intestine, and lung. The main metabolic pathway for styrene is
oxidation by microsomal monooxygenase to styrene-7,8-oxide,
followed by (1) rapid enzymatic hydration to styrene glycol or
(2) conjugation with glutathione.
Styrene glycol may be transported to the kidneys as a
glucuronic acid conjugate which is either excreted as such or
hydrolyzed in the bladder to release styrene glycol. Successive
oxidation of the released styrene glycol yields mandelic acid and
phenylglyoxylic acid, the major urinary metabolites of styrene in
man (Harkonen, 1978; Liebman, 1975; HSDB, 1989c). The
glutathione conjugate of styrene oxide is further metabolized to
the diastereoisomeric hydroxymercapturic acids N-acetyl-S-(l-
phenyl-2-hydroxy ethyl) cysteine and N-acetyl-S-(2-phenyl-2-
hydroxyethyl) cysteine (Delbressine, 1981).
Lof et al. (1986a) monitored the blood levels of styrene,
styrene-7,8-oxide and styrene glycol in 10 men occupationally
exposed to styrene (average concentration 99 mg/cu m) in two
glass-fiber reinforced plastics factories. The relative uptake
of inspired styrene was about 63%, and the concentration of
styrene glycol in the blood was linearly related to styrene
uptake during the previous 5 hr. The concentration of styrene-
7,8-oxide was at the detection limit of 0.02 /imol/1 in most
samples.
50
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Styrene metabolism appears to be inducible. Relative to
unexposed controls, the rates at which styrene and styrene glycol
are cleared from the blood and styrene glycol conjugates are
formed are significantly higher in individuals with prior
exposure to styrene (Lof et al., 1986b).
5.4.2 Activation
Styrene-7,8-oxide, a metabolic intermediate of styrene, is
biologically reactive and can bind to cellular macromolecules.
It is formed by the monooxygenase-mediated epoxidation of styrene
(Harkonen, 1978).
5.4.3 Host Factors
Biotransformation of styrene into styrene-7,8-oxide is
selectively increased by phenobarbital-type inducers of
microsomal mixed-function oxidases (MFO) and inhibited by
inhibitors of MFO. Combined occupational exposure to styrene and
acetone has caused monooxygenase induction in chemical workers
(Dolara et al., 1983). Metabolism of styrene is suppressed by the
administration of toluene or trichlorethylene.
5.5 PROTEIN ADDUCTS
5.5.1 Characterization
SO alkylates DNA and several amino acids in vitro.
particularly cysteine. The order of binding to free amino acids
is cysteine » histidine > lysine > serine. When human blood and
serum proteins were incubated with 3H-SO in vitro. HPLC and GC-MS
analysis of the modified protein hydrolysates yielded as the
predominant product two cysteine adducts, i.e., the alpha- and
beta-substitution products. An excess of the a-substituted
product is formed with cysteine and glutathione. The 6-
substitution product is more abundant in N-7-SO-guanosine adducts
(Hemminki, 1986a,b).
5.5.2 Rate of Formation
Rate constants for the reaction of styrene or styrene-7,8-
oxide could not be found in the literature.
51
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5.5.3 Dose-Response
Nordqvist et al. (1985) investigated the dosimetry in the
mouse of radiolabeled styrene and SO based on determination of
binding to DNA, hemoglobin and plasma proteins. Maximum binding
in serum proteins was obtained within 30 min after administration
of SO. The degree of alkylation of macromolecules in vivo by SO
is 0.1-0.2 times that resulting from EO administration, even
though the chemical reactivity of EO is somewhat lower than that
of SO. However, the biological halflife of SO in the mouse after
intraperitoneal (i.p.) injection of 200 mg/kg B.W. was 3.4
minutes, about one third that of EO. With styrene, maximum
binding was obtained 5 hours after treatment. The subsequent
decline of binding of radioactivity appeared to reflect the
turnover of serum proteins (t1/2 of serum albumin = ca. 1 day in
the mouse).
Alkylation of the N-terminal valine of hemoglobin was
determined by the modified Edman procedure as described by Mowrer
et al. (1986). The pentafluorophenylisothiohydantoin derivatives
of the N2-(2-hydroxy-2-phenylethyl)valine adducts (HOStVal) were
quantified by GC-MS using negative chemical ionization. This SO-
Hb adduct constituted about 3% of the total radioactivity bound
to the protein (3 nmol/g Hb at 1.1 mmol/kg B.W.). The degree of
alkylation increased non-1inearly with dose. However, this is
not surprising considering the conditions of the experiment.
Both the metabolic activation and detoxification of styrene were
saturated at two of the four dose levels used. Also, the time
delay before sacrifice was not the same for all exposed animals.
Animals receiving the two "low" doses were sacrificed after 5
hours, while those receiving the two high (i.e., metabolically
saturating) doses were sacrificed after 2 hours, insufficient
time for maximum binding to have occurred.
An NIH-funded human monitoring study is currently being
conducted at Columbia University. Blood samples from workers at
boat building facilities in Maine and Connecticut have been
collected, but the analyses for styrene-hemoglobin adducts have
not yet been performed.
5.5.4 Background Levels
Although specific background levels of styrene adducts were
not reported in the literature, such background levels are
expected to be very low.
5.5.5 Methods of Detection
Heminki (1986a) modified proteins in vitro with tritiated
styrene or styrene oxide. Enzymatic digests of the modified
52
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proteins were separated by HPLC and the fractions were collected
for determination of radioactivity. Styrene oxide-cysteine
adducts were quantified by GC-MS.
Although the majority of Hb-styrene adducts appear to be
formed at cysteine (Hemminki, I986a and b), a more suitable
adduct for exposure monitoring is N2-(2-hydroxy-2-phenylethyl)
valine, which represents 3% of the styrene bound to rat Hb in
vivo (Nordqyist et al., 1985). The latter adduct may be readily
measured using the modified Edman degradation technique followed
by GC-MS analysis. It should be possible to detect much lower
levels of HOStVal using this method than either HOEtVal or
HOPrVal, because HOStVal elutes in a portion of the chromatogram
relatively free of background interferences (Mower et al., 1986).
5.6 RESEARCH NEEDS
Studies of human exposure to styrene have been proposed by
Nordqyist, et al. (1985) and Perera (1987). Such a molecular
epidemiological study of occupational exposure to styrene is
currently being conducted in boat-building facilities in Maine
and Connecticutt, where the heaviest exposures occur during hull
and deck lamination. In addition to hemoglobin adducts, this
study proposes to characterize and validate four other biomarkers
of exposure: lymphocyte DNA adducts, micronuclei, sister
chromatid exchanges, and unscheduled DNA synthesis. , At this
time, blood samples have been collected from styrene-exposed
workers and are in the process of being analyzed. This study
should be followed closely, since its findings could (1)
demonstrate the feasibility of using Hb adducts (specifically,
HOStVal) to monitor human exposure to styrene and (2) define the
relationship between the formation of protein-styrene adducts and
DNA-styrene adducts in humans.
Serum albumin-styrene adducts also warrant further study.
At low levels of exposure, binding is very much higher with
plasma proteins than it is with Hb (Nordqyist et al., 1985).
Such findings are not unexpected considering that (1) the highly
reactive major metabolite of styrene, styrene,7,8,oxide, has a
short biological half-life; and (2) serum albumin is synthesized
in the same cells (i.e., the hepatocyte) in which the reactive
metabolites of styrene are generated. However, no reports of
specific styrene-albumin adducts were found in the literature,
and further research is required in this area.
53
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6. GROUP ii CHEMICALS:
4-AMINOBIPHENYL
6.1 MANUFACTURE AND USE
4-Aminobiphenyl (4-ABP) (CAS # 92-67-1), also called p-
aminodiphenyl, 4-biphenylamine, 4-phenylaniline, anilonobenzene
and xenylamine, currently has no commercial use in the U.S. and
is no longer manufactured in most countries, including the U.S.,
because of its carcinogenicity (Merck, 1983). However,
measurable occupational exposure to 4-ABP may still occur during
the manufacture of dyes using 2-ABP, an intermediate that
contains 4-ABP as a contaminant. Formerly used as a rubber anti-
oxidant, 4-ABP's only current use is as a research chemical and
as an analytical reagent for the detection of sulfates. (NTP,
1985; Skipper et al., 1986).
6.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Measurable exposure to 4-ABP occurs in cigarette smokers
(Tannenbaum et al., 1986; Bryant et al., 1987; Skipper et al.,
1987; Perera et al., 1987). Significant exposure to
4-biphenylamine may also still occur in the workplace. 4-ABP is
formed as an unwanted side-product in the manufacture of other
amines or dyes and could result in exposures exceeding the
nanogram amounts found in cigarette smoke (Skipper et al., 1986).
Trace amounts of 4-ABP occur in certain azo dyes (FD&C yellow #6
or tartrazine, and FD&C yellow #5 or sunset yellow,) used in a
number of food, drug and cosmetic products, and these dyes may
contain larger quantities of 4-ABP "subsidiary dyes" that could
yield more 4-ABP on metabolism. Also, 4-ABP and 4-nitrobiphenyl
(which yields 4-ABP upon enzymatic nitroreduction) may occur in a
number of combustion products (HSDB, 1989d).
6.3 KNOWN HEALTH EFFECTS
Only the N-hydroxy metabolite of 4-ABP is mutagenic in the
Ames test, inducing both frameshift and base-pair substitution
mutations (Lazear and Louie, 1978).
4-ABP is a confirmed human carcinogen (NTP, 1985). It
causes bladder cancer in the dog, which exhibits almost no
acetylase activity toward aromatic amines, and in humans, which
exhibit at least two phenotypes, i.e., "slow acetylators" and
"fast acetylators". In species with active acetylation (i.e.,
rat, mouse and hamster), 4-ABP induces tumors of the liver,
intestine, and mammary gland, but bladder tumors are rarely
observed (Tannenbaum et.al., 1986).
54
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In humans, bladder cancer induction by aromatic amines such
as 4-ABP is believed to be initiated by the reaction of free N-
hydroxylamine or nitrenium ion with DNA in target cells of the
bladder. The N-hydroxylamines formed in the liver are most
probably transported to the bladder as N-glucuronides which are
subsequently hydrolysed by the low pH of urine (Bryant et al.,
1987). The major 4-ABP-DNA adduct, N-(deoxyguanosin-8-yl)-
acetyl-4-ABP, has recently been monitored by a competitive,
avidin-Biotin amplified ELISA using rabbit antibodies that can
distinguish between the adduct and the free compound (Roberts et
al., 1986).
6.4 METABOLISM
6.4.1 Detoxification
Acetylation appears to be the detoxification route in man.
4-Acetamidobiphenyl is the acetylated urinary metabolite of 4-ABP
(Johnson et al., 1980). The covalent binding of an acetylated
metabolite of 4-ABP to a single tryptophan in serum albumin near
one of its fatty acid binding sites suggests the possibility of
an important role for this protein in carcinogen transport or
detoxification (Skipper et al., 1985).
6.4.2 Act ivat ion
4-ABP is converted to N-hydroxy-4-aminobiphenyl by arylamine
N-hydroxylase, a cytochrome P-450-dependent enzyme (Masson et
al., 1983). N-hydroxylation of the parent amine appears to be
the critical step in the metabolic activation of 4-ABP and
various other aromatic amines to the carcinogenic species.
N-Hydroxy-4-aminobiphenyl is the major metabolite of 4-ABP in
liver fractions from rat, mouse, guinea pig, rabbit and hamster
(McMahon et al., 1980). 4-ABP may also be activated within the
target tissue itself, i.e., the bladder mucosa or urothelium in
dogs, rabbits and man.
N-hydroxylamine metabolites bind to DNA and proteins. The
major DNA adduct is N-(deoxyguanosin-8-yl)-4-aminobiphenyl
(Beland et al., 1983). The reactive N-nitroso compound, which is
formed during the co-oxidation of hemoglobin and N-hydroxy-4-ABP,
forms sulfinamide adducts with cysteine in Hb (Green et al.,
1984; Ringe et al., 1988). N-sulfonyloxy-N-acetyl-4-
aminbbiphenyl, the probable ultimate electrophile responsible for
the formation of 3-(tryptophanyl-Nl-yl)4-ABP in serum albumin, is
formed by sulfotransferase-catalyzed esterification of N-hydroxy-
N-acetylaminobiphenyl (Skipper et al., 1985). The Hb-4-ABP
adduct represents that portion of 4-ABP that is N-hydroxylated
directly, while the albumin adduct reflects that portion which is
acetylated prior to N-hydroxylation (Skipper et al., 1984).
55
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6.4.3 Host Factors
Slow acetylators are known to be at increased risk for
bladder cancer which may be induced by a number of aromatic
amines, including 4-ABP. Thus, it is possible that the
acetylator status of the host will affect the ratio of Hb adducts
to serum albumin adducts formed with 4-ABP. If both adducts
could be monitored simultaneously, one might expect the ratio of
the two to be higher in slow acetylators than in fast
acetylators.
6.5 PROTEIN ADDUCTS
6.5.1 Characterization
In rats treated with 4-ABP, a single major serum albumin
adduct, 3-(tryptophan-Nl-yl)-4-ABP, is formed in vivo by reaction
of N-sulfonyloxy-N-acetyl-4-aminobiphenyl at the single
tryptophan residue at position 214 in rat serum albumin (RSA), a
site that is homologous in rat and man (Skipper et al., 1985).
A single major 4-ABP adduct (an acid-labile cysteine
sulfinamide) is also formed in both rat (Green et al., 1984) and
human hemoglobin (Ringe et al., 1988). In man, the Hb-4-ABP
adduct is a sulfinamide of cysteine 936, the only reactive
cysteine residue in Hb. It is formed by reaction with the N-
nitrosobiphenyl that is generated within the erythrocyte by
cooxidation of Hb and N-hydroxyaminobiphenyl (Figure 5). Some
observed values of this Hb-ABP adduct are listed in Table 6.
N-hydroxy-aminobiphenyl may also bind directly to DNA to
form the major DNA-4-ABP adduct N-(deoxyguanosin-S-yl)-4-ABP.
6.5.2 Rate of Formation
Specific values of k for the reaction of 4-ABP metabolites
with proteins were not found in the literature. However, it may
be inferred from the high percentage (5%) of a dose of 4-ABP
binding to hemoglobin that the apparent rate constants for the
formation of the N-hydroxy and N-nitroso derivatives will be
relatively large.
6.5.3 Dose-Response
Skipper et al. (1985) identified a single major serum
albumin adduct in rats treated with 100 mg/kg tritiated 4-ABP
intragastrically. In rats treated with 2-80 mg/kg 4-ABP,
56
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P450 OX.
Cyt. P450 red
OxyHb
metHb
Hb-SH
N-s-Hb
0
II
Hb —S —OH-
H^or OH'
in vitro
Figure 5.
Proposed scheme for the formation in vivo and
the hydrolysis in vitro of acid-sensitive
hemoglobin-^-ABP adducts (after Skipper et
al., 198.
57
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approximately 0.02% of the dose was bound to tryptophan in serum
albumin and cleared with a half-life of 4.7 days, essentially the
same as that of RSA (Skipper et al., 1984). The adduct, 3-
tryptophanyl-4-ABP, occurred at the single tryptophan residue of
RSA at position 214 (this site is homologous in rat and man),
probably as a result of reaction with the metabolite N-
sulfonyloxy-N-acetyl-4-aminobiphenyl. This electrophile is known
to be formed by sulfotransferase-catalyzed esterification of N-
hydroxy-N-acetylaminobiphenyl. Thus, the serum albumin adduct
reflects the contribution of N-acetyltransferase activity as well
as N-hydroxylation to the overall metabolism of 4-ABP.
A single major 4-ABP adduct was also found in rat
hemoglobin. The adducted amino acid residue was identified as
cysteine. In single dose experiments, the dose-response was
linear from 0.5 /*g to 5000 /ig/kg (Figure 6), with approximately
5% of the dose being bound to Hb in the form of an acid-labile
cysteine adduct. Background levels of unknown origin were
observed in untreated rats at the level of 6 ± 2 pmol/g Hb.
Chronic administration of tritiated 4-ABP (11.4 ttq once every 48
hr for 75 days) led, after roughly 60 days (the lifetime of Hb in
the rat), to accumulation of radioactivity to a plateau level 30
times greater than that obtained after a single dose (Figure 7).
The decline in Hb-adduct levels after the termination of dosing
was also consistent with the rate of protein turnover (Green et
al., 1984).
Using gas chromatography with electron capture detection
(EC), Skipper et al. (1986) were able to examine the binding of
chronic doses of 4-ABP as low as 50 ng/kg (around 10-20
ng/animal) and found that the fraction of dose bound to Hb was as
great as it was with larger doses.
Because real samples contain EC-sensitive materials that
interfere with analysis, the levels of 4-ABP-Hb adducts in human
smokers and non-smokers have been monitored using negative
chemical ionization mass spectrometry (Tannenbaum al., 1986;
Skipper et al., 1987; Bryant et al., 1987; Perera et al., 1987).
Bryant et al.(1987) found the NCIMS method sensitive down to
levels below 10 pg or 50 fmol 4-ABP/10 ml blood. Levels of 4-ABP
released from Hb samples were significantly higher in smokers of
at least 1 pack/day (mean=154 pg/g Hb) than in non-smokers
(mean-28 pg/g Hb), and there was no overlap between the two
groups.
In a study of 22 healthy smokers and 24 non-smokers, Perera
et al.(l987) found a highly significant positive correlation
between 4-ABP-Hb adducts and DNA adducts in smokers but not in
non-smokers. The mean level of 4-ABP-Hb adducts (pg/g Hb) was
154.5 ± 49.3 in smokers (range=75-256, n=19), and 32.2 ± 12.3 in
non-smokers (range=7-51, n=18).
58
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100
10
o
6
c
£ 10
CD
U
3
•o
Oi
COi
000
I
05 50 50 500
Dose o' 4-ominobiphenyl {^g
5000
Figure 6. Hemoglobin adduct formation as a linear function of
single, intraperitoneal doses of 4-aminobiphenyl administered to
rats. Adduct levels were measured as free 4-ABP released by mild
hydrolysis in vitro (after Green et al., 1984).
59
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20
40
60 t80
Time (doys)
100
120
K^
(J
Figure 7. Accumulation and elimination of hemoglobin adducts
with chronic administration of 4-aminobiphenyl.
Adducts were measured as bound [3H]ABP (2.05 p.C) in
the total blood volume of a rat (based on 64% of its
body weight) dosed by gavage with 11.4 fig every other
day for 75 days. With the cessation of dosing, adduct
levels declined at a rate commensurate with the life
span of hemoglobin in rats, i.e., approximately 60
days (after Green et al., 1984).
60
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In a group of 40 smokers and 25 non-smokers living in or
near Turin, Italy, the mean level of 4-ABP-Hb adducts (pg/g Hb)
was 180 ± 85 in smokers, and 51 ± 22 in non-smokers. In a group
of 19 smokers and 18 non-smokers in New York, the values were 290
± 130 and 37 ± 13. The levels of adducts in samples taken at 3
different Boston stop-smoking clinics were considerably lower
than in the Turin and New York groups, which consisted of ongoing
smokers. In these studies, as well as those mentioned earlier,
all the smokers smoked at least 20 cigarettes per day, and there
was no overlap with adduct values measured in controls (i.e.,
non-smokers). However, for smokers consuming fewer than 20 cpd,
adduct levels have been measured which fall into the non-smoker
range (Skipper et al., 1987).
Tannenbaum et al.(1986) have reviewed the use of Hb adducts
in monitoring 4-ABP exposure in smokers. The range of 4-ABP
exposure for smokers (15-35 ng/day) the authors calculated from
Hb adduct data was of the same order of magnitude as that
expected (20-100 ng/day) for a 70 kg man smoking one pack of
cigarettes containing 1-5 ng 4-ABP/cigarette. Considering the
numerous assumptions made and the necessity of using
toxicokinetic data from animal experiments in the calculations
for human exposure, the reasonable agreement between expected and
observed adduct levels suggests that this method has great
potential for monitoring human exposure to 4-ABP and similar
compounds.
6.5.4 Background Levels
Background levels of 4-ABP-Hb adducts (Table 6) have been
detected in rats, dogs and man, but not in monkeys or fish. The
detected background levels are considered to be consistent with
low-level ubiquitous contamination of air, food, or water. Green
et al. (1984) observed background levels of unknown origin in
untreated rats at the level of 6 ± 2 pmol/g Hb. Background
levels in humans range from 0 to 0.25 ng (0-1.5 pmol) per 10 ml
sample of whole blood, compared to 0.4-2.0 ng (2.4-11.8 pmol)
per 10 ml whole blood in rats (Skipper et al., 1986).
6.5.5 Methods of Detection
The 4-ABP-Hb adduct is sensitive to mild hydrolysis by
either 0.1 N HC1 in acetone (the customary method of dissociating
heme from Hb and precipitating globin) or 0.1 N NaOH, with the
free amine being released into solution. The free amine may then
be extracted into hexane, derivatized and analyzed by GC-MS
(Figure 8). Acid hydrolysis has been used on radiolabeled
samples analyzed by HPLC or LSC (Green et al., 1984; Skipper et
al., 1984), but base hydrolysis is preferred when samples are to
be analyzed by GC, because it yields a much cleaner chromatogram.
61
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Table 6. EXPOSURE AND BACKGROUND LEVELS OF
HEMOGLOBIN-AMINOBIPHENYL ADDUCTS
(Unless otherwise specified, adduct levels are expressed
as pg/g Hb)
Exposure
Adduct Level
Reference
0.5-5000 M9/kg approx 5% of the Green et al. (1984)
(single doses) administered dose.
Cigarette Smoking 65-265 Tannenbaum et al. (1986)
(mean 163 ± 71)
Cigarette smoking 75-256 Perera et al. (1987)
(mean 154.5 ± 49.3)
>20 cigarettes/day 154, avg. Bryant et al. (1987)
>20 cigarettes/day 180 ± 85 Skipper et al. (1987)
(n=40)
>20 cigarettes/day 290 ± 130 Skipper et al. (1987)
(n=19)
Species
Background Level
Reference
Rat
Rat
Man (n=38)
Man
Man (n=24)
Man (n=25)
Man (n=18)
1015
400-2000
pg/10 ml
whole blood.
7-99
(mean 47 ± 25)
28
7-51
(mean 32.2 ± 12.3)
51 ± 22
37 ± 13
Green et al., 1984
Skipper et al. (1986)
Tannenbaum et al. (1986)
Bryant et al.
Perera et al.
Skipper et al.
Skipper et al.
(1987)
(1987)
(1987)
(1987)
The study by Green et al. (1984) was done with rats using
radiolabeled 4-ABP. Adducts were measured as bound
radioactivity in the total blood volume of a rat, based
on 6.4% of its body weight. In all other studies, the
cysteinyl Hb-4-ABP adduct was determined by GC-MS after
being released by treatment with mild acid or base.
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BLOOD SAMPLE
HEMOLYSATE
(supernatant)
Centrifuge and wash
erythrocytes.
Lyse in distilled H2O.
Pellet cell debris.
Wash supernatant.
Add internal standards
Release adduct with mild
base treatment. (See text
for alternative step.)
(precipitate)
T
HEME
FREE AMINES
GLOBIN
Extract amines into hexane.
Add derivatizing agent.
Concentrate sample on
rotary evaporator.
QUANTIFY BY GO-MS
Figure 8. Procedure for analyzing arylamine-hemoglobin
adducts by GC-MS.
63
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Using gas chromatography with electron capture detection
(EC), Skipper et al. (1986) have been able to accurately
determine levels of the cysteinyl sulfinamide adduct down to 0.1
ng (0.6 pmol) in 10 ml of whole rat blood. However, in spite of
its enormous sensitivity, EC is insufficiently selective. Real
samples contain enough EC-sensitive material to make adduct
analysis by electron capture detection unfeasible below 100 pg/g
Hb (Bryant et al., 1987).
Negative-ion chemical ionization mass spectrometry (NCIMS),
on the other hand, provides both the sensitivity and selectivity
required for the detection and quantification of adducts at
levels found in human blood samples. The NCIMS method is
sensitive down to levels below 10 pg (50 fmol) 4-ABP/10 ml whole
blood (Bryant et al., 1987).
Because the albumin-ABP adduct occurs at such low levels in
vivo and is not acid labile, GC techniques are inadequately
sensitive to monitor its formation. Efforts are under way to
develop an immunoassay for this adduct that would have the
necessary selectivity and sensitivity (Skipper et al., 1986), but
the antibodies developed thus far recognize the biphenyl portion
of the molecule and therefore cannot distinguish between the
adduct and 4-ABP or its metabolites.
6.6 RESEARCH NEEDS
It is now possible to detect 18 femtomoles of the DNA adduct
N-(guanosin-S-yl)-aminobiphenyl using a competitive avidin-biotin
ELISA (Roberts et al. 1986). However, highly sensitive and
specific immunoassays do not yet exist for the analysis of 4-ABP-
protein adducts. With some refinement, existing antibodies
directed against the biphenyl portion of the molecule might be
used to measure the acid-hydrolyzed Hb adduct. However,
quantification of 3-(tryptophan-Nl-yl)-4-ABP in albumin will
require the development of antibodies that are highly selective
for the intact protein adduct.
In a recent study Bryant et al.( 1988) concluded that there
is an association between levels of 4-ABP-hemoglobin adducts and
relative risk for bladder cancer. The nature of this association
could be explored further if practical measurements of the
albumin adduct could also be made in vivo. Immunoassay research
on ABP-protein adducts, still in the developmental stage at
present, should eventually produce antibodies that will permit
the measurement of the albumin adduct derived from the N-acetyl
metabolite as well as the hemoglobin adduct derived from the N-
hydroxy metabolite of 4-ABP. When such analytical methods become
available, it may be possible to demonstrate a biochemical basis
for the observed association between acetylator phenotype and
risk for the development of bladder cancer.
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7. GROUP II CHEMICALS:
BENZIDINE
7.1 MANUFACTURE AND USE
Benzidine (BZD) (CAS # 92-87-5), also called (1,1'-
biphenyl)-4,4'-diamine, p-diaminodiphenyl, and 4,4'-blaniline,
has been manufactured only for captive consumption (i.e., in-
house use) since OSHA regulations effectively banned its
commercial production in the U.S. in 1973. Annual U.S.
production of BZD, which formerly amounted to many millions of
pounds, dropped dramatically after the compound was found to be a
human carcinogen. In 1979 and 1980, U.S. production and imports
of BZD were approximately 500 Ibs and 9000 Ibs, respectively
(NTP, 1985). In recent years, there have been no imports of
benzidine.
Benzidine has been used for more than 60 years as an
intermediate in the production of azo dyes (e.g., Congo red;
Direct blacks, browns, blues, and greens), sulfur dyes, fast
color salts, naphthols and other dyeing compounds (NTP, 1985).
Imports of benzidine-based dyes have risen sharply since the
curtailment of domestic production. Direct azo-dyes are used in
paper, rubber, plastic, and leather products. Benzidine is also
used as a reagent for hydrogen peroxide in milk, and for
detection of blood stains; a stain in microscopy; and a
stiffening agent in rubber compounding (Sax and Lewis, 1987).
The dihydrochloride is used for the quantitative determination of
sulfates, and as a reagent for metals (Merck Index, 1983).
7.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Benzidine has not been reported to occur in nature. The
chemical may be released into the environment as emissions and in
wastewater during its production and use as an intermediate in
the manufacture of direct azo dyes. Benzidine-based dyes may be
converted to benzidine in streams into which these dye wastes
have been discharged. Overall, however, no significant exposure
to BZD is expected to occur in the general population (HSDB,
1989e).
Previously, exposure to benzidine was primarily
occupational, occurring via dermal absorption, inhalation, and
ingestion in workers connected with its production and conversion
into direct azo dyes. However, BZD is currently produced in the
U.S. for captive consumption only with strict regulations that it
be maintained in isolated or closed systems which would limit its
65
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release. Benzidine is regulated as a human carcinogen by OSHA.
Thus, there is no TLV for BZD, and all exposures are kept to an
absolute minimum (IARC, 1982; ACGIH 1986c).
Nevertheless, low-level occupational exposure to BZD may
still occur in the form of the benzidine-based dyes of which more
than 250 have been reported. Benzidine-based dyes may contain
unreacted BZD as a contaminant (< 1-1254 ppm), and the dyes
themselves may be metabolized to BZD. NIOSH estimated that about
700 people were exposed to benzidine and that approximately
79,000 workers in 63 occupations were potentially exposed to
benzidine-based dyes (IARC, 1982).
7.3 KNOWN HEALTH EFFECTS
The U.S. EPA has published a review of the health effects of
benzidine and benzidine-based dyes (U.S. EPA, 1980). More
recently, the Agency for Toxic Substances and Disease Registry
has reviewed the toxicology of benzidine (ATSDR, 1988a).
Symptoms of acute benzidine poisoning include cyanosis,
methemoglobinemia, headache, mental confusion, nausea and
vertigo. Ingestion of benzidine may produce vomiting, and damage
the liver and kidneys. Inflammation and papillomata (both
sessile and pedunculated) of the bladder may precede the
appearance of malignancy (HSDB, 1989e).
The FDA, U.S. EPA and OSHA have declared benzidine to be a
carcinogen in humans as well as animals. Epidemiologic studies
have established that there is a high incidence of bladder tumors
among workers exposed to benzidine, especially during its
production. Benzidine also represents an occupational hazard to
workers involved in the manufacture of azo dyes for textile,
leather, and paper products (ACGIH, 1986c).
Like 4-ABP, BZD induces hepatic and mammary tumors in
species that rapidly acetylate aromatic amines (hamsters, guinea
pigs, mice and rats), and bladder tumors in species that display
acetylation polymorphism (rabbits and humans) or relatively low
acetylase activity (dogs). In rats, BZD also causes Zymbal's
gland tumors. The elimination pattern also correlates with
target organ specificity. In rodents, a greater proportion of
absorbed BZD is excreted in the bile, while larger animals such
as dog and monkey excrete BZD primarily in the urine (Williams et
al., 1985).
A variety of bulky carcinogen-DNA adducts may be similarly
genotoxic. Supporting this possibility is the finding by Talaska
et al. (1987) that each of two different BZD-DNA adducts observed
in mice exhibited approximately equal correlations with
chromosomal aberrations. Also, the activated t24 human bladder
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carcinoma oncogene contains a GC->TA transversion, a single-point
mutation favored by many different suspected bladder carcinogens
(Tabin et al., 1982; Reddy et al., 1982).
7.4 METABOLISM
7.4.1 Detoxification
In experiments with rats and dogs, clearance of [14C]-BZD
from the blood exhibits multiphasic kinetics, suggesting that BZD
is distributed in two or more compartments in the body.
In a study of exposed workers, the major metabolites of BZD
found in the urine were the parent amine (4-6%), and the
arylamides monacetylbenzidine (2-5%) and diacetylbenzidine
(5-10%). The rest of the excreted material was in the form of
conjugates, including those of 3-hydroxy benzidine (U.S. EPA,
1980).
Metabolic conjugation of N-hydroxy arylamides to form N-
glucuronyl-oxy ethers represents a major pathway for biliary.and
urinary excretion of aromatic amine carcinogens. Although these
conjugates are generally considered to be stable detoxification
products, N-deacetylation (either enzymatic or alkaline-induced)
can yield the corresponding N-glucuronyloxy arylamines which may
undergo heterolytic cleavage to form a nitrenium/carbenium
cation-glucuronyl lactonate anion pair. Enzymatic formation of
N-glucuronyloxy arylamines by direct O-glucuronidation of N-
hydroxy arylamines does not appear to occur (Kadlubar and Beland,
1985).
Benzidine-based dyes may be transported to sites of
metabolism and excretion in a bound form on plasma proteins,
especially serum albumin. The pattern of binding to different
plasma proteins, as revealed by crossed immunoelectrophoresis,
may be characteristic for a given dye (Emmett et al., 1985).
Martin and Kennelly (1985) have reviewed the metabolism of
biphenyl-based dyes. Many genotoxic dyes are hydrolyzed in vivo
and metabolized to BZD, primarily by azoreductases of intestinal
bacteria, but also, to a lesser extent, by liver azoreductases.
Using freshly voided human feces, Cerniglia et al. (1986)
demonstrated the anaerobic reduction of Direct Black 38 and the
formation of benzidine, monoacetylbenzidine, 4-aminobiphenyl and
4-acetylaminobiphenyl. Thus, apart from reversible binding to
plasma proteins, the detoxification pathways for such dyes will
be similar to those for benzidine and 4-aminobiphenyl.
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7.4.2 Activation
Kadlubar and Beland (1985) have reviewed the metabolic
activation pathways of arylamines and arylamides, some of which
are described below. " In most cases, a nitrenium ion intermediate
is the proposed ultimate electrophile. Aryl nitrenium ions may
result from the heterolytic cleavage of (1) protonated N-hydroxyl
arylamines after either protonation or O-esterification, and (2)
N-hydroxyl arylamides after either O-esterification or
intramolecular N,0-acyltransfer. The simple deprotonation of an
aryldiimine would also give rise to an aryl nitrenium ion
(Yamazoe et al., 1986). With the exception of diimine formation,
N-hydroxylation appears to be a necessary step in the activation
of arylamines to reactive species that bind to macromolecules.
In BZD-treated rodents, a major DNA adduct, N-
(deoxyguanosin-8-yl)-N'-acetylbenzidine, and a minor DNA adduct,
N-(deoxyguanosin-8-yl)-N,N'-diacetylbenzidine, are formed in vivo
(Soileau, 1987) . The probable proximate electrophiles are N-
hydroxy-N'-acetylbenzidine (HOBZDAc) and N-hydroxy-N,N'-
diacetylbenzidine (HOAcBZDAc), respectively. The initial
acetylation of BZD yields N-acetylbenzidine, which is then N-
hydroxylated to form HOBZDAc. HOBZDAc may react directly with
DNA or be further acetylated to HOAcBZDAc prior to adduct
formation (Martin and Kennelly, 1985). The corresponding
nitrenium ions may be formed directly from these two metabolites
via protonation and heterolytic cleavage, or they may result from
the hydrolysis of a conjugated (0-esterified) derivative. With a
pKb between pH 5 and 6, 1-10% of the N-hydroxy derivative is in
the protonated form even under neutral conditions. Species
susceptibility to urinary bladder carcinogenesis correlates well
with increased urine acidity and decreased frequency of
urination. Thus, protonated N-hydroxy arylamines may be ultimate
carcinogens for bladder cancer.
N-acetylation of an N-hydroxy arylamine is a well documented
metabolic pathway for aromatic amines. Rearrangement of the
resulting N-hydroxy arylamide (or 0-acetylation of an N-hydroxy
arylamine) yields the unstable and highly reactive N-acetoxy
arylamine which may undergo heterolytic cleavage to form singlet
nitrenium/carbenium cation-acetate anion pairs. Subsequent
reaction with methionine or N2-guanine gives the ortho-
substituted product while reaction with C8-guanine gives the N-
substituted product. Metabolic formation of N-acetoxy arylamines
appears to be a major pathway for both mutation induction and
initiation of carcinogenesis.
Sulfuric acid conjugation of the N-hydroxy arylamide of
benzidine is mediated by 3'-phosphoadenosine-S'-phosphosulfate
(PAPS)-dependent sulfotransferases and yields N-sulfonyloxy-N-
acetylbenzidine. The latter benzidine metabolite is probably
responsible for those adducts of DNA, protein and glutathione
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(GSH) that retain the N-acetyl group (i.e., 10-20% of the total
DNA adducts). N-acetylated DNA-benzidine adducts involve both N-
substitution at the C position of guanine and ortho-ring
substitution with the exocyclic N2 atom of guanine. N-acetylated
protein and GSH adducts involve predominantly ortho-ring
substition of the arylamide with the sulfur atom in methionine or
cysteine, respectively.
At least one activation pathway does not begin with N-
hydroxylation. Diimines are formed directly by peroxidative
metabolism of aryldiamines. The enzyme prostaglandin H synthase
(PHS) is widely distributed in extrahepatic tissues and is
present at high levels in dog and human urinary bladder
epithelium. PHS can co-oxidize benzidine to benzidine-diimine or
4,4'-diiminobiphenyl which, after simple deprotonation (pKa
around 2) to generate the nitrenium ion, readily binds to
sulfhydryl groups in protein or GSH and to the C8 position of
guanine to form ortho- and N-substituted products, respectively.
In vitro studies have demonstrated the PHS-dependent formation of
N-(deoxyguanosin-S-yl)-benzidine, which is also formed in vivo in
the dog urothelium (Zenser et al., 1983; Yamazoe et al., 1986).
7.4.3 Host Factors
Among benzidine-exposed workers, those with slow acetylator
phenotype were at a significantly higher risk for bladder cancer
(Meigs et al., 1986). The results of in vitro experiments have
established that benzidine is, in fact, a substrate for
polymorphic N-acetylation by human liver (Peters et al., 1989).
A survey of benzidine-exposed workers indicated that those
with lower than normal serum properdin levels were also more
likely than others to develop bladder tumors (ACGIH, 1986c).
Properdin is a minor serum protein (<0.03%) with complement-
dependent cytolytic properties.
7.5 PROTEIN ABDUCTS
7.5.1 Characterization
As with other aromatic amines, the most readily measured
protein adducts of benzidine are the acid/base labile cysteinyl
sulfinamides formed by co-oxidation of an N-hydroxy metabolite
and hemoglobin with subsequent binding of the nitroso product to
the reactive cysteine residue of Hb. There are at least 3
sulfinamide-type Hb adducts in benzidine-treated rats. The
identity of the major benzidine adduct in rat Hb may be inferred
from its cleavage product, monoacetylbenzidine, to be the
cysteine sulfinamide of N-hydroxy-N'-acetylbenzidine. Benzidine
itself was a minor cleavage product reflecting the presence of N-
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hydroxybenzidine in the erythrocyte. A third minor cleavage
product was not identified. Thus, N-hydroxy-N'-acetyl-benzidine
is indicated as the predominant proximate electrophile in rat
hemoglobin as well as in rat liver DNA (Neumann 1987).
7.5.2 Rate of Formation
No data were available in the literature concerning rate
constants of reaction between BZD and specific nucleophiles.
7.5.3 Dose-Response
The Hb-binding of benzidine in rats was determined by
Pereira and Chang (1981) to be around 22.5 pmol/g Hb/jumol dose/kg
and by Neumann (1984a) to be around 60 mmol/mol Hb/mmol dose/kg.
The first value represents the total binding of radiolabeled
compound after precipitation of globin in acidic acetone, a
procedure which it is now known would have removed the major
adduct (i.e., the cysteine sulfinamides) prior to analysis. The
second, considerably higher figure is, therefore, the more
accurate because it is based on measurements of the cleavage
product (i.e., monoacetylbenzidine) of the major Hb adduct (i.e.,
the cysteine sulfinamide of N-hydroxy-N'-acetylbenzidine).
In subsequent experiments, Neumann (1987) calculated a
hemoglobin binding index (HBI) in the rat of 24 mmol bound/mol
Hb/mmol dose/kg. This value was based on total hydrolyzable
adducts which included three cleavage products:
monoacetylbenzidine (HBI=19 ± 3.5), the parent amine (HBI=2.4 ±
0.1), and an unidentified product (HBI=3.0 ± 0.1). Thus, N-
hydroxy-N'-acetyl-benzidine is indicated as the predominant
proximate electrophile in rat hemoglobin as well as in rat liver
DNA.
In single dose experiments with [14C]-benzidine-treated
female rats, adduct levels (i.e., HBI) declined linearly in a
semi-logarithmic plot, with a biological half-life of 11.5 days.
Approximately 1-2% of the bound radioactivity remained after 70
days (the average life-span of rat RBC's is 63 days), indicating
that, for an extended period of time after dosing, Hb continued
to be labeled by benzidine that was released from some deep
compartment and metabolically activated. Thus, the use of Hb
adducts as dose monitors for BZD and other aromatic amines may
require that the elimination rate and accumulation
characteristics of each compound be determined separately
(Albrecht and Neumann, 1984; Neumann, 1984a).
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7.5.4 Background Levels
No data on benzidine-protein adducts in humans was found in
the 1iterature.
7.5.5 Methods of Detection
The same methods used to analyze 4-ABP-Hb adducts would be
applicable to the cysteinyl sulfinamide adducts of BZD in Hb.
7.6 RESEARCH NEEDS
All protein adduct data currently available for benzidine
are derived exclusively from animal studies. Information on
benzidine-protein adducts in humans is needed. However, at
current low levels of exposure, it may prove difficult to locate
a human population in which BZD-Hb adducts can be identified and
quantitated.
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8. GROUP II CHEMICALS:
METHYLENEBIS (2-CHLOROANILINE)
8.1 MANUFACTURE AND USE
Methylenebis(2-chloroaniline) or MBOCA (CAS # 101-14-4) is
also called 3,3'-dichloro-4,4'-diaminodiphenylmethane; 4,4'-
methylenebis(2-chloroaniline); 4,4'-methylenebis(2-chloro-
benzenamine); di-(4-amino-3-chlorophenyl)methane; methylene
bis(o-chloroaniline) or MOCA (HSDB, 1989f). It is no longer
produced in the U.S., but it is available in the form of tan-
colored pellets and is widely used by numerous small companies in
the polyurethane industry as a curing agent in the production of
polyurethane elastomers and epoxy resin systems containing
isocyanates (Merck Index, 1983; Sax, 1987).
8.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Experiments in dogs demonstrated that percutaneous
absorption is a viable route of entry for MBOCA. Between 2.4 and
10% of a dermal dose was absorbed into the systemic circulation
in 24 hr. (Manis et al., 1984).
Exposure to MBOCA is no longer likely to occur outside the
workplace. In consideration of MBOCA's carcinogenicity, large
companies, e.g., the auto industry, have voluntarily switched to
alternative compounds. The recommended threshold limit value for
MBOCA in 1983-84 was 0.02 ppm or 0.22 mg/cu m time weighted
average (TWA) (ACGIH, 1986d). However, as there are no OSHA
standards for MBOCA, it is probable that substantial occupational
exposure still occurs in scattered, specialized polyurethane
facilities across the country. Routine urinalysis is the current
method of monitoring exposure to MBOCA (NTP, 1985; Zeighami,
1987) . The existence of active monitoring programs and the
Polyurethane Manufacturers Association should facilitate the
identification of exposed populations.
8.3 KNOWN HEALTH EFFECTS
The N-hydroxy metabolite of MBOCA generated in a rat liver S9
system was mutagenic in the Salmonella tvphimurium mutagenicity
assay (Kuslikis et al., 1988). MBOCA is carcinogenic in rats,
mice and dogs (Manis et al., 1984).
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8.4 METABOLISM
8.4.1 Detoxification
In rats, a single dose of [UC]-MBOCA (0.49 mg/kg iv) was
distributed primarily to the small intestine, liver, adipose
tissue, lung, kidney, skin and adrenals. Subsequently, a
biphasic decline in radioactivity was observed in all tissues
except the small intestine, adipose and skin which exhibited a
transient increase. (Partitioning of MBOCA into fatty tissues is
consistent with its octanol/water partition coefficient of 8912'
which indicates a moderate potential for bioconcentration.)
Although very lipophilic, [C]-MBOCA was completely eliminated
within 48 hours, primarily (73.4%) via the feces (Tobes et al.,
1983). Approximately one-third of the MBOCA administered to rats
is excreted in the urine in the form of at least 10 different
metabolites, only 1-2% of which is identifiable as MBOCA (Farmer
et al., 1981).
In dogs, radiolabeled MBOCA administered intravenously
(i.v.) was rapidly and extensively metabolized and excreted in
both bile and urine (32% and 46% of the dose, respectively, in 24
hr). After 24 hr, only 0.4-0.5% of the total urinary excretion,
which amounted to 1.3% of the dose, was parent compound. The
highest tissue concentrations of radioactivity were found in
liver, kidney and fat (Manis et al., 1984).
As is the case with several other aromatic amine carcinogens
(e.g., 4-ABP, BZD and 2-NA), the major canine urinary metabolite
of MBOCA is an ortho-hydroxysulfate conjugate (Manis and
Braselton, 1984). Thus, sulfation of ring-hydroxylated
metabolites is a major detoxification mechanism.
In contrast to the situation in rats and dogs, the urine of
exposed workers contained only the parent compound MBOCA; levels
in urine were < 1500 nmol/1. (Farmer et al., 1981).
8.4.2 Activation
When hydrolyzed by arylsulfatase in vitro, the ortho-hydroxy
sulfate conjugate of MBOCA binds to both DNA and protein. Thus,
while conjugation with sulfate may be a detoxification mechanism
in dogs, the ortho-hydroxy arylamine metabolite may be reactive
(Manis and Braselton, 1984).
In vitro studies with canine liver and kidney slices
demonstrate that acid-labile conjugates (e.g., glucuronides) may
be produced in extra-bladder tissues. In vivo, these conjugates
may be hydrolyzed in acidic urine; the arylamine might then be
absorbed by the urothelium and activated to protein and DNA
binding species (Manis and Braselton, 1986).
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8.4.3 Host Factors
Presumably, the same factors that apply with other aromatic
amines.
8.5 PROTEIN ADDUCTS
8.5.1 Characterization
In the study of Braselton et al. (1988), MBOCA was the major
if not the only cleavage product detected in Hb of MBOCA-treated
rats and guinea pigs. Considering that these animals are known
to be "fast acetylators", the predominance of the N-hydroxy
metabolite might not have been expected. However, such findings
are consistent with the observation by Neumann (1987) that the
ratio of hemoglobin binding indices for identifiable acetylated
vs. non-acetylated cleavage products went down from 19:2.4 for
benzidine to 1.5:2.0 for 3,3'-dichloro-benzidine, a compound
structurally similar to MBOCA. Apparently, chlorination inhibits
acetylation of bicyclic aromatic amines.
8.5.2 Rate of Formation
No information on rate constants of reaction between
MBOCA and protein was found in the literature.
8.5.3 Dose-Response
Cheever et al. (1988) administered [14C]-MBOCA to rats in a
single oral dose of 281 jumol/kg (75 mg/kg) . The level of bound
radioactivity, which was equivalent to approximately 3.6 nmol
MBOCA/g globin after 24 hours, had declined to 0.93 nmol/g globin
29 days after treatment, suggesting that the label would persist
beyond the 60-day lifetime of rat Hb. The level of radioactivity
in rat liver DNA was much higher than that in rat Hb and decayed
at a similar rate. These results are difficult to interpret
because (1) non-specific binding of radioactivity was measured
rather than specific adducts, and (2) globin was precipitated in
cold 1% HC1 in acetone prior to counting, thereby removing any
cysteinyl-MBOCA sulfinamides which may well constitute the major
adduct with Hb.
Braselton et al. (1988) demonstrated that the metabolites N-
hydroxy- and monohitroso-MBOCA form acid-labile adducts with rat
and human Hb in vitro in a dose-dependent manner. Subcutaneous
administration of 4-500 mg/kg MBOCA to rats and guinea pigs
resulted in the dose-related in vivo formation of MBOCA-Hb
adducts. These adducts remained elevated in the blood for more
than 42 days.
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The adduct levels observed by Braselton et al. (1988) did
not reach maximum until 1 week or more after dosing, suggesting
an extended period of adduct formation as MBOCA was released from
a deep compartment. Adduct levels were also much lower than
might have been anticipated. The hemoglobin binding index (HBI)
) of MBOCA in rats was calculated to be 0.33 mmol adduct/mol
Hb/mmol dose/kg body weight. This is substantially lower than
the value of 2.0 mmol/mol/mmol/kg calculated by Neumann (1987)
for 3,3'-dichlorobenzidine in rats. Possibly, the methylene
group makes MBOCA less reactive than dichlorobenzidine;
however,the ethenyl group in 4-dimethylaminostilbene (HBI=147)
does not have that effect. Another possibility is that major
losses occurred during sample preparation in the Braselton study.
Globin was precipitated by dropwise addition to ice cold acetone
containing 0.2% HC1 (one fifth the strength commonly used to
precipitate globin and hydrolyse cysteine sulfinamide adducts)
and the supernatant was not tested for the presence of either
unprecipitated globin or MBOCA.
The levels of MBOCA (i.e., the diamine cleavage product)
released from gently hydrolyzed Hb adducts may be much higher in
dogs, rabbits and humans than in rapidly acetylating animals such
as the rat and guinea pig. As mentioned earlier, substitution at
the ortho position by chlorine appears to depress the formation
of monoacetylated, but not hydroxylated, benzidine-Hb adducts
(Neumann, 1987).
8.5.4 Background Levels
Braselton et al. (1988) detected no MBOCA-Hb adducts in
control animals. No other information on background levels of
MBOCA-protein adducts was found in the literature.
8.5.5 Methods of Detection
Braselton et al. (1988) precipitated globin from adducted Hb
in ice-cold acetone containing 0.2% HCl and washed the
precipitate with acetone (X3) and ether (X2) to eliminate non-
covalently bound MBOCA. The globin was then hydrolyzed in 1.0 N
HCl for 2 hours. The solution was adjusted to pH 9-10 with 10 N
NaOH, then sequentially extracted into ether (X2), and 1 N HCl.
After again adjusting the pH of the solution to 9-10, the
cleavage products were extracted (X2) into hexane and converted
to the bis-heptafluorobutyl derivative. The identity of the acid
released material was then determined by GLC with electron
detection and confirmed by GC-MS using electron impact
ionization. The minimum detectable amount of MBOCA-bisHFB was
0.5 pg, and the ECD signal was linear form 0.5 to 80 pg.
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8.6 RESEARCH NEEDS
The results of only two in vivo studies of Hb-MBOCA adducts
have been reported in the literature to date, and these raise
questions of methodology and interpretation. Free MBOCA should
be removed from Hb samples by extensive dialysis rather than
acidic washes to avoid losses of acid-labile HB-MBOCA adducts
prior to globin precipitation.
Specific Hb-MBOCA adducts (e.g., acid-labile cysteine
sulfinamides) need to be identified and quantified in low-dose
animal experiments using the same methods which have already been
applied so successfully to the study of 4-ABP-Hb adducts.
Rabbits would be a good choice of species because (1) blood would
be more readily available for time course studies, and (2)
rabbits, like humans, are variable acetylators. Time course
studies are needed to elucidate the kinetics of accumulation and
elimination of MBOCA-Hb adducts.
The Braselton group at Michigan State University in Lansing
is currently preparing more MBOCA-Hb adduct data for publication
and is seeking funding for a human monitoring study in that
state.
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9. GROUP II CHEMICALS:
0-TOLUIDINE
9.1 MANUFACTURE AND USE
O-Toluidine (CAS # 95-53-4), also called 2-aminotoluene, o-
methylaniline, and 2-methylbenzenamine, is still manufactured in
the U.S. (Table 7), usually by the reduction of o-nitrotoluene,
for a relatively pure product, or by the reduction of crude
nitrotoluene for a mixed product containing p-toluidine (SAX,
1987). In 1975, close to a million grams were produced (HSDB,
1989g); no current production figures were available.
Of the 3 isomeric forms of toluidine, only the ortho and
para forms are important in industry. O-toluidine is used
primarily as a chemical intermediate in the synthesis of numerous
dyes and other intermediates (e.g., rhodine base). It is used in
printing textiles blue-black and in making various colors fast to
acids (Merck, 1983). It is also used in the preparation of ion-
exchange resins, in the laboratory analysis of glucose, and, as
an antioxidant, in the manufacture of rubber (HSDB, 1989g).
9.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Inhalation is the principal route of exposure to o-
toluidine, although dermal absorption of the chemical may also
occur (Sax, 1984, p.2593). Some 3,761 workers were potentially
exposed to 2-aminotoluene in 1981-83 (HSDB, 1989g). The greatest
potential for exposure exists among makers of dyes and pigments.
The OSHA standard for o-toluidine is 5 ppm as an 8 hr TWA and the
TLV in air is 2 ppm, but exposures in manufacturing facilities
are likely to be much lower. Smokers are exposed to o-toluidine
at a rate of approximately 32 ng/cigarette, depending on brand
and type of tobacco (NTP, 1985; Zeighami, 1987)
2-Aminotoluene is not expected to bioaccumulate or persist
long in air or surface waters (HSDB, 1989g).
9.3 KNOWN HEALTH EFFECTS
Acute effects include methemoglobinemia, hematuria, CNS
depression and damage to kidneys and bladder. In rodents,
o-toluidine produces cancers of various organs, including the
urinary bladder. An increased incidence of bladder cancer has
also been observed in workers exposed to o-toluidine, but
confounding exposures to other carcinogenic chemicals make it
impossible to identify o-toluidine specifically as the causative
agent. IARC rates o-toluidine as a Group 2A human carcinogen
(NTP, 1985).
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Table 7. U.S. PRODUCTION OP O-TOLUIDINE
(2-AMINOTOLUENE)
MANUFACTURERS
SITES OP PRODUCTION
Eastman Kodak Co Lab &
Research Prods Div,
Hq, Rochester, NY
(716) 458-7951
El Dupont DeNemours & Co,
Hq, Wilmington, DE
(800) 441-7515
First Chem Corp,
Hq, Jackson, MS
(601) 969-0217
GFS Chemicals,
Hq, Columbus, OH
(614) 881-5501
Spectrum Chemical Mfg
Gardena, CA
(800) 772-8786
Rochester, New York
Deepwater, New Jersey
Pascagula, Mississippi
Columbus, Ohio
(research quantities only)
Gardena, California
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9.4 METABOLISM
9.4.1 Detoxification
o-Toluidine is metabolized to various aminomethylphenols and
excreted as acid-hydrolyzable conjugates (HSDB, 1989g).
9.4.2 Activation
Similar to other aromatic amines (aromatic amine metabolism
is covered in some detail in the section on benzidine).
9.4.3 Host Factors
The same as for other aromatic amines. Also, 2-aminotoluene
may be released in humans as a metabolite of the local
anesthetics aptocaine and prilocaine (Casarett and Doull, 1986,
p. 110).
9.5 PROTEIN ADDUCTS
9.5.1 Characterization
The only protein adducts of o-toluidine that have been
analyzed to date are the acid-labile, sulfinic acid amides of the
reactive cysteine of hemoglobin.
9.5.2 Rate of Formation
The rate constants of reaction between o-toluidine and
proteins or amino acids have not been reported in the literature.
However, the hemoglobin binding index of o-toluidine has been
determined (Neumann, 1987), and is much lower than that of other
aromatic amines. (See "Dose-Response" below).
9.5.3 Dose-Response
The HBI of o-toluidine, as determined 24 hr after
administration of 0.6 mmol/kg of the compound to female Wistar
rats, was 4.0 mmol adduct/mol Hb/mmol dose/kg (Neumann, 1987).
This figure represents only the hydrolyzable fraction (presumably
cysteine sulfinamides) and not total binding of metabolites. By
comparison, the corresponding values for 4-ABP and benzidine were
344 and 24, respectively.
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As measured by GC-NCIMS, levels of the same hydrolyzable
adducts in human hemoglobin samples from smokers and non-smokers
averaged 290 ± 130 and 170 ± 98 pg/g Hb, respectively, in a Turin
study (Skipper et al., 1987). Although an upward trend was
evident in smokers compared to non-smokers, there was
considerable overlap between the two groups, largely due to high
background levels.
In another Turin study, Bryant et al. (1988) found that
levels of o-toluidine-Hb adducts were associated with tobacco
type as well as smoking status. In this study, mean adduct
levels were 188 ± 19, 290 ± 19, and 329 ± 23 pg/g Hb in non-
smokers, smokers of blond (flue-cured) tobacco, and smokers of
black (air-cured) tobacco, respectively. Mainstream smoke from
black tobacco contains higher levels of aromatic amines than does
that from blond tobacco. Blond tobacco is smoked primarily in
the U.S.
9.5.4 Background Levels
The mean value for non-smokers (i.e., "non-exposed"
controls) in the Turin study (170 ± 98 pg/g Hb) is almost twice
as high as that obtained in a Boston study (89 ± 31 pg/g Hb)
(Skipper et al., 1987); however, the difference was not
statistically significant. Possible sources of these background
adducts include passive smoking and urban air pollution.
9.5.5 Methods of Detection
O-Toluidine-Hb adducts are analyzed by GC-NCIMS, as
described previously for other aromatic amines.
9.6 RESEARCH NEEDS
To date, the literature contains no report of a study using
protein adducts to monitor occupational exposure to o-toluidine.
In those human exposure monitoring studies that have been done,
cigarette smoking is the source of exposure and o-tuluidine-Hb
adducts are measured to supplement data on 4-aminobiphenyl-Hb
adducts, the primary focus of the studies.
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10. GROUP III CHEMICALS:
N-Nitrosonornicotine and 4-(Methyl-
nitrosamino)-l-(3-pyridyl)-1-butanone
10.1 MANUFACTURE AND USE
N-nitrosonornicotine (NNN) (CAS # 16543-55-8) and 4-
(methylnitrosamino)-l-(3-pyridyl)-1-butanone (NNK) are tobacco-
specific nitrosamines. NNN and NNK are among the most important
carcinogens in tobacco smoke. Quantitatively, they are also the
major known carcinogens present in so-called "smokeless"
cigarettes. NNN is a yellow, water-soluble oil that is neither
produced nor used commercially in the U.S. Its only known use is
as a research chemical (NTP, 1985; HSDB, 1989h).
10.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Uptake of NNN and NNK occurs almost exclusively as a result
of exposure to tobacco, tobacco smoke, or other nicotine-
containing materials. NNN is formed by the nitrosation of
nicotine, the major alkaloid in tobacco. The concentration of
NNN in commercial U.S. tobacco products varies from 1.9 to 88.6
ppm, which is one of the highest reported values for an
environmental nitroso compound. Approximately 140 ng/cigarette
has been measured in the mainstream smoke of one popular U.S.
brand; roughly half is formed during the burning of tobacco, the
other half originates from unburned tobacco (NTP, 1985; HSDB,
1989h).
10.3 KNOWN HEALTH EFFECTS
In rodents, depending on the dose and the route of
administration, NNN & NNK produce tumors of the nasal cavities,
esophagus, forestomach, trachea, lung and liver (NTP, 1985). Of
the two chemicals, NNK is the more potent carcinogen. Activation
of a K-ras protooncogene has been demonstrated in NNK-induced,
murine lung tumors (Belinsky et al., 1988). The Carcinogen
Assessment Group of the U. S. EPA includes NNN on its list of
potential human carcinogens.
10.4 METABOLISM
10.4.1 Detoxification
No relevant data were found in the literature.
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10.4.2 Activation
The major activation pathway of NNN and NNK is
a-hydroxylation, i.e., hydroxylation of the carbons adjacent to
the nitrosamine group. Depending on which of two possible
a-carbons is hydroxylated, the product can be either a
methylating agent or 4-(3-pyridyl)-4-oxobutyl-diazohydroxide, a
proposed ultimate carcinogen of NNN and NNK that forms bulky
adducts with Hb (Carmella and Hecht, 1987) and DNA (Hecht et al.,
1988) . The methylating species of NNK metabolite forms the
promutagenic DNA adduct O -methylguanine which accumulates in
target tissues of rats during repeated exposure to NNK (Belinsky
et al., 1986).
In the Clara cell of the lung, where the highest
accumulation of O6-methylguanine occurs, alkylation efficiency
increases dramatically with decreasing doses of NNK, suggesting
the existence in this cell of a low K,,, (i.e., high affinity)
pathway for the activation of NNK that becomes saturated at high
doses. A similar pathway appears to exist in the respiratory,
but not the olfactory/ nasal mucosa. Extrapolation from high-
dose alkylation data would greatly underestimate carcinogenic
risk at low doses, if this low K,,, pathway were not taken into
consideration (Belinsky et al., 1987; Swenberg et al., 1987).
Cell proliferation is another factor that should be taken
into account in low dose extrapolations. In rats chronically
treated with high doses of NNK (i.e., 16-50 mg/kg), the incidence
of induced malignant tumors was 45% in the olfactory mucosa, but
only 5% in the respiratory mucosa (Belinsky .et al., 1987;
Swenberg et al., 1987). Although the levels of Os-methylguanine
were similar in the two regions, cytotoxicity-related cell
proliferation was observed only in the olfactory mucosa. Low,
sub-cytotoxic doses tend to induce benign tumors of both the
respiratory and olfactory mucosa.
10.4.3 Host Factors
The activation pathway of NNN and NNK (i.e., ot-
hydroxylation) has been measured in human tissues, where a 100-
fold interindividual variation is observed. The endogenous
formation of nitrosamines is another possible source of
individual variability. However, due to the absence of human
monitoring data, it is not known to what extent, if any, NNN
and/or NNK are formed endogenously in smokers and non-smokers
(Carmella and Hecht, 1987).
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10.5 PROTEIN ADDUCTS
10.5.1 Characterization
In vivo, the N-hydroxy metabolites of both NNN and NNK
form a mixture of as yet unidentified adducts at nucleophilic
centers in rat hemoglobin. Treatment of either NNN- or NNK-
adducted globin with dilute NaOH or HC1 releases a portion of the
bound material in the form of 4-hydroxy-l-(3-pyridyl)-1-butanone
(HPB). The probable ultimate electrophile, 4-(3-pyridyl)-4-
oxobutyldiazohydroxide, is produced by a-hydroxylation of either
NNN or NNK and binds to Hb to yield an acid/base-labile adduct
(Carmella and Hecht, 1987; Carmella et al., 1987).
Probable sites of formation for these globin adducts include
cysteine, histidine, lysine, valine, aspartate and glutamate.
The authors (Carmella and Hecht, 1987; Carmella et al., 1987)
suggest that the parent adduct may be either an aspartate or
glutamate ester formed by reaction of these residues with the
electrophilic metabolite 4-(3-pyridyl)-4-oxobutyldiazohydroxide.
10.5.2 Rate of Formation
No relevant data were available in the literature.
10.5.3 Dose-Response
In rats treated with radiolabeled NNK, total binding (10-720
fmol/kg) was linear over a 100-fold dose range (0.03-3.9 p.mol/kg)
and amounted to 0.1% of dose 24 hours after treatment. Label was
detectable in globin for 6-8 weeks after treatment. Treatment of
globin with aqueous NaOH released 10-15% of the bound
radioactivity as HPB (about 0.02% of dose 24 hours after
treatment), which was detectable for 6 weeks following a single
dose. With chronic dosing, globin adducts accumulated and
reached steady state after 35 days. After cessation of
treatment, radioactivity was lost from globin with a half-life of
12 days (Carmella et al., 1987; Carmella and Hecht, 1987).
In rats treated with radiolabeled NNN, the globin adducts
formed represented approximately 0.09% of the dose. The total
radioactivity bound to globin had a half-life of 10 days. A
smaller percentage (around 1.6-2.4%) of these NNN-globin adducts
were released as HPB upon treatment with aqueous base.
Furthermore, these adducts were detectable for only 1 week after
treatment (Carmella and Hecht, 1987; Carmella et al., 1987).
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10.5.4 Background Levels
Considering that tobacco products are the only source of
exposure to NNN and NNK, one would not expect detectable
background levels of adducts with these compounds. However, the
possibility of endogenous production of NNN and/or NNK has not
yet been ruled out.
10.5.5 Methods of Detection
Carmella and Hecht (1987) isolated globin in the standard
way. Packed red blood cells were washed and lysed. The
resulting mixture was centrifuged to remove cell debris and the
Hb-containing supernatant was dialyzed overnight before being
added dropwise to ice-cold 1% HC1 in acetone. The globin thereby
precipitated was then dried at 35"C.
Acid/base-labile adducts were then liberated by dissolving
the globin in 0.1 N NaOH. After the pH was readjusted to 6, the
precipitated globin was removed by centrifugation and the
supernate was extracted with chloroform and concentrated to
dryness. The identity of the cleavage product 4-hydroxy-l-(3-
pyridyl)-1-butanone (HPB) was determined by HPLC and confirmed by
GC-MS after derivatization with
bistrimethylsilyltrifluoroacetamide.
10.6 RESEARCH NEEDS
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. Unfortunately,
Carmella and Hecht (1987) calculate that adduct levels in humans
should be in the range of fmol/ml, which is near the limit of
detection of most current methods.
It is possible, however, that the level of HPB-yielding
globin adducts was underestimated. Significant losses may have
occurred during sample preparation, since the globin was
precipitated in acetone containing 1% HC1 and dried down before
being treated with 0.1 N NaOH to release labile adducts as HPB.
Even if levels of HPB-yielding NNN-Hb adducts in humans prove to
be as low as predicted, NNN-exposure might still be monitored
using one of the unidentified Hb adducts that made up 98% of the
bound material, provided, of course, they are not all methylation
products. There is also the possibility that NNN adducts are
present in serum albumin at higher levels than in Hb, perhaps as
an ester of the N-terminal aspartate.
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A survey of the literature suggests that the analysis of
serum albumin adducts using the modified Edman degradation has
not yet been attempted, even though such an approach, if
successful, might be useful for monitoring exposures to chemicals
that bind poorly to Hb. The reactivity of the amino groups of
valine and aspartic acid should be quite similar; however, the
carboxyl group of aspartate could, conceivably, compete for the
Edman reagent and alter the cyclization reaction in such a way as
to prevent cleavage. The appropriate experiments should be
performed to resolve these questions.
Before tobacco-specific nitrosamines can be considered any
further as candidates for protein adduct-based exposure
monitoring, specific NNN/NNK adducts will have to be identified
and characterized in exposed humans. Reports of human NNN/NNK-Hb
adducts have not yet appeared in the literature. The methodology
is available, however, for the detection, identification and
comparison of DNA and protein adducts in humans exposed to
NNN/NNK, i.e., cigarette smokers.
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11. GROUP III CHEMICALS:
BENZO(A)PYRENE
11.1 MANUFACTURE AND USE
Benzo(a)pyrene (B(a)P) (CAS # 50-32-8), also called
3,4-benzpyrene, is a carcinogenic polycyclic aromatic hydrocarbon
(PAH). Although not produced commercially in the U.S., B(a)P is
a ubiquitous environmental contaminant. Its only use in pure
form is as a positive control for laboratory mutagenicity and
carcinogenicity studies (HSDB, 1989i).
11.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Sir Percival Potts described the first recorded case of
occupational cancer in the late eighteenth century after
observing a high incidence of scrotal cancer in chimney sweeps
who had been exposed since childhood to contact with soot from
coal fires (Doull et al., 1980).
It is now known that B(a)P (among other carcinogens) is
present in fossil fuels and coal tar (Sax, 1987), and is a
product of the incomplete combustion of organic materials.
Today, occupational exposure to B(a)P occurs at airports, tarring
facilities, refuse incinerators, power plants, and coke
manufacturing facilities. The general public is exposed to B(a)P
via cigarette smoke (2-122 ng/cigarette), certain food sources
(0.1-50 ppb) and air pollution. OSHA indirectly limits exposure
to B(a)P by requiring that occupational exposure to coal tar
pitch volatiles not exceed an 8-hour TWA of 0.2 mg/cu m. (1 ppm
= approx 10.32 mg/cu m) (NTP, 1985).
11.3 KNOWN HEALTH EFFECTS
The health effects of B(a)P and other PAH's have been
reviewed by the U.S. EPA in two 1984 Health Effects Assessment
documents (U.S. EPA, 1984a,b).
Both a local and a systemic carcinogen in animals, B(a)P has
produced tumors in all species tested following oral, dermal or
inhalation exposure (NTP, 1985). In humans, numerous
epidemiological studies have shown a clear association between
occupational exposure to PAH-containing materials (e.g., coke
oven emissions, soots, tars, and neutral oils) and increased
cancer risk (U. S. EPA, 1984b). NIOSH concludes that these
materials are carcinogenic to humans. The Carcinogen Assessment
Group of U.S. EPA classifies B(a)P as a Group B2 carcinogen.
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11.4 METABOLISM
11.4.1 Detoxification
The principal route of oxidative metabolism of B(a)P and
most other PAH's is effected by the aryl hydrocarbon hydrolase
(AHH) of the endoplasmic reticulum and nuclear envelope (Hodgson
and Guthrie, 1980). Hydroxylation reactions usually decrease
toxicity, increase water solubility and provide sites for
conjugate formation. Alternatively, initial arene epoxide
metabolites of B(a)P may rearrange nonenzymatically to form
phenolic derivatives, or be enzymatically converted to
dihydrodiols (by epoxide hydratase) or to glutathione conjugates
(by glutathion S-transferase). Clearance of Hb adducts in mice
is linear and consistent with the 40-day mean lifespan of the
murine erythrocyte (Calleman, 1982).
11.4.2 Activation
In a review of the metabolism of PAHs, Phillips and Grover
(1984) note that B(a)P is metabolized primarily in the liver,
although it may also be activated to protein binding forms by co-
oxidation with lipoxygenase, unsaturated fatty acids and heme-
compounds. The ultimate carcinogen, which is thought to be the
reactive, bay region, vicinal diol epoxide, (+)-anti-7B,8a-
dihydroxy-9a,10a-epoxy-7,8,9,19-tetrahydrobenzo(a)pyrene (BPDEI),
is the product of the following sequence of reactions.
First, the 7,8 double bond is oxygenated by a mixed-function
oxidase to yield the 7,8-epoxide, which is subsequently hydrated
by epoxide hydratase to yield the 7,8-dihydro-7,8-dihydroxy
derivative. Then, a second MFO-mediated oxygenation at the 9-10
double bond produces the putative ultimate carcinogen (Hodgson
and Guthrie, 1980).
Unless they are detoxified by conjugation reactions,
reactive isomers of BPDE are able to form adducts with nucleic
acids and protein. In the major BaP-DNA adducts, isomers of BPDE
are covalently bound to the 2-amino group of guanine; BPDE-DNA
adducts induce base substitution mutations, mainly G:C to T:A
transversions (Maher et al., 1987).
11.4.3 Host Factors
There are wide interindividual variations in metabolism of
PAH's in human populations (Weston et al., 1989). Genetic
variation at the Ah locus, which determines the inducibility of
AHH, may be particularly important (U. S. EPA, 1984a).
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11.5 PROTEIN ADDUCTS
11.5.1 Characterization
The existence of BPDE adducts in Hb is inferred from the
release from acid-treated globin of BPDE-tetrols. However, the
adducted amino acids have not been identified. Furthermore,
acid-released tetrols account for only 1-2% of the total B(a)P
bound to Hb in vivo, as determined by radioactivity measurements.
For the most part, the remaining adducts have yet to be
identified.
11.5.2 Rate of Formation
No values for the rate constant k for reaction of B(a)P with
protein or amino acids were found in the literature.
11.5.3 Dose-Response
In mice exposed to 1-100 pmol [3H]-B(a)P/g body weight,
Balhorn et al. (1985) demonstrated a linear relationship between
exposure dose and serum albumin adducts (i.e., bound
radioactivity), which vere cleared at a rate consistent with the
halflife of serum albumin in the mouse (tl/2=l day). Binding of
B(a)P to hemoglobin was nearly 3 orders of magnitude lower than
binding to serum albumin.
Some portion of the minor adduct formed by reaction of the
BPDE-I metabolite with protein is converted to free tetrols upon
mild treatment with acid (Koreeda et al., 1978). Using
HPLC/fluorescence of acid-released tetrols, Shugart (1985) was
able to detect BPDE-Hb adducts in mice 24 hr after a single
topical dose of B(a)P. The formation of BPDE-1-HB adducts was
linear with dose up to 800 jxg B(a)P (which corresponded to 0.02
pmol tetrols/mg globin) on a log-log plot, and correlated with
the degree of adduct formation in skin DNA. The author concluded
that the Hb adduct was formed almost exclusively by interaction
with anti-BPDE, the same ultimate carcinogenic form of B(a)P that
interacted with DNA in the target organ. Twenty four hours after
intraperitoneal administration of 400 jug BaP, measured levels of
BPDE-1-tetrols were 0.05 pmol/mg globin (Shugart and Kao, 1985;
Shugart, 1986).
Weston et al. (1989) have applied HPLC and synchronous
fluorescence spectroscopy to the detection of benzo(a)pyrene-
7,10/8,9-tetrahydrotetrol released by mild acid hydrolysis from
the Hb of smokers. Their results suggested levels of bound
carcinogen in smokers were around 1 ng BPDE/g Hb, even though
cigarettes contain 2-30 times as much B(a)P as 4-ABP.
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Wallin et al. (1987) used both HPLC/LSC and a competitive
ELISA to measure B(a)P tetrols released by acid treatment of Hb
from rats treated with 1 mg [3H]B(a)P by i.p. injection 48 hrs
earlier. Covalent binding was observed in liver DNA (64.4
pmol/mg), liver protein (78.3 pmol/mg), lung protein (60
pmol/mg), skin protein (40.6 pmol/mg) and globin (2.3 pmol/mg).
Covalent binding in all tissues except skin was even lower (by
30-50%) after topical administration of 1 mg [3H]-B(a)P (1.8
pmol/mg globin).
When in vitro modified BPDE-1 globin was treated with dilute
acid, about 40% of the radioactivity was released, more than 95%
of which was cochromatographed with two UV peaks corresponding to
BPDE-1 tetrols. Only about 2-3% (0.057 pmol/mg) of the
radioactivity bound to in vivo-modified. mouse globin was
released by hydrolysis in 0.12 N HC1, and less than half (45%) of
this was BPDE-1 tetrols (0.023 pmol/mg). The levels of acid-
released tetrols observed in these experiments were below the
limit of detection by competitive ELISA, even though the antibody
could detect as little as 0.10 pmol tetrol/mg globin in
hydrolysates of in vitro modified BPDE-1 globin. The authors
concluded that the majority of B(a)P-Hb adducts are not the
result of diol epoxide binding and have yet to be identified.
Lee and Santella (1988) also used antibody 8E11 to measure
BPDE-globin adducts formed in vivo in mice treated with B(a)P.
However, prior to analysis, the globin was enzymatically digested
(rotated for 2-3 days at 37°C with insoluble protease), and the
modified peptides were isolated on an immunoaffinity column of
antibody 8E11 coupled to CNBr-activated Sephadex 4B (recovery
around 66%). In a competitive ELISA with digested BPDE-I-
modified bovine serum albumin (BSA), 50% inhibition occurred at
400 fmol of adduct compared to 1450 frool for non-digested, BPDE-
modified BSA.
In mice administered 0.33, 1.0 and 3.0 mg [3H]-B(a)P i.p.,
< 10% of the total radioactivity associated with Hb was from
globin. Around 80% was from the heme fraction and the remainder
was from free B(a)P metabolites. The ELISA values for BPDE
adducts in enzymatically digested globin were 1.20, 2.63 and 4.34
nmol/g, respectively, which represented 90-100% of the [3H]-
B(a)P-globin adducts determined by radioactivity over the same
dose range.
The monoclonal antibody, 8E11, used in the above studies was
originally raised against albumin-bound BPDE-DNA adducts. It
cross-reacts with a variety of B(a)P metabolites and adducts. At
a dilution of 1:100,000, the I50 for BPDEI-1-tetrols, BPDE-1-DNA,
and BPDE-1-protein was 250, 350, and 3000 fmol, respectively
(Wallin et al., 1987). At a dilution of 1:30,000, the I50 for
BP-9,10-diol, BP-7,8-diol, BPDE-I-tetrol, BPDE-I-BSA (digested),
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BPDE-I-BSA (non-digested), 4-OH-BP and 5-OH-BP was 150, 250, 350,
400, 1450, 42,700 and >1000,000 fmol, respectively (Lee and
Santella, 1988).
11.5.4 Background Levels
B(a)P-Hb adducts were not detectable in non-exposed mice
(Lee and Santella, 1988). No data were available in the
literature on background levels of B(a)P-Hb adducts in humans.
11.5.5 Methods of Detection
Benzo(a)pyrene and other polynuclear aromatic hydrocarbons
(PAH) are intrinsically fluorescent. HPLC combined with
synchronous fluorescence spectroscopy is a highly specific method
for detecting B(a)P adducts in both human hemoglobin and DNA
(Weston et al., 1989). Also, since fluorescence spectroscopy is
not a destructive process, samples analyzed by this method may be
subsequently derivatized and re-analyzed by GC-MS for validation
(Weston et al., 1989).
The (very) minor B(a)P adduct that is formed by reaction of
the BPDE-I metabolite with Hb is converted to free tetrols upon
mild treatment with acid (Koreeda et al., 1978). These BPDE-Hb
adducts are measured indirectly by HPLC/fluorescence spectrometry
of the corresponding acid-released tetrols (Shugart, 1985a and
1985b). However, Shugart observed wide variations in the
recovery of tetrols from modified mouse Hb. Because the protein
has a strong buffering capacity, the hydrolysis conditions for Hb
are difficult to control. In addition, a significant percentage
(as much as 15%) of the "free" tetrols may remain noncovalently
bound to protein residues even after extensive dialysis
(Pastorelli et al., 1988). However, the vast majority of B(a)P-
globin adducts are relatively stable to acid, and at least one of
peptide adducts, like the tetrol-releasing adduct(s), is derived
from anti BPDE.
Can et al. (1989) have recently made improvements in the
laser induced fluorimetry of anti-BPDE-adducted peptides at room
temperature . First, they ran enzymatically digested globin on
an immunoaffinity column to enrich the sample in BPDE-peptide
adducts and remove the background protein fluorescence due
principally to aromatic amino acids. Then, applying a temporal
discrimination technique, they also reduced the water Raman
signal. The latter is only 40-50 nm away from the peak emission
for the pyrene fluorophore, which occurs at 379 nm in the
synchronous spectral mode. This arrangement allowed them to
achieve a detection limit of 10 picomolar with a signal to noise
ratio of 3.
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A particularly sensitive form of this method is fluorescence
line narrowing spectrometry (FLNS), in which the analytes are
dissolved in a glass-forming solvent, cooled to cryogenic
temperatures (4-20° Kelvin), and excited with a pulsed tunable
dye laser. The resulting vibronic fluorescence bands are much
narrower than usual, making it possible to selectively measure
analytes in mixtures, e.g., intact DNA-PAH adducts, and PAH in
solvent-refined coal. Jankowiak et al. (1988) have recently used
a new form of FLNS to measure intact DNA-PAH adducts with a
detection limit of approximately 1 femptomol, or about 3 modified
bases in 108 (20 /xg of DNA) . In addition to exhibiting high
sensitivity and selectivity, FLNS also ensures rapid sample
throughput, and applies to a wide range of biomolecules,
including globin-PAH and polar metabolites in urine. Of course,
the equipment is quite expensive, and the method, while an
important research tool, is not well suited for mass screening.
In general, antibody techniques have not proven to be highly
successful means of monitoring B(a)P exposure in humans (Weston
et al., 1987). Intact protein adducts may not be accessible to
the antibody, and hydrolyzed adducts require cleanup and analysis
with HPLC, which may reduce sample yield by 30-40% Still, the
latter methods have been validated in animal studies over a 10-
fold dose range (Lee and Santella, 1988) and, with some
refinement, may be applicable to monitoring human exposures to
PAHs.
Endogenous antibodies against a range of PAH-DNA adducts are
known to be formed in the sera of humans. However, only 30% of
occupationally exposed study subjects have tested positive thus
far.
11.6 RESEARCH NEEDS
Due to the low levels of B(a)P-Hb adducts that can be found
in human samples, increased sensitivity is required before
synchronous fluorescence spectroscopy will be of value as a tool
in epidemiological studies. Can et al. (1989) are currently
investigating the feasibility of direct fluorometry of intact
anti-BPDE derived globin adducts using their temporal
discrimination technique to remove spectral interferences and
hope to achieve analytical sensitivity limits of less than 1 fmol
of adduct.
However, from an exposure assessment perspective, a more
productive approach may be to seek more abundant B(a)P adducts.
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. Do B(a)P adducts
form at the amino nitrogen of valine - the major BPDE-DNA adducts
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are formed at the 2-amino group of guanine - and, if so, can they
be analyzed by the modified Edman procedure? Such questions
could be answered by in vitro experiments designed to determine
which amino acids are arylated by which metabolites of B(a)P.
Finally, a much greater effort should be made to identify
and quantify specific B(a)P-serum albumin adducts. In mice, the
highest level of B(a)P adducts occurs in liver proteins (Wallin
et al., 1987), of which newly synthesized albumin is one, and
total binding of radiolabeled B(a)P is almost three orders of
magnitude higher in serum albumin than in Hb (Balhorn et al.,
1985). A similar degree of magnification might reasonably be
expected for the BPDE-I adduct in serum albumin. Also, if B(a)P
metabolites react with the amino group of the N-terminal aspartic
acid residue, then certain acid-stable B(a)P-albumin adducts
might be detectable using the modified Edman procedure.
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12. GROUP III CHEMICALS:
1-NITROPYRENE
12.1 MANUFACTURE AND USE
The common PAH 1-nitropyrene (1-NP) (CAS # 5522-43-0) may
also be called 3-nitropyrene (Sax, 1984). The chemical is
neither produced nor used commercially in the U.S. but is a
widespread environmental contaminant. Nitropyrene is not listed
in the Merck Index (1983), Hawley's Condensed Chemical Dictionary
or the Hazardous Substances Database of the Toxnet system of
databases.
12.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Nitro polycyclic aromatic hydrocarbons (including 1-NP) are
widespread environmental contaminants which may pose a
significant human health hazard. They may be found in airborne
particulates, diesel emissions, coal fly ash, carbon black
photocopier toners and smoke from nitrate-fortified cigarettes.
1-NP is the principal nitro PAH found in diesel exhaust. Upon
inhalation, the majority of the 1-NP adsorbed onto diesel
particles is fully available for distribution throughout the body
and for further metabolism and possible activation (Jackson et
al., 1985).
12.3 KNOWN HEALTH EFFECTS
1-NP is mutagenic in bacterial and mammalian cell cultures
(Maher et al., 1987) and carcinogenic in rats (Jackson et al.
1985) .
12.4 METABOLISM
12.4.1 Detoxification
No data were available in the literature regarding routes of
detoxification. However, 1-NP is probably eliminated in bile and
urine as conjugates of ring-hydroxylated and N-hydroxylated
metabolites.
12.4.2 Activation
The metabolism and biological activity of nitro PAHs has
been reviewed by Beland et al. (1985). 1-NP may be converted to
1-aminopyrene by the action of bacterial, microsomal or cytosolic
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nitroreductases. A subsequent metabolite, N-hydroxy-l-
aminopyrene binds directly to DNA to form N-deoxyguanosin-8-yl)-
1-aminopyrene, the only major DNA-l-NP adduct (Maher et al.,
1987) , thereby accounting for the mutagenicity exhibited by 1-NP
in the Ames test.
The oxidative metabolism of 1-NP by different P-450 isozymes
leads to the formation of two K-region epoxides and a variety of
ring hydroxylation products. Both epoxides can bind to DNA and
to glutathione in vitro (Djuric et al., 1986). Although binding
to glutathione is fastest in the presence of glutathione
transferases, non-enzymatic binding occurs at a measurable rate.
The same glutathione conjugates are also found in the bile of
rats administered 1-NP. The probability exists that both
reductive and oxidative metabolites of 1-NP also form protein
adducts in vivo.
12.4.3 Host Factors
No data were found in the literature regarding host factors
in the formation of 1-NP adducts. However, it is reasonable to
assume that variations in nitroreductase activity and/or
oxidative metabolism will have both qualitative and quantitive
effects on the formation of 1-NP adducts.
12.5 PROTEIN ADDUCTS
12.5.1 Characterization
Although no fully characterized NP-protein adducts have been
described in the literature, there is reason to believe that the
major adduct in Hb is a cysteinyl sulfinic acid amide of a ring-
oxidized, N-hydroxylamine metabolite (Johnson et al., 1988).
12.5.2 Rate of Formation
No data were available in the literature regarding rate
constants of reaction between 1-NP metabolites and protein.
12.5.3 Dose-Response
In the inhalation studies of Jackson et al. (1985), 0.6% of
a radioactive dose was bound to lung proteins (and none to DNA)
24 hr after administration. Johnson et al. (1988) observed a
linear relationship between dose and formation of 1-NP-Hb adducts
in rats treated by gavage with 0.01- 1000 ng [3H]l-NP/kg body
weight. Approximately 0.06% of the total radioactivity was
bound, at least 80% of which was released into solution during
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precipitation of the globin with acidic acetone. Reverse-phase
HPLC analysis yielded a single major peak which did not co-elute
with 1-aminopyrene. Although not yet fully characterized, the
adduct appears to be formed from a ring oxidized arylhydroxyl-
amine. No other reports of specific nitropyrene-protein adducts
were found in the literature.
12.5.4 Background Levels
No data were available in the literature regarding
background levels of 1-NP.
12.5.5 Methods of Detection
Specific 1-NP-protein adducts have yet to be identified.
Experiments to date have usually involved radiolabed compounds,
and the methods of analysis have been limited to HPLC and liquid
scintillation counting.
12.6 RESEARCH NEEDS
This chemical needs further study in all biomarker-related
areas, beginning with the identification of the major adduct with
Hb.
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13. GROUP IV CHEMICALS:
VINYL CHLORIDE
13.1 MANUFACTURE AND USE
Vinyl chloride (VC) (CAS # 75-01-4), also called
chloroethene and chloroethylene, is the most important of the
industrial vinyl monomers. As Table 8 shows, VC is manufactured
at several sites in the U.S., especially in Louisiana. It was
the 19th highest-volume chemical produced in the U.S. in 1985
(Sax, 1987) at 9.48 billion Ibs (HSDB, 1989J). It is
manufactured by the dechlorination of ethylene dichloride or by
reaction of acetylene and hydrogen chloride (Sax, 1987). Most of
the VC produced in the U.S.—over 9 billion Ibs. in 1988 (C&EN,
1989)—is used in the plastics industry for the manufacture of
polyvinyl chloride (PVC) and copolymers. It is also used in
adhesives for plastics, in the organic synthesis of other
organic chemicals (Merck, 1983) such as chloroacetaldehyde,
methyl chloroform and 1,1,1-trichloroethane (HSDB, 1989J).
13.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Vinyl chloride monomer is not known to occur in nature.
Discharges from the plastics industry are the major source of
environmental VC, most of which eventually ends up in the
atmosphere. Emissions are not to exceed 10 ppm. The maximum
contaminant level allowed in drinking water is 2 ;ug per liter.
The TLV for VC recommended by the ACGIH (1986e) is 5 ppm
(approximately 10 mg/ cu m). However, because VC is an
established human carcinogen, atmospheric levels in the work-
place are usually below the 8 hr TWA of 1 ppm promulgated by OSHA
in 1983. Thus, while the size of the population with
occupational exposure to VC should be quite large, the levels of
exposure may be very low (HSDB, 1989J).
Inhalation is the major route of VC exposure. Respiratory
absorption of VC occurs rapidly, the percentage retained (around
42% in man) being independent of the concentration inhaled at low
exposures (ATSDR, 1988).
13.3 KNOWN HEALTH EFFECTS
The health effects of VC have been reviewed by the U. S. EPA
(1984c). More recently, the Agency for Toxic Substances and
Disease Registry reviewed the toxicology of vinyl chloride in a
report that cites 234 references (ATSDR, 1988b).
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TABLE 8. U.S.PRODUCTION OF VINYL CHLORIDE
MANUFACTURERS
SITES OF PRODUCTION
BF Goodrich Chemical Group,
Cleveland, OH
Borden Chemicals and Plastics,
Geismar, LA
Dow Chemical USA
Midland, MI
Formosa Plastics Corporation USA
Florham Park, NJ
Georgia Gulf Corporation
Atlanta, GA
Occidental Chemical Corporation
Dallas, TX
Avon Lake, Ohio
Henry, Illinois
Pedricktovm, New Jersey
Plaquemine, Louisiana
Deer Park, Texas
Louisville, Kentucky
Niagara Falls, Ontario
Shawnington, Quebec
Scottford, Alberta
Geismar, Louisiana
Oyster Creek, Texas
Plaquemine, Louisiana
Baton Rouge, Louisiana
Point Comfort, Texas
Plaquemine, Louisiana
Deer Park, Texas
Niagara Falls, New York
PPG Industries Inc, Chemicals Group Lake Charles, Louisiana
Pittsburgh, PA
Vista Chemical Company
Houston, TX
Lake Charles, Louisiana
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Short-term exposures to sufficiently high concentrations can
cause narcotic effects such as dizziness, incoordination,
headache, unconsciousness and even death. Chronic exposure to
low concentrations may produce so-called "vinyl chloride
disease", which is characterized by acro-osteolysis, dermatitis,
thrombocytopenia, angioneurosis, and hepatitis-like liver damage
(U.S EPA, 1984C).
Although rare forms of neoplasia, both hepatic and
extrahepatic angiosarcomas are readily induced by VC exposure in
rats, mice and hamsters (U.S. EPA, 1984c; ATSDR, 1988). VC-
induced liver cancer in the mouse is highly correlated with the
activation of an H-ras oncogene by a single-point mutation
(Stowers et al., 1987). In humans, hepatic angiosarcomas occur
almost exclusively among VC reactor cleaners (Williams et al.,
1985) .
13.4 METABOLISM
13.4.1 Detoxification
In rats, rapid uptake by the tissues leads to equilibration
between atmospheric and tissue concentrations in approximately 15
minutes. Distribution and metabolism are also rapid, with the
parent compound being concentrated in the fatty tissues and the
metabolites becoming concentrated in the liver and kidneys.
At exposure levels below 50 ppm, the major route of
metabolism is a pathway involving alcohol dehydrogenase (ADH),
whereby VC is sequentially oxidized to 2-chloroethanol, 2-
chloroacetaldehyde (CAA) and 2-chloroacetic acid. Formation of
the last product is limited by the conjugation of CAA with
ubiquitous sulfhydryl groups. CAA may also be formed by the
peroxidation and dehydration of 2-chloroethanol, or by the
spontaneous rearrangement of 2-chloroethylene oxide (CEO). Both
CAA and CEO form conjugates with hepatic glutathione which are
subsequently metabolized to the mercapturic acid N-acetyl-S-(2-
hydroxyethyl)cysteine or the dicarboxylic acid thiodiglycolic
acid and excreted in the urine. The latter substance has been
used to monitor occupational exposure to VC (ATSDR, 1988).
13.4.2 Activation
Although the microsomal oxidation of VC is a minor pathway
at low concentrations, it is the major route of metabolism at
higher concentrations. This pathway leads to the formation of the
highly reactive epoxide 2-chloroethylene oxide (CEO) which binds
predominantly to DNA (Bolt, 1984). The major DNA adduct is
probably 7-N(2-oxoethyl) deoxyguanosine. This adduct may cyclize
to involve the Os position as well as the N7 position. In vitro
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experiments (Ottenwalder et al., 1983) have shown that CEO is
stable enough to diffuse out of the hepatocytes in which it is
formed into sinusoidal cells. If such a process occurs
the subsequent alkylation of DNA in liver epithelial cells might
account for the induction by vinyl chloride of
hemangioendotheliomas of the liver.
Sequential oxidation via the alcohol dehydrogenase pathway,
or rearrangement of CEO, leads to the formation of another
reactive metabolite, 2-chloroacetaldehyde , which binds to
protein (Bolt, 1984), probably to form S-(2-oxoethyl)cysteine
adducts (Svensson and Osterman-Golkar, 1986). It has been
suggested that protein binding of VC metabolites at sulfhydryl
groups may account for the hepatotoxicity of vinyl chloride (Bolt
et al., 1982).
13.4.3 Host Factors
Alcohol consumption may potentiate the effects of VC
exposure. As a substrate for ADH, ethanol will competitively
inhibit the detoxification of vinyl chloride by that enzyme.
Animal experiments confirm that more liver tumors are induced by
the co-administration of VC plus ethanol than by the
administration of VC alone (ATSDR, 1988) . Animal data also
demonstrate that MFO-induction potentiates the hepatotoxicity of
VC and suggest that humans may be made more sensitive to VC by
exposure to environmental pollutants and drugs that induce MFO
(ATSDR, 1988b).
13.5 PROTEIN ADDUCTS
13.5.1 Characterization
The reactive epoxide metabolites of VC introduce 2-oxoethyl
groups at the sulfhydryl groups of cysteine and the 1-N and 3-N
positions of histidine (Osterman-Golkar et al., 1977).
13.5.2 Rate of Formation
No values of the rate constants of reaction between VC and
protein were found in the literature.
13.5.3 Dose-Response
The only dose-response data that could be found in the
literature are reported by Walles et al. (1988). In mice treated
with 100, 250 or 500 ppm VC, levels of N-(2-oxoethyl)histidine
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were 5.4, 40.0 and 40.0 nmol/g Hb, respectively, indicating that
metabolic activation of VC was saturated at 250 ppm.
13.5.4 Background Levels
No published information is available. However, some
background 2-hydroxyethyl adducts may result from the reaction of
endogenous glycol aldehyde with protein.
13.5.5 Methods of Detection
To date, published studies of the known 2-oxoethyl adducts
of cysteine and histidine have employed GC-MS analysis of
appropriate fractions of total protein hydrolysates (Bolt et al.,
1982; Bolt, 1984; Osterman-Golkar et al., 1977). In protein
hydrolysates, all VC adducts will be detected as 2-
hydroxyethylations indistinguishable from those produced by
ethylene oxide and ethylene dichloride. The modified Edman
degradation procedure has, apparently, not yet been applied to
the analysis of VC-protein adducts.
13.6 RESEARCH NEEDS
No reliable method currently exists for biological
monitoring of exposure to vinyl chloride at concentrations below
5 ppm (ATSDR, 1988) . VC is metabolized too rapidly for urinary
levels of the unchanged compound to be of any use. There is some
correlation between exposure dose and levels of urinary
thiodiglycolic acid, the major urinary metabolite of VC, but that
correlation is too weak to be reliable at low levels of exposure.
Consequently, a protein adduct-based method of exposure
monitoring would be particularly useful in the case of vinyl
chloride. Unfortunately, little information was found in the
literature regarding the identification and quantification of
specific VC-protein adducts, indicating that more research is
needed in all areas. In particular, it may be worthwhile to
apply the modified Edman degradation procedure to the analysis of
2-oxoethyl- and 2-hydroxyethyl-valine in the Hb of animals
exposed to vinyl chloride.
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14. GROUP IV CHEMICALS:
ETHYLENE DICHLORIDE
14.1 MANUFACTURE AND USE
Ethylene dichloride (EDC) (CAS # 107-06-2) or 1,2-
dichloroethane was the 14th highest volume chemical produced in
the U.S. in 1985 (Sax, 1987) and is the largest volume
chlorinated organic chemical currently being produced. In 1988,
estimated production of EDC was almost 13.7 billion Ibs (C&EN,
1989) . Ethylene dibromide, by contrast, is rapidly being phased
out. EDC is manufactured either by the direct catalytic
chlorination of ethylene or by the catalytic oxychlorination of
acetylene with HC1 and oxygen. It is also produced as a
byproduct of the chlorohydrin process for the manufacture of
ethylene oxide. Production of EDC from 1980 through 1986 has
been recorded at 11.1, 10.0., 7.6, 11.5, 7.3, 12.1, and 12.9
billion pounds, respectively (HSDB, 1989k). Ten companies
currently produce EDC at sites located predominantly in Texas and
Louisiana (Table 9).
The great majority of EDC is used in the production of vinyl
chloride. It is also used in the synthesis of trichlorethylene,
vinylidene chloride and trichlorethane (Sax, 1987), as a solvent
for fats, oils, gums, waxes, resins and especially rubber (Merck
Index, 1983); as a lead scavenger in antiknock gasoline; and in
paint, varnish and finish removers. Other uses include metal
degreasing; ore flotation; and fumigation of grains, orchards,
upholstry and carpets (Sax, 1987).
14.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
EDC does not occur naturally in the environment. However,
significant atmospheric releases occur from its production and
use as a chemical intermediate, lead scavenger, extraction and
cleaning solvent, diluent for pesticides, grain fumigant and in
paint, coatings and adhesives. Due to its high vapor pressure,
most EDC in soil and water will ultimately migrate into the
atmosphere, where photooxidation is its primary fate.
Nevertheless, drinking water is a potential source of human
exposure, particularly near point sources or hazardous waste
sites. Limited accumulation of EDC has been documented in human
fat and milk (ATSDR, 1988).
The primary route of exposure is by inhalation in the
workplace where the TLV is 10 ppm (Sax, 1987). The highest
exposures might be expected among workers in the chemical and
allied products industry. However, monitoring data on
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TABLE 9. U.S. PRODUCTION OF ETHYLENE DICHLORIDE
MANUFACTURERS
SITES OF PRODUCTION
BF Goodrich Co
BF Goodrich Chem Group
Cleveland, OH
Borden Chemicals
Geismar, LA
Dow Chemical Co,
Midland, MI
Formosa Plastics Corp USA
Florham Park, NJ
Georgia-Gulf Corp
Atlanta, GA
PPG Indust, Inc,
Chem Div,
Pittsburgh, PA
Spectrum Chemical Mfg.
Gardena, CA
Union Carbide Corp
Ethylene Oxide Derivatives Div.
Taft, LA
Vista Chemical Co,
Houston, TX
Vulcan Chemicals,
Div of Vulcan Materials Co,
Birmingham, AL
La Porte, Texas
Calvert City, Kentucky
Geismar, Louisiana
Freeport, Texas
Oyster Creek, Texas
Plaguemine, Louisiana
Baton Rouge, Louisiana
Point Comfort, Texas
Plaguemine, Louisiana
Lake Charles, Louisiana
Gardena, California
Taft, Louisiana
Lake Charles, Louisiana
Geismar, Louisiana
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occupational exposure are generally inadequate. Methods are
available to detect and quantify EDO in human blood and urine.
However, exposure to EDC is routinely monitored by determination
of the chemical in the breath (ATSDR, 1988).
14.3 KNOWN HEALTH EFFECTS
In 1984, the U.S. EPA published a report on the health
effects of EDC (U.S. EPA, 1984d). More recently, the toxicology
of EDC has been reviewed by the Agency for Toxic Substances and
Disease Registry (ATSDR, 1988).
Inhalation of EDC vapors can produce irritation of the
respiratory tract and conjunctiva, corneal clouding, equilibrium
disturbances, narcosis and abdominal cramps (Merck, 1983). EDC
is carcinogenic in mice and rats when administered dermally or by
gavage, but not when administered by inhalation (U.S. EPA,
1984d). To date, however, no epidemiological evidence exists to
establish an association between inhalation exposure to EDC and
cancer in humans (ATSDR, 1988) . Both the U.S. EPA and the FDA
have listed EDC as an animal carcinogen (NTP, 1981).
14.4 METABOLISM
14.4.1 Detoxification
The available information on EDC metabolism has been derived
from studies on rodents. Some EDC is expired unchanged or as
CO., but most is excreted in the urine, primarily as chloroacetic
acid, S-carboxymethylcysteine and thiodiacetic acid (ATSDR,
1988). Both S-carboxymethyl-cysteine and thiodiacetic (or
thiodiglycolic) acid are metabolites of the 2-oxoethyl adduct of
glutathione formed by reaction with chloroacetaldehyde (ATSDR,
1988). Thus, EDC appears to be detoxified in much the same way
as vinyl chloride, i.e., via the alcohol dehydrogenase pathway
and by conjugation with glutathione.
14.4.2 Activation
Dihaloalkanes such as EDC are unique in that conjugation
with glutathione (GSH) can be a mechanism of activation rather
than detoxification. Reaction of DNA with S-(2-chloroethyl)GSH
and episulfonium intermediates results in the formation of the
bulky DNA adduct S-[2-(N7-guanyl)ethyl]GSH (Guengerich et al.,
1987). Although N7-purine lesions are not generally mutagenic—
aflatoxin B: is the single known exception—because they are not
in the DNA base-pairing region, there is evidence that the S-[2-
(N7-guanyl)ethyl]GSH adduct is related to genetic damage. The
episulfonium ion may be•detoxified by conjugation with a second
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GSH molecule. Microsomal oxidation of EDC produces 2-
chloroacetaldehyde (CAA) and 2-chloroethylene oxide (CEO), both
of which introduce 2-oxoethyl groups onto nucleophilic sites of
macromolecules.
14.4.3 Host Factors
No information on host factors governing EDC toxicity was
found in the literature. However, since the two chemicals share
the same reactive metabolites, the toxicity of EDC and VC can
reasonably be expected to be similarly affected by such factors
as alcohol consumption and exposure to MFO-inducers.
14.5 PROTEIN ADDUCTS
14.5.1 Characterization
Vinyl chloride, a metabolite of EDC, may form 2-hydroxyethyl
adducts directly with cysteine by Michael addition. The epoxide
metabolite CEO and its spontaneous rearrangement product CAA form
2-oxoethyl adducts with the SH-group of cysteine and with the
nitrogen atoms of histidine and N-terminal valine. When Hb is
treated with sodium borohydride prior to analysis, all 2-oxoethyl
groups are reduced to 2-hydroxyethyl (HOEt) groups (Svensson and
Osterman-Golkar, 1986).
14.5.2 Rate of Formation
No values for the rate constants of reaction of EDC with
protein were found in the literature.
14.5.3 Dose-Response
Twenty-two hours after intraperitoneal injection of mice
with 14C-labeled EDC, Svensson and Osterman-Golkar (1986)
measured 8 pmol of N-(3-HOEt)histidine/g Hb per 0.1 mmol EDC/kg
body weight, all of which appeared to be derived from the
corresponding 2-oxoethyl adduct. Hydroxyethyl adducts were
approximately 60 times higher in cysteine than in histidine and
were derived predominantly (92%) from the corresponding 2-
oxoethyl adducts. The s-value of an alkylating agent is a
measure of its selectivity in reactions with nucleophilic centers
of varying strength. The high ratio of cysteine alkylation to
histidine alkylation observed for 2-oxoethyl groups in the
hemoglobin of DCE-treated mice is consistent with
chloroacetaldehyde (s-value - 1.3) rather than chloroethylene
oxide (s=0.8) as the major electrophilic metabolite (Svensson and
Osterman-Golkar, 1986)..
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Nothing was found in the literature concerning EDC-serum
albumin adducts. However, ethylene dibromide (EDB), another 1,2-
dihaloethane which may reasonably be expected to exhibit chemical
and metabolic behavior at least qualitatively similar to that of
EDC, has been shown to bind to serum albumin (139 nmol/g), but
not appreciably to other plasma proteins, in rats given daily
oral doses (25 mg/kg) of the radiolabled compound for 12
consecutive days (Ansari et al., 1988). EDB also binds to human
serum albumin in vitro (280 pmoles/mg) when incubated in the
presence of rat liver microsomes and a NADPH generating system to
yield an as yet unidentified adduct.
14.5.4 Background Levels
No data were found in the literature concerning background
levels of EDC-protein adducts. However, as with vinyl chloride,
some background 2-oxoethyl or 2-hydroxyethyl adducts may result
from the reaction of endogenous glycol aldehyde with protein.
14.5.5 Methods of Detection
The 2-oxoethyl adducts produced by EDC are typically
measured by HPLC/GC-MS analysis of a total protein hydrolysate.
The degree of 2-oxoethylation may be determined as the difference
between the degree of 2-hydroxylation of reduced and unreduced
Hb. Although 2-oxoethyl adducts would be expected to form at the
N-terminal valine of hemoglobin, no reports were found in the
literature of these adducts being analyzed using the modified
Edman degradation procedure.
14.6 RESEARCH NEEDS
Further studies are required to identify specific EDC
adducts formed with human Hb and serum albumin, develop methods
of detecting and quantifying those adducts, and establish in vivo
dose-response relationships. Assuming that human occupational
exposure to EDC is sufficiently high to produce detectable levels
of protein adducts, some alternative to total protein analysis
will have to be found in order to make monitoring those adducts
feasible on a large scale. A first step in this direction may be
to apply the modified Edman degradation procedure to the analysis
of EDC-Hb adducts, since the study by Svensson and Osterman-
Golkar predates the development of this method. Finally, if
exposure to EDC is to be quantified, study populations will have
to be carefully chosen to minimize confounding exposures, since
EDC-Hb adducts cannot be distinguished from VC-Hb adducts.
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15. GROUP IV CHEMICALS:
ACRYLONITRILE
15.1 MANUFACTURE AND USE
Acrylonitrile (AN) (CAS f 107-13-1), also called vinyl
cyanide, 2-propenenitrile and cyanoethylene, was the 38th highest
volume chemical produced in the U.S. in 1985 (Sax, 1987) at 2.337
billion Ibs (HSDB, 19891). The chemical is manufactured by
several plants in Texas (Table 10). Methods of manufacture
include (1) catalytic reaction of propylene oxygen with ammonia,
(2) catalytic addition of hydrogen cyanide to acetylene, and (3)
dehydration of ethylene cyanohydrin (Sax, 1987).
AN is an industrial monomer used extensively in the
manufacture of synthetic fibers, rubbers, and resins for a
variety of consumer goods. Most is used in the production of
acrylic and modacrylic fibers, acrylonitrile-butadiene-styrene
(ABS) and styrene-acrylonitrile (SAN) copolymers, nitrile
elastomers and latexes such as butadiene-acrylonitrile
copolymers, and adiponitrile, an intermediate in the production
of nylon. It is also used to produce acrylamide (NTP, 1985; Sax,
1987) .
15.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Because acrylonitrile is a volatile chemical, exposure
occurs primarily by inhalation but also occurs by absorption
through the skin. The sizeable production and use of AN would
suggest that the exposed population is also quite large. The
greatest potential exposures to AN are expected to occur among
acrylic resin makers, organic chemical synthesizers, pesticide
workers, and rubber, synthetic fiber, and textile makers (NTP,
1985). However, the actual levels of exposure may be low, since
the OSHA standard of 2 ppm (4.5 mg/cu m) requires personal
protective equipment, training, medical surveillance, signs and
labeling, and engineering controls (AC6IH, 1986f). The general
population may be exposed to low, but undetermined quantities of
airborne AN from industrial emissions and cigarette smoke (NTP,
1985).
15.3 KNOWN HEALTH EFFECTS
Acrylonitrile, a well-known neurotoxicant (Farooqui and
Ahmed, 1983a), is acutely toxic via the metabolic release of
cyanide. Inhalation of AN can cause nausea, vomiting, diarrhea,
weakness, headache, and jaundice (Dreisbach, 1980). Skin contact
with AN has caused epidermal necrolysis.
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TABLE 10. U.S PRODUCTION OF ACRYLONITRILE
MANUFACTURERS
SITES OF PRODUCTION
American Cyanamid Co.
Chemical Prod Div.
Wayne, NJ
E.I.du Pont de Nemours & Co., Inc.
Petrochems Dept., Polymer Ints Dept.
Beaumont, TX
Monsanto Co.
Monsanto Fibers & Ints Co.
Chocolate Bayou, TX
BP America, Inc.
BP Chemicals America, Inc.
Cleveland, OH
Sterling Chemicals
Texas City, TX
Fortier, LA
Beaumont, TX
Chocolate Bayou, TX
Green Lake, TX
Lima, OH
Texas City, TX
YEAR
PRODUCTION
SOURCE
(all figures in millions of Ibs)
1986
1987
1988
2.182 X 10E9 Ibs
2.452 X 10E9 Ibs
2.576 X 10E9 Ibs
C&EN, 1989
C&EN, 1989
C&EN, 1989
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Although AN is weakly mutagenic in Salmonella test strains
after metabolic activation, negative results have been reported
in rodents (Hogy and Guengerich, 1986). Nevertheless, AN does
cause cancers of the.brain, forestomach, and Zymbal's gland in
rats. However, tumors are not observed in the liver where most
of the reactive metabolites are generated (Hogy and Guengerich,
1986). The results of an epidemiological study suggest that
workers exposed to AN may have an increased risk of developing
lung and brain tumors (O'Berg, 1980).
15.4 METABOLISM
15.4.1 Detoxification
Cyanide produced by the hydrolysis of the epoxide metabolite
of AN is converted by the enzyme rhodanese to the less toxic
thiocyanate, which is then excreted in the urine (Geiger et al.,
1983) . However, conjugation with glutathione (GSH) appears to be
the major route by which AN is detoxified. The double bond of
the parent compound can react by Michael addition with the
sulfhydryl group of GSH to form S-(2-cyanoethyl)glutathione.
This major reaction may occur non-enzymatically or it may be
mediated by GSH S-transferase. To a lesser extent, GSH may also
react in vivo with the epoxide metabolite, 2-cyanoethylene oxide
(CNEO), to form S-(2-cyanohydroxyethl)-glutathione (Van Bladeren
et al., 1981). Both GSH conjugates are then further metabolized
to mercapturic acids and excreted in the urine.
15.4.2 Activation
AN may be oxidized by cytochrome P-450 to yield the
relatively stable epoxide 2-cyanoethylene oxide (CNEO). CNEO can
either hydrolyze to glycoaldehyde and highly toxic cyanide or it
can rearrange to 2-cyanoacetaldehye. CNEO, but not AN itself,
binds to DNA, while both the parent compound and (to a lesser
extent) the epoxide form protein adducts (Hogy and Guengerich,
1986). In vivo studies confirmed that the epoxide is
sufficiently stable to migrate from the liver to extrahepatic
organs where it can interact with DNA to a limited extent
(Farooqui and Ahmed, 1983a; Hogy and Guengerich, 1986).
15.4.3 Host Factors
Animal studies suggest that the target tissue for AN-related
tumor induction (i.e., the brain) is determined by known
variations in tissue-specific rates of DNA repair, rather than by
the site of metabolite generation (Farooqui and Ahmed, 1983a;
Hogy and Guengerich, 1986).
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15.5 PROTEIN ADDUCTS
15.5.1 Characterization
The major AN-protein adduct formed in vitro has been
identified as S-(2-cyanoethyl)cysteine (Geiger et al., 1983).
15.5.2 Rate of Formation
No relevant information was available in the literature.
15.5.3 Dose-Response
Duverger et al. (1982) demonstrated that most of the binding
of AN to rat liver proteins in vitro was the result of direct
alkylation by AN, although some binding resulting from the
microsomal activation of AN was also detected.
Consistent with acrylonitrile's reactivity towards
sulfhydryl groups is the observation that roughly 5% of a dose
incubated in vitro with isolated hepatocytes becomes bound to
protein, primarily in the form of S-(2-cyanoethyl)cysteinyl
residues (Geiger et al., 1983a).
Binding of [14C]-AN to rat hemoglobin in vivo (presumably,
via the reactive cysteine residue) is also extensive and
coincides with rapid GSH depletion. Three hours after oral
administration of 46.5 mg/kg AN, covalent binding of AN to Hb was
> 2.0 fimol/ml erythrocytes (Faroogui and Ahmed, 1983b) .
Fennell et al. (1989) administered 6, 10, or 28 mg/kg [2,3-
14C]-AN to rats and removed blood at 6 hr (4 mg/kg) or 24 hr (10
or 28 mg/kg). The three treatments resulted in adduct levels of
96, 1180, and 3670 nmol equivalents/g globin, respectively.
Chromatography of acid-hydrolyzed globin on Dowex 50 revealed 7
radioactive peaks, the major one corresponding to S-(2-carboxy-
ethyl)cysteine, the hydrolysis product of S-(2-cyanoethyl)-
cysteine. The remaining adducts are currently being
characterized.
15.5.4 Background Levels
No relevant information was available in the literature.
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15.5.5 Methods of Detection
At present, the major AN-protein adduct (and the only one so
far identified), S-(2-cyanoethyl)cysteine/ can only be detected
and quantified by analysis of total protein hydrolysates.
Hydrolysis of this adduct prior to amino acid analysis or GC-MS
would yield S-(2-carboxyethyl)cysteine (Geiger et al., 1983).
Fennell et al. (1989) are currently developing a GC-MS assay for
S-(2-carboxyethyl)cysteine.
15.6 RESEARCH NEEDS
No studies of acrylonitrile-protein adducts in humans, and
very few in animals, were found in the literature. More in vivo
research is required in all areas, including adduct
identification and dose-response relationships.
It is especially important, from the standpoint of exposure
monitoring, to determine the chemical identities and relative
levels of AN-protein adducts formed in AN-exposed animals by
reaction with cyanoethylene oxide, and by direct cyanoethylation
(i.e., Michael addition). Hb adducts resulting from reaction
with the epoxide would, presumably, include N-alJcylvalines that
might be detectable using the modified Edman degradation
procedure. By contrast, adducts formed by direct cyanoethylation
can, at present, only be detected and quantified by GC-MS
analysis of total protein hydrolysates, pending development of
that assay by Fennell et al. If AN-valine adducts cannot be
detected in Hb of exposed animals, then they should be sought in
serum albumin.
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16. GROUP iv CHEMICALS:
ACRYLAMIDE
16.1 MANUFACTURE AND USE
Acrylamide (CAS # 79-06-1) or propenamide is made from
acrylonitrile by reaction with sulfuric acid (Sax, 1987). U.S.
production of acrylamide was 140 million pounds in 1985 (Calleman
and Costa, 1989). It is used as a grouting agent and in the
manufacture of polymers for water treatment.
16.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
In industry, acrylamide exposure is mainly via dermal, next
via inhalation, and last via oral routes (Sax, 1984). The OSHA
standard TLV-TWA is 0.03 mg/cu.m., or approximately 0.01 ppm
(Sax, 1987). The use of acrylamide as a grouting agent and inx
polymers for water treatment may also result in human exposure to
the compound (Calleman and Costa, 1989).
16.3 KNOWN HEALTH EFFECTS
Acrylamide is an irritant to skin (erythema and peeling of
the palms), eye and mucous membranes (Sax, 1984). Long known to
be neurotoxic in mammals, acrylamide is readily absorbed through
unbroken skin and causes peripheral neuropathy (numbness,
tingling and touch tenderness), and CNS paralysis. Although the
compound does not appear to be mutagenic, it is clastogenic and
carcinogenic in rodents (Dearfield, et al., 1988).
16.4 METABOLISM
16.4.1 Detoxification
Acrylamide, a small, highly water-soluble molecule, is
rapidly absorbed, distributed throughout the body, and
metabolized, primarily by conjugation with GSH (Dearfield, et
al., 1988). The majority of a dose is excreted within 24 hours.
16.4.2 Activation
Acrylamide is a direct-acting agent, reacting with both DNA
and sulfhydryl groups in protein by Michael addition at the
double bond (Dearfield, et al., 1988; Bailey et al., 1987).
Glycidamide, a reactive epoxide metabolite of acrylamide, also
binds to sulfhydryl groups (Calleman and Costa, 1989).
Ill
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16.4.3 Host Factors
No relevant information was found in the literature.
16.5 PROTEIN AODUCTS
16.5.1 Characterization
Two acrylamide-Hb adducts have been identified. In rats,
the parent compound forms the Hb adduct S-(3-amino-3-oxopropyl)
cysteine by Michael addition (Bailey et al., 1987). Hydrolysis
of Hb with 6 N HC1 converts the adduct to S-(2-carboxyethyl)
cysteine. Glycidamide, the epoxide metabolite of aery1amide,
forms S-(2-hydroxy-3-amino-3-oxopropyl)cysteine, which is
hydrolyzed to S-(2-carboxy-2-hydroxyethyl) cysteine during protein
analysis (Calleman and Costa, 1989).
16.5.2 Rate of Formation
No relevant information was found in the literature.
16.5.3 Dose-Response
GC-MS analysis of S-(2-carboxyethyl)cysteine is capable of
detecting aery1amide exposures of 0.5 mg/kg in rats, but it is
not yet sensitive enough to be useful in monitoring human
exposure (Bailey et al., 1987).
16.5.4 Background Levels
No relevant information was found in the literature.
16.5.5 Methods of Detection
The cysteine adducts of aery1amide are measured by GC-MS
analysis of total protein hydrolysates.
16.6 RESEARCH NEEDS
Insufficient information is available on dose-response
relationships in animals and acrylamide adduct levels in exposed
and unexposed humans. Also, before Hb adducts can be used to
monitor human exposure to acrylamide, more abundant adducts must
be found or else more sensitive methods of analysis must be
developed. The possiblity that levels of acrylamide adducts are
substantially higher in serum albumin than in Hb should be
112
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investigated. Efforts should also be made to detect and
characterize an N-terminal valine adduct formed by the
glycidamide metabolite of acrylamide. Such a finding could
greatly facilitate all subsequent research.
Acrylamide exposure cannot be distinguished from
acrylonitrile exposure using the hydrolysis products of the
cysteine adducts, because the amide and nitrile groups are both
converted into carboxyl groups during protein hydrolysis.
However, if the metabolites glycidamide and glycidonitrile both
form N-terminal valine adducts, the milder pH changes that occur
during the modified Edman degradation procedure might leave the
amide and nitrile groups of the respective adducts intact.
Alternatively, the amide and nitrile groups might be
differentially derivatized prior to analysis.
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17. GROUP IV CHEMICALS:
CHLOROFORM
17.1 MANUFACTURE AND USE
Chloroform (CAS # 67-66-3) or trichloromethane is produced
in the U.S. by reaction of chlorinated lime with acetone,
acetaldehyde or ethanol or by carefully controlled chlorination
of methane (Sax, 1987). In 1988, 524 million Ibs of chloroform
was produced (C&EN, 1989) by some half a dozen companies at eight
plants across the U.S. (Table 11.). Chloroform is principally
used as a reactant in the manufacture of freon (fluorocarbon-22),
fluorocarbon plastics, dyes, drugs and pesticides; and as an
extractant and industrial solvent in the dye and drug industries
(NTP, 1985; Sax, 1987). Chloroform has been largely replaced as
an anesthetic by safer compounds. In 1976, the FDA banned the
use of chloroform in drug, cosmetic and food packaging products
(Merck, 1983).
17.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
The major sources of chloroform released to the environment
are pulp and paper mills, drinking water chlorination, pharma-
ceuticals, ethylene dichloride manufacture and trichlorethylene
photodegradation (ATSDR, 1988a). Most discharged chloroform
migrates into the atmosphere where it has a half-life of about 3
months, but it can also leach through soil into groundwater where
is expected to persist for relatively long periods of time. The
general population is exposed to chloroform (64-396 /jg/day)
primarily by inhalation, but also by ingestion of drinking water
and various foods (ATSDR, 1988). Absorption of chloroform intake
by inhalation and ingestion is estimated to be 49-77% and 100%,
respectively (U.S. EPA, 1984e).
Occupational exposure is expected to be highest among
production workers (ORNL, 1987). Due to the absence of published
studies, estimated levels of occupational exposure are unavail-
able. Exposure is likely to be low, however, and involve
fugitive emissions from reasonably closed systems (HSDB, 1989m).
OSHA has established a TLV of 10 ppm in air (50 mg/cu m) and a
PEL of 50 ppm for 10 minutes (Sax, 1987) .
17.3 KNOWN HEALTH EFFECTS
The toxicology of chloroform has been reviewed by the U. S.
EPA (1984e) and by the Agency for Toxic Substances and Disease
Registry (ATSDR, 1988).
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TABLE 11. U.S. PRODUCTION OF CHLOROFORM
MANUFACTURERS
SITES OF PRODUCTION
Baker JT Chem Co.
Phillipsburg, NJ
Dow Chem Co.
Midland, MI
LCP Chem & Plastics Inc.
Edison, NJ
Occidental Petroleum Corp.
Spectrum Chemical Mfg.
Gardena, CA
Vulcan Materials Co.
Birmingham, AL
Phillipsburg, NJ
(reprocess only)
Freeport, Texas
Plaguemine, LA
Moundsv i11e, WV
Belle, WV
Gardena, CA
Geismar, LA
Wichita, KS
YEAR
1983
1984
1985
1986
1987
1988
PRODUCTION
362
405
275
422
462
524
MILLION
MILLION
MILLION
MILLION
MILLION
MILLION
LBS
LBS
LBS
LBS
LBS
LBS
SOURCE
C&EN,
C&EN,
C&EN,
C&EN,
C&EN,
C&EN,
1989
1989
1989
1989
1989
1989
115
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The primary symptoms of subchronic exposure to chloroform
(22-237 ppm) in humans are depression, gastrointestinal disturb-
ances (e.g., flatulence and nausea), headache and frequent,
burning urination (U.S. EPA, 1984e).
While chloroform is not mutagenic in E. coli or Salmonella
typhimurium with or without metabolic activation, it caused
cancers of the liver, kidney, thyroid and mammary glands in
rodents. Chloroform is classified by the EPA as a probable human
carcinogen (U.S. EPA, 1984e).
Although chloroform induces hepatocellular and kidney
carcinomas, respectively, in mice and rats, its non-mutagenicity
and its poor binding to DNA suggest that an epigenetic mechanism
is responsible for the induction of these cancers. Thus, DNA
adducts are not the appropriate biomarkers for monitoring
exposure to chloroform (or other epigenetic carcinogens).
However, protein adducts may be.
17.4 METABOLISM
17.4.1 Detoxification
Humans metabolize chloroform to CO2 in a dose-dependent
manner (ATSDR, 1988). For example, 35%, 50% and 100% of oral
doses of 1.0, 0.5 and 0.1 g were so metabolized in one study.
In another study, 17% of a dose was also eliminated unchanged in
expired air.
17.4.2 Activation
In both liver and kidney, the reactive, microsomal
metabolite of chloroform is phosgene. In vitro studies using
renal cortical slices (Bailie et al., 1984) and Hb incubated with
rat liver microsomes (Pereira et al., 1984) suggest that phosgene
is capable of reacting with thiol groups in glutathione and
proteins and cyclizing to form a 2-oxothiazolidine derivative.
Reaction of phosgene with cysteine yields 2-oxothiazolidine-4-
carboxylic acid.
17.4.3 Host Factors
Uptake and storage of chloroform in adipose tissue is
expected to increase with excess body weight and obesity (ATSDR,
1988) .
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17.5 PROTEIN ADDUCTS
17.5.1 Characterization
Phosgene, the reactive metabolite of chloroform, reacts with
cysteine, either free (Bailie et al., 1984) or in Hb (Perera et
al., 1984), to yield a 2-oxothiazolidine derivative. On further
metabolism and/or hydrolysis, 2-oxothiazolidine derivatives
formed with either GSH or free cysteine should yield 2-hydroxy-
thiazolidine-4-carboxylic acid. However, the presence of this
chloroform-protein adduct has not yet been conclusively
demonstrated in vivo.
Periera et al. (1984) identified N-hydroxymethyl cysteine
and 2-hydroxythiazolidine-4-carboxylic acid as the major and
minor products, respectively, detected by GC-MS analysis. The
authors proposed that the 2-oxothiazolidine adduct was converted
to these products during borohydrate reduction and proteinase K
hydrolysis. The presence of this pair of analytes in an
enzymatically digested Hb sample would indicate exposure to one
or more trihalomethanes. It should be noted, however, that there
is some controversy over the identification of N-hydroxymethyl-
cysteine, because the hydroxymethylamino moiety should have been
too unstable to be isolated as such.
17.5.2 Rate of Formation
No relevant data were available in the literature.
17.5.3 Dose-Response
The covalent binding index (CBI)for chloroform determined in
vitro is 14.2 pmole bound/g Hb/jimole dose/kg body weight (Pereira
and Chang, 1981). The extent of in vivo binding of radiolabeled
chloroform to rat hemoglobin is linearly dependent upon dose in
the range 0.1 to 100 /umol/kg (Pereira and Chang, 1982a,b) . At
higher doses, binding increases at a reduced rate. Hemoglobin
adduct levels in a 20-30 mg sample of Hb resulting from a single
dose of 0.1 /imol chloroform/kg were below the level of detection.
However, the binding of 10 daily doses of either 0.1 or 1.0
Mmol/kg was additive, resulting in 15.6 and 167 pmol bound/g Hb,
respectively. Hemoglobin adducts in mice were eliminated at a
rate consistent with the lifespan of erythrocytes in the rat
(Pereira and Chang, 1982a,b).
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17.5.4 Background Levels
No relevant information was available in the literature.
17.5.5 Methods of Detection
The major chloroform adduct in rat Hb modified in vitro
was identified by GC-MS after amino acid analysis and thin-layer
chromatography of an enzyme (proteinase K) digest (Pereira et
al., 1984).
17.6 RESEARCH NEEDS
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 on chloroform-protein
adducts is needed in all areas. However, the analysis of total
protein hydrolysates is not appropriate for routine monitoring
and hydrolyzable chloroform-protein adducts are not likely to
exist. Of course, it may be possible, now or in the future, to
develope an immunoassay for the detection and quantification of
an identified chloroform-protein adduct. It is questionable,
however, whether the potential human health risk represented by
current levels of exposure to chloroform justify such an
approach.
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18. GROUP V CHEMICALS:
BENZENE
18.1 MANUFACTURE AND USE
Benzene (CAS # 71-43-2) was the 16th highest volume chemical
produced in the U.S. in 1985 (Sax, 1987). Annual production in
the U.S. is in excess of 1 billion gallons—1.6 X 10* gallons
were produced in 1988 (C&EN, 1989)—and accounts for over 30% of
total benzene production worldwide (ATSDR, 1988). Table 12 is a
list of the many U.S companies and production plants engaged in
the manufacture of benzene. Over 90% of the benzene produced in
the U.S. is derived from petroleum sources (HSDB, I989n).
Methods of manufacture include catalytic reforming of petroleum,
pyrolysis of gasoline, fractional distillation of coal tar, and
hydrodealkylation of toluene (Sax, 1987).
Benzene is widely used in the synthetics industry as a
chemical intermediate, primarily in the manufacture of ethyl
benzene (an intermediate in the synthesis of styrene), cumene
(the starting material for phenols, phenol derivatives and
acetone used in the manufacture of various resins and fibers) and
cyclohexane (a chemical used to make nylon fibers and resins)
Previously, benzene was an important organic solvent, and a minor
component of unleaded gasoline. However, both of these uses are
currently being phased out (ATSDR, 1988).
18.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Benzene is produced by natural as well as by manmade
sources, and is ubiquitous in the environment. Although the
heaviest exposures occur in occupational settings, particularly
at petroleum refineries and tire manufacturing facilities, the
largest numbers of people are exposed to benzene via automobile
exhaust and cigarette smoke (ATSDR, 1988). People living near
landfills may also be exposed to benzene that has leached into
the groundwater. Approximately half of all benzene-containing
wastes from the petroleum industry is disposed of by landfilling.
The most common route of exposure is the lungs, where
approximately 30% of the benzene may be absorbed into the blood
(ATSDR, 1988). The average daily uptake of benzene by inhalation
has been estimated to be about 0.6 mg for urbanites (Arfellini et
al., 1985) and 0.5 mg for smokers (ATSDR, 1988). Exposure to
benzene may also occur via ingestion and dermal absorption.
According to one estimate (NTP, 1985), approximately 250 /xg/day
may be ingested with food and water.
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TABLE 12. U.S. PRODUCTION OF BENZENE
MANUFACTURERS
SITES OF PRODUCTION
Amerada Hess Corp,
New York, NY
American Petrofina Inc
Dallas, TX
Amoco Corp,
Chicago XL
Ashland Oil, Inc,
Ashland, KY
Atlantic Richfield Co
Subsidiary: Lyondell
Petrochemical Co,
Houston, TX
BP Chemicals America, Inc.
Subsidiary: Sohio Oil Company,
Cleveland, OH
Cain Chemical, Inc,
Houston, TX;
Chevron Corp,
Subsidiary: Chevron Chemical Co,
Aromatics and Derivatives Div
Houston, TX
The Coastal Corp,
Subsidiary: Coastal Refining
and Marketing, Inc,
Houston, TX
Dow Chemical USA,
Midland, MI
Exxon Corp, New York, NY
Exxon Chemical Co Division,
Exxon Chemical Americas,
Houston, TX
Independent Refining Corp,
Houston, TX
St Croix, Virgin Islands
Big Springs, Texas
Port Arthur, Texas
Texas City, Texas
Cat1ettsburg, Kentucky
Channelview, Texas
Lima, Ohio
Alliance, Louisiana
Chocolate Bayou, Texas
Corpus Christi, Texas
Philadelphia, Pennsylvania
Port Arthur, Texas
Corpus Christi, Texas
Westville, New Jersey
Freeport, Texas
Plaguemine, Louisiana
Baton Rouge, Louisiana
Baytown, Texas
Winnie, Texas
120
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TABLE 12. U.S. PRODUCTION OF BENZENE (cent.)
Koch Industries, Inc.
Subsidiary:Koch Refining Co
Mobil Oil Corp, New York, NY
Mobil Chemical Co, Division,
Petrochemicals Division,
Houston, TX
Phillips Petroleum Co,
Subsidiary: Phillips 66 Co,
Chemicals and Catalysts Div
Subsidiary: Phillips Puerto
Rico Core Inc, Hato Rey, PR
Salomon, Inc, New York, NY,
Subsidiary: Hill Chemical Co
Shell Oil Co,
Shell Chemical Co, Div,
Houston, TX
Southland Corp,
Subsidiary: Citgo Petroleum Corp,
Tulsa, OK
Spectrum Chemical Mfg.,
Gardena, CA
Sun Co, Inc,
Subsidiary: Sun Refining and
Marketing Co,
Philadelphia, PA
Texaco Inc,
Subsidiary: Texaco Chemical Co,
Bellaire, TX
Union Pacific Corp, Subsidiary:
Champlin Petroleum Co,
Fort Worth, TX
Unocal Corp,
Los Angeles, CA
Subsidiary: Union Oil Co
of California
USX Corp,
Subsidiary: Marathon Oil Co,
Findley, OH
Corpus Christi, Texas
Beaumont, Texas
Chalmette, Louisiana
Sweeny, Texas
Guayama, Puerto Rico
Houston, Texas
Deer Park, Texas
Odessa, Texas
Wood River, Illinois
Lake Charles, Louisiana
Gardena, California
Middlesex, New Jersey
Marcus Hook, Pennsylvania
Toledo, Ohio
Delaware City, Delaware
El Dorado, Kansas
Port Arthur, Texas
Corpus Christi, Texas
Beaumont, Texas
Lemont, Illinois
Texas City, Texas
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18.3 KNOWN HEALTH EFFECTS
The toxicology of benzene has been reviewed by the U.S. EPA
(1984f) and, more recently, by the Agency for Toxic Substances
and Disease Registry (ATSDR, 1988).
The acute toxicity of benzene from inhalation or ingestion
is relatively low, and lethal exposures have been rare. Central
nervous system effects become evident around 50 ppm in air, and
are the usual result of non-lethal exposure. Acute toxic
responses may include irritation of mucous membranes, depression,
restlessness, excitement, convulsions and even death following
respiratory collapse (Merck, 1983).
In chronic exposure, hematopoietic and lymphoid tissues are
the most sensitive target organs; bone marrow depression and
aplastic anemia are the major symptoms of low and high dose
exposure, respectively (Kalf et al., 1987). The U.S. EPA
classifies benzene as a Class A human carcinogen (NTP, 1885).
However, while benzene is clastogenic in vivo, the compound
induces neither mutagenesis nor unscheduled DNA synthesis, both
common effects of most chemical carcinogens (Arfellini et al.,
1985). Evidence suggests that, rather than initiate, benzene may
promote leukemogenesis via the immunosuppressive activity of its
metabolites (Kalf et al., 1987).
18.4 METABOLISM
18.4.1 Detoxification
Benzene tends to distribute itself in fatty tissues and
blood; over 50% of an absorbed dose may be translocated to the
bone marrow (ATSDR, 1988) . The main site of benzene metabolism
is the liver, where the chemical is first converted to an epoxide
(i.e., benzene oxide) by the action of a cytochrome P-450 enzyme.
Phenol, the major metabolite of benzene (approximately 30% of
dose) may then be formed by non-enzymatic rearrangement of
benzene oxide.
After benzene is metabolized to phenolic compounds, the
latter may be conjugated with sulfate, glucuronic acid or
glutathione to form sulfate esters, glocuronides or mercapturic
acids, respectively (ATSDR, 1988). These water-soluble
conjugates, especially the sulfate esters of phenol, constitute
the major urinary metabolites of benzene. Another urinary
metabolite, the ring-opened product trans,trans-muconic acid, may
be a good indicator of benzene exposure. However, only very
recent exposures could be so monitored because urinary excretion
of benzene metabolites is essentially complete 48 hours after
exposure.
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18.4.2 Activation
Benzene induces the enzymes required for its own metabolism
and it must be metabolized before its toxicity can be expressed.
The benzene oxide produced in the liver may be transformed by
epoxide hydratase into 1,2-dihydrodiol as a prelude to catechol
formation. Further metabolism may yield hydroquinone, catechol,
p-benzoquinones and semiquinones (Kalf et al., 1987). The last
two, particularly p-benzoquinone, are generally considered to be
the toxic metabolites of benzene because of their known ability
to bind to cellular macromolecules such as DNA and protein.
Hydroquinone may be activated in the bone marrow to p-
benzoquinone by cooxidation with prostaglandin H synthase or heme
compounds (Schlosser et al., 1989). Muconaldehyde, a hematoxic
ring-opened metabolite of benzene, is a bifunctional agent that
is capable of crosslinking macromolecules in vitro (Mylavarapu
et al., 1989).
In rodents, at least, the toxic metabolites of benzene
appear to be generated more efficiently at lower exposure
concentrations. If true in humans as well, this would have
important implications for risk assessment for benzene (Sabourin
et al., 1989; Medinsky et al., 1989).
18.4.3 Host Factors
Alcohol consumption potentiates benzene toxicity by
accelerating the hydroxylation of benzene and the conversion of
phenol into toxic metabolites.
18.5 PROTEIN ADDUCTS
18.5.1 Characterization
Specific, well-characterized, benzene-protein adducts have
not yet been described in the literature. However, considering
that glutathione conjugates p-benzoquinone in a predominantly
non-enzymatic process, any protein adduct between cysteine
residues and p-benzoquinone is a likely candidate. Unpublished
preliminary results of work being done at the Lovelace Inhalation
Toxicology Research Institute suggest that S-phenyl cysteine may
be formed in Hb of benzene-exposed rats; this adduct was not
detected, however, in humans occupationally exposed to benzene.
18.5.2 Rate of Formation
No relevant information was found in the literature.
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18.5.3 Dose-Response
The only ongoing research on benzene-Hb adduct formation is
taking place at the Lovelace Inhalation Toxicology Research
Institute in Albuquerque, New Mexico (Henderson et al., 1989).
Preliminary results with rats and mice exposed to [14C]-benzene
suggest that Hb adducts may have potential as markers of exposure
to benzene, but not as markers of benzene toxicity.
Since their last report, Henderson et al. have identified
the major Hb adduct in benzene-exposed rodents as S-phenyl-
cysteine, but have been unable to detect this adduct in
hemoglobin from Chinese workers exposed to 25 ppm benzene
(unpublished). They plan next to look for benzene adducts in
serum albumin.
18.5.4 Background Levels
No relevant information was found in the literature.
18.5.5 Methods of Detection
Currently, GC-MS analysis of total protein hydrolysates is
the only method available for the detection of benzene-protein
adducts.
18.6 RESEARCH NEEDS
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. With respect to this objective, it is
recommended that the ongoing research at the Lovelace Inhalation
Toxicology Research Institute be followed closely.
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19. GROUP V CHEMICALS:
FORMALDEHYDE
19.1 MANUFACTURE AND USE
Formaldehyde (HCHO) (CAS # 50-00-0), also called
oxymethylene, formic aldehyde, and methanal, was the 24th highest
volume chemical produced in the U.S. in 1985 (Sax, 1987).
Approximately 5.6, 5.5, 5.7 and 6.7 billion Ibs of HCHO (37% by
wt) were produced in 1985, 1986, 1987 and 1988, respectively
(C&EN, 1989). HCHO is manufactured in numerous plants across the
country (Table 13) and made commercially available as a 37-50%
solution (formalin) stabilized with 10-15% methanol to prevent
polymerization (HSDB, 1989o). It is used as a disinfectant, an
antiseptic, a deodorant, a tissue fixative, or an embalming
fluid. Polymers of HCHO are used as adhesives in particle board,
plywood and in urea-formaldehyde insulation (Sax, 1987).
19.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Potential exposure to HCHO is mainly through inhalation and
skin absorption. Occupational exposure during production is
probably very low, because synthesis takes place in closed,
automated systems (NTP, 1985). In addition, OSHA has recently
lowered the TLV for formaldehyde to 1 ppm (Sax, 1987) .
The general population is exposed to HCHO through its use in
construction materials, wood products, textiles, home
furnishings, paper, cosmetics and Pharmaceuticals. HCHO also
occurs in automobile,exhaust and cigarette smoke (NTP, 1985).
Over the years, HCHO-containing building products decompose, with
the liberation of free formaldehyde. Indoor air concentrations
up to 1.9 ppm have been found in mobile homes (Dreisbach, 1980).
Employees in recently remodeled offices may also be exposed to
enough HCHO to produce symptoms of so-called "tight building
syndrome" (Thrasher et al., 1989).
19.3 KNOWN HEALTH EFFECTS
Formaldehyde is cytotoxic; it reacts chemically with most
substances in the cell. The primary symptoms of acute exposure
to the chemical in the air are respiratory tract and eye
irritation and may occur in some individuals at concentrations
well below 1 ppm. Other symptoms include laryngeal edema, and
skin sensitivity reactions with urticarial swelling, proteinuria
and hematuria (Dreisbach, 1980). The toxicity of HCHO is
potentiated by absorption to particles such as those
characteristic of Los Angeles-type smog (Doull et al., 1980).
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TABLE 13. U.S PRODUCTION OF FORMALDEHYDE
MANUFACTURERS
SITES OF PRODUCTION
Borden Chem Co.
Div of Borden Inc.
Columbus, OH
BTL of Ohio, Inc,
BTL Specialty Resins
Toledo, OH
Chembond Corp.
Eugene, OR
Du Pont, E I De Nemours & Co.
Chem & Pigments Dept.
Wilmington, DE
GAF Corp
Chem Products,
Wayne, NJ
Georgia-Pacific Corp
Atlanta, GA
Demopolis, AL
Dibol, TX
Fayetteville, NC
Fremont, CA
Kent, WA
La Grande, OR
Louisville, KY
Missoula, MT
Sheboygan, WI
Springfield, OR
Geismar, LA
Vicksburg, MS
Hampton, SC
Houston, TX
Malvern, AR
Andalusia, AL
Moncure, NC
Springfield, OR
Winnfield, LA
Belle, WV
Grasselli, NJ
Healing Springs, NC
La Porte, TX
Toledo, OH
Calvert City, KY
Texas City, TX
Albany, OR
Columbus, OH
Conway, NC
Crosset, AR
Luskin, TX
Russelville, SC
Taylorsville, MS
Vienna, GA
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TABLE 13. U.S PRODUCTION OF FORMALDEHYDE (cont.)
MANUFACTURERS
SITES OF PRODUCTION
Hercules, Inc.
Wilmington, DE
Hoechst Celanese
Somerville, NJ
International Minerals
and Chemicals,
Pitman-Moore Inc.
Allentown, PA
Monsanto Co.
St Louis, MO
Occidental Petroleum Corp.
Hooker Chem Corp., Subsid.
Ourez Div.
North Tonawanda, NY
Reichold Chems, Inc.
White Plains, NY
Rogue Valley Polymers
White City, OR
Western, Inc, DB
Virginia, MN
Wright Chem Corp.
Riegelwood, NC
Louisiana, MO
Bishop, TX
Newark, NJ
Rock Hill, SC
South Whitehall Township, PA
Alvin, TX
Springfield, MA
Toronto, Canada
White City, OR
Las Vegas, NM
Virginia, MN
Acme Station, NC
127
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At high inhalation doses (~6-15 ppm) only, HCHO induces
nasal cancers (i.e, squamous cell carcinoma) in rats, but not in
mice (Swenberg et al., 1985b). Between 4 and 5 ppm HCHO in air
is intolerable to most humans (Doull et al., 1980, p. 625).
Also, rats are obligate nose-breathers while humans are
"optional" nose-breathers. The induction by inhaled HCHO of
squamous cell carcinoma exhibited a threshold somewhere between
2.0 and 5.6 ppm. Covalent binding of HCHO to DNA in rat nasal
mucosa was nonlinearly dependent on airborne concentration,
suggesting that DNA repair may have become saturated at higher
doses. The discrepancy between the rat data and the mouse data
may be accounted for by the observation that mice more
effectively reduced their rate of respiration (and, hence, their
uptake of HCHO) in response to sensory irritation.
HCHO-DNA adducts are highly unstable, and the assumption
that HCHO-DNA adducts are directly relevant to nasal
carcinogenesis has not yet been validated in the laboratory. The
nasal tumors induced in rats seem to correlate more closely with
the cytotoxicity and cell proliferation induced at high absolute
air concentrations than they do with total dose expressed as a
concentration-duration product (Swenberg et al., 1985b). Thus,
although some controversy exists (Cohn vs Casanova-Schmitz,
1985), the rat data do not appear to be particularly relevant to
the human situation.
19.4 METABOLISM
19.4.1 Detoxification
Formaldehyde occurs naturally in all biological tissues. It
is produced endogenously from serine, glycine and other compounds
containing N-, O-, and S-methyl groups and, in its activated form
(N5,N10-methylene-tetrahydrofolate), it is an intermediate in the
biosynthesis of purines, thymine, methionine and serine.
Consequently, specific metabolic pathways exist for its
detoxification (Heck and Casanova-Schmitz, 1984; Swenberg et al.,
12985b).
Most exogenous HCHO is rapidly oxidized, principally in
erythrocytes and the liver, first to formic acid, then to CO2,
which is either metabolically incorporated into biomolecules or
excreted in exhaled breath. The half-life of HCHO is only 1 min
in several species, but the t:/2 for formic acid is species-
dependent. Some HCHO is also excreted in the urine as formic
acid and as a cysteine adduct resulting from the further
metabolism of an HCHO-GSH conjugate. Although HCHO does
covalently bind to cellular macromolecules, HCHO-DNA adducts are
highly unstable and most DNA-protein cross-links formed by HCHO
are rapidly repaired.
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19.4.2 Activation
Formaldehyde is a direct-acting compound.
19.4.3 Host Factors
No relevant information was found in the literature.
19.5 PROTEIN ABDUCTS
19.5.1 Characterization
Although HCHO does bind to proteins, no descriptions of
specific, characterized protein adducts were found in the
literature. However, formaldehyde, with its structural
similarity to phosgene, can be expected to react with cysteine in
Hb to form a thiazolidine derivative which, upon hydrolysis,
would yield an N-Methyl cysteine adduct (Pereira et al., 1984).
In addition, a stable cross-linked formaldehyde adduct between
deoxyguanosine and N-acetylcysteine has recently been formed in
vitro and characterized by proton NMR spectroscopy as Na-
methylene-(Na-acetylcystein-S-yl)deoxyguanosine (Fennell et al.,
1988) .
19.5.2 Rate of Formation
No relevant information was found in the literature.
19.5.3 Dose-Response
The covalent binding of formaldehyde to form DNA-protein
cross-links is nonlinearly dependent upon dose (Swenberg et al.,
1985b). Pathways leading to the detoxification or removal of
formaldehyde are significantly more efficient at low levels of
exposure. Levels of DNA-protein cross-links in monkeys are about
one-tenth of those in rats receiving the same dose of
formaldehyde (Casanova et al., 1989)
19.5.4 Background Levels
No relevant data were found in the literature.
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19.5.5 Methods of Detection
No relevant information was found in the literature.
19.6 RESEARCH NEEDS
At this time, biomonotoring research data on HCHO is
deficient in all areas from adduct identification to methods
development.
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20. GROUP V CHEMICALS:
2,4-TOLUENE DIISOCYANATE
20.1 MANUFACTURE AND USE
The chemical 2,4-toluene diisocyanate (CAS # 584-84-9) or
2,4-tolylene diisocyanate is produced by reaction of 2,4-
diaminotoluene with phosgene. The product is a highly reactive
chemical capable of reacting with a large number of compounds,
including itself, to form polymers of variable size. It is
widely used in the manufacture of rigid polyurethane products for
consumer use (Sax, 1987).
20.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Potential human exposure to TDI occurs mainly through
inhalation during manufacture and production of the chemical. In
addition, firefighters may be exposed to TDI that is released by
pyrolysis of polyurethane foams and may represent up to 1% of the
total weight of the foams themselves (NTP, 1985). Full-time
workers in redecorated offices may also be exposed to low levels
of TDI emitted from isocyanate-containing adhesives in the
remodeling materials (Thrasher et al., 1989). Because TDI is
such a potent allergen, OSHA has set its TLV at 5 ppb (parts per
billion), 8-hr TWA, and 20 ppb (0.14 mg/cu m) for 10 minutes
(NTP, 1985).
20.3 KNOWN HEALTH EFFECTS
The isocyanate functional groups of TDI react extensively
with -OH, -SH, or -NH groups on proteins to produce a variety of
adverse health effects, the most important of which, from an
occupational standpoint, is irritation and sensitization of the
respiratory tract (Karol, 1986). The literature on the
respiratory effects of isocyanates has been reviewed by Karol
(1986) . Some building-related illness may be related to TDI
released from building materials, especially in recently
renovated offices (Thrasher et et al., 1989).
TDI is a carcinogen in rats and female mice (NTP, 1985).
When administered by gavage, the chemical produced subcutaneous,
pancreatic, liver and mammary neoplasms in rats and circulatory
and liver neoplasms in female mice. TDI was not carcinogenic in
male mice. No positive studies on the carcinogenicity of TDI by
the inhalation route have been reported.
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20.4 METABOLISM
20.4.1 Detoxification
No relevant information was found in the literature, but
conjugation by GSH seems a likely pathway of TDI detoxification.
20.4.2 Activation
No relevant data were available in the literature. TDI
reacts with albumin in vitro without metabolic activation (Jin
and Karol, 1988) .
20.4.3 Host Factors
No relevant data were available in the literature.
20.5 PROTEIN ADDUCTS
20.5.1 Characteri z at ion
No relevant data were available in the literature.
20.5.2 Rate of Formation
No relevant data were available in the literature.
20.5.3 Dose-Response
Although no specific TDI-protein adducts appear to have been
described in the literature, the isocyanate functional groups of
TDI are reported to react extensively with -OH, -SH, or -NH
groups on proteins (Karol, 1986).
Recently, methods have been proposed for monitoring
occupational exposure to TDI which focus not on the abundance of
TDI-protein adducts evidently formed in vivo, but on the
endogenous antibodies formed against these modified proteins
(Karol et al., 1987). Guinea pigs exposed for 3 hours per day
for 5 days to 0.12-10 ppm of TDI vapor exhibited a concentration-
dependent antibody response 22 days after the initial exposure.
Although the antibody response was highly specific, the linearity
of the dose-response curve was limited to a narrow range of dose
(1-30 mg/kg), and the response was not detectable 30 days after
exposure. Human serum albumin, heavily modified in vivo with
isocyanates, has been used to detect antibodies in sera of
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patients with isocyanate lung disease (Jin and Karol, 1988).
20.5.4 Background Levels
No relevant data were available in the literature.
20.5.5 Methods of Detection
Unspecified TDI-serum albumin adducts have been detected
indirectly by assaying for endogenous antibodies to those adducts
formed in vivo in exposed animals and humans (Karol et al., 1987
and Jin and Karol, 1988).
20.6 RESEARCH NEEDS
Apart from the detection of antibodies to endogenous TDI-
protein adducts in exposed animals and humans, no protein adduct
research appears to have been done on TDI. Research is needed in
all areas from adduct identification to method development.
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 would be any
adducts formed at the N-terminal amino acids of either Hb or
serum albumin.
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21. GROUP V CHEMICALS:
7,12-DIMETHYLBENZANTHRACENE
21.1 MANUFACTURE AND USE
The compound 7,12-DimethyIbenzanthracene (DMBA) (CAS #
57-97-6), also called 9,10-dimethyl-l,2-benzanthracene and 1,4-
dimethyl-2,3-benzphenanthrene, is not used commercially in the
U.S. and no production figures are available. Two U.S. companies
— Aldrich Chemical in Madison, Wisconsin and Eastman Kodak in
Rochester, New York — manufacture small quantities of DMBA for
use as a research chemical, e.g., in experimental medicine to
induce various malignant tumors for testing anti-neoplastic drugs
(HSDB, 1989p).
21.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
DMBA is a synthetic chemical that does not occur naturally.
While many other polynuclear aromatic hydrocarbons are products
of incomplete combustion and therefore occur in vehicle exhaust,
emissions from coal- and oil-burning stoves and furnaces, etc.,
there is no evidence that DMBA is formed in this process (HSDB,
1989p). Given the absence of commercial production and use,
there does not seem to be much potential for human exposure to
DMBA outside the research laboratory.
21.3 KNOWN HEALTH EFFECTS
DMBA is a potent breast carcinogen. For reasons as yet
unknown, a methyl group adjacent to the bay-region greatly
enhances the tumorigenicity of benzanthracene, a weak carcinogen
in its own right. DMBA, one of the most potent synthetic PAH
carcinogens known, is a much more powerful initiator than B(a)P
(HSDB, 1989p).
21.4 METABOLISM
21.4.1 Detoxification
DMBA is readily absorbed and tends to localize primarily in
body fat and fatty tissues such as the breast. It is also
rapidly taken up by the liver, where it is converted into more
polar metabolites and excreted via the bile into the feces. DMBA
may undergo microsomal oxidation at the 7- or 12-methyl group,
and at the 3,4 and 8,9 double bonds to yield a variety of
products which may be subsequently conjugated prior to excretion
(HSDB, 1989p).
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21.4.2 Activation
Like most PAHs, DMBA must be metabolized to an epoxide
intermediate before its carcinogenicity can be expressed. While
both bay-region and non-bay-region diol-epoxides contribute to
the binding of DMBA to DNA in vivo, the biologically active
intermediate appears to be the bay-region, anti-3,4-diol 1,2-
epoxide (Phillips and Grover, 1984). The sulfate ester of a
major microsomal metabolite of DMBA, 7-hydroxy-12-methyl-
benz[a]anthracene (HMBA), covalently binds to cysteine and lysine
and methionine residues in protein. Both metabolically formed
and synthetic sulfates of HMBA react with DNA in a similar manner
(Watabe et al., 1983).
Fujimori (1982) showed that DMBA, non-covalently bound to
protein, could be activated in vitro to covalently-binding
species by irradiation at 365 nm. The author suggest that,
although the unique photoproducts formed are not similar to known
metabolites of DMBA, they may have some significance in
connection with photo-carcinogenesis.
21.4.3 Host Factors
The ratio between the various metabolites (and DNA adducts)
of DMBA may be affected by species and dose (Morse et al., 1987)
as well as by dietary factors such as selenium and protein
(Singletary and Milner, 1987).
21.5 PROTEIN ADDUCTS
21.5.1 Characterization
Three protein adducts have been identified in vitro; S-(12-
methylbenz[a]anthracene-7-methyl)cysteine, N-(12-methylbenz[a]-
anthracene-7-methyl)lysine, and S-(12-methylbenz[a]anthracene-7-
methyl)homocysteine, a methionine adduct. The ultimate
electrophile appears to be the sulfate ester of 7-hydroxy-12-
methylbenz[a]anthracene (Watabe et al., 1983).
21.5.2 Rate of Formation
No relevant information was found in the literature.
21.5.3 Dose-Response
Watabe et al. (1983) demonstrated in vitro that 7-hydroxy-
12-methylbenz[a]anthracene (7-HMBA), a major metabolite of DMBA
in the rat liver, can be activated to a protein-binding form in
135
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the presence of a hepatic soluble supernatant fraction (S105)
contain-ing a PAPS-generating system and a cytosolic
sulfotransferase. Three amino acid adducts were identified: S-
(12-methyl- benz[a]anthracene-7-methyl)cysteine, N-(12-
methylbenz[a]anthracene-7-methyl)lysine, and S-(12-
methylbenz[a]anthracene-7-methyl) homocysteine.
The same adducts were formed when synthetic HMBA sulfate was
reacted with cysteine, lysine and methionine. The sulfuric acid
ester group of 7-HMBA sulfate may act as a leaving group, in
which case the product would be a stable 7-methylenecarbonium ion
which could react with nucleophilic sites of macromolecules. The
adrenal necrosis induced by both DMBA and 7-HMBA may be the
result of extensive protein-binding to the reactive 7-HMBA
sulfate formed in the adrenals, which are well known sites of
steroid sulfate biosynthesis.
No in vivo studies of DMBA-protein adducts were found in the
literature.
21.5.4 Background Levels
No relevant information was found in the literature.
21.5.5 Methods of Detection
In the in vitro study by Watabe et al. (1983), modified
protein was isolated from the microsomal incubation medium,
hydrolyzed with 10 N HC1 and analyzed by HPLC and mass
spectrometry.
21.6 RESEARCH NEEDS
In vivo research on DMBA-protein adducts is needed in all
areas from adduct identification to methods development.
However, the apparent absence of an exposed population makes DMBA
a poor choice for use in monitoring chemical exposure in humans.
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22. GROUP V CHEMICALS:
EPICHLOROHYDRIN
22.1 MANUFACTURE AND USE
Epichlorohydrin (EC) (CAS # 106-89-8), also called
chloropropylene oxide, chloromethyloxirane, and l-chloro-2,3-
epoxypropane, is a highly volatile epoxide used primarily as an
intermediate in the manufacture of epoxy and phenoxy resins
(HSDB, 1989q). It is also used as a solvent for cellulose esters
and ethers, paints, varnishes, nail enamels and lacquers (Merck,
1983; Sax, 1987). Annually in the U.S., several hundred million
pounds of EC (HSDB, 1989q) is collectively produced by four
different companies at five sites (Table 14).
22.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Human exposure to EC is probably confined to production and
manufacturing facilities, where current levels of occupational
exposure are probably less than I ppm (Zeighami et al., 1987).
The current TLV is 2 ppm in air (ACGIH, 1986g).
22.3 KNOWN HEALTH EFFECTS
A strong irritant and sensitizer, EC is toxic by inhalation,
ingestion and skin absorption (Sax, 1987). EC vapors can
irritate the eye. Chronic exposure may result in damage to the
lung and kidneys and can produce temporary sterility (Sax, 1984).
The nephrotoxicity of EC may be due to the metabolic formation
and subsequent deposition of oxalic acid crystals (U.S. EPA,
1983) .
EC is a weak carcinogen in the mouse. Overall, however, the
evidence for the chemical's carcinogenicity in humans is
inadequate. One study of exposed factory workers showed an
excess of respiratory cancer, but confounding exposures precluded
identification of a probable causative agent (NTP, 1985).
22.4 METABOLISM
22.4.1 Detoxification
Most EC is conjugated with GSH and excreted in the urine or
metabolized to CO2 and exhaled (Gringell et al., 1985).
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TABLE 14. U.S.PRODUCTION OF EPICHLOROHYDRIN
MANUFACTURERS SITES OF PRODUCTION
Aldrich Chemical Co, Inc, Milwaukee, WI
Milwaukee, WI
Chemical Dynamics Corporation South Plainfield, NJ
Plainfield, NJ
The Dow Chemical Company Freeport, TX
Midland, MI
Shell Oil Co, Deer Park, TX
Shell Chem Co, Div, Norco, LA
YEAR PRODUCTION SOURCE
1977 1.32 X 10E11 G HSDB, 1989q
(2.91 X 10E8 Ibs)
1982 1.52 X 10E11 G HSDB, 1989q
(3.35 X 10E8 Ibs)
1984 2.00 X 10E11 G HSDB, 1989q
(4.41 x 10E8 Ibs)
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22.4.2 Activation
EC is a direct-acting alkylating agent that binds to
macromolecules in tissues, primarily in liver, kidney and
forestomach (Gringell et al., 1985). The parent compound binds
non-enzymatically to GSH and SH-groups in proteins (U.S. EPA,
1983). However, there is evidence that a metabolically activated
form of EC also binds to macromolecules.
EC binds to DNA, RNA and protein in vivo in rats and mice
after i.p. administration of 0.805 mg/kg of radiolabeled EC, a
dose 200-fold lower than the LD50 (Mazzullo, et al., 1984). In
pooled organs, the extent of binding followed the order stomach
>kidney >liver >lung for DNA, and kidney >liver >lung >stomach
for protein. The binding ratio protein/DNA in liver, kidney,
lung and stomach, respectively, was 10, 21.4, 8.3 and 2.0 for the
rat and 5, 22.4, 8.8 and 2.2 for the mouse (Mazzullo, et al.,
1984) .
The observed discrepancies between (a) DNA binding and
protein binding in the stomach as compared to other organs, and
(b) the protein/DNA binding ratio in the liver of rats versus
mice suggest that some portion of the binding of EC to
macromolecules in vivo may be mediated by activating enzymes of
variable effectiveness in different organs. If no metabolic
activation had been involved, then binding to both macromolecules
in both rodent species should have consistently reflected the
distribution of the direct-acting agent (Mazzullo, et al., 1984).
22.4.3 Host Factors
No relevant information was found in the literature.
22.5 PROTEIN ADDUCTS
22.5.1 Characterization
EC binds in vitro about 40 times more strongly to cysteine
than to any other amino acid (Hemminki, 1986a). No descriptions
of specific EC-protein adducts were found in the literature.
However, it is possible that S-(2,3-dihydroxypropyl)cysteine,
which occurs as a urinary metabolite (Gringell et al., 1985), may
also occur in proteins. Such a possibility is improved by the
fact that the reaction between EC and GSH can occur without the
mediation of glutathione-S-transferase.
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22.5.2 Rate of Formation
No relevant information was found in the literature.
22.5.3 Dose-Response
No relevant information was found in the literature.
22.5.4 Background Levels
No relevant information was found in the literature.
22.5.5 Methods of Detection
No relevant information was found in the literature.
22.6 RESEARCH NEEDS
More research is needed in all areas from adduct
identification to methods development. As an epoxide, EC would
be expected to form N-terminal valine adducts in Hb. Priority
should be given to the search for such adducts. All subsequent
research on EC-protein adducts would be greatly facilitated by
the identification of an adduct that was measurable using the
modified Edman degradation procedure.
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23. GROUP V CHEMICALS:
BENZYL CHLORIDE
23.1 MANUFACTURE AND USE
Benzyl chloride (CAS # 100-44-7), also called a-
chlorotoluene, (chloromethyl)benzene and tolyl chloride, is made
at a few sites in the U.S. (Table 15) by passing chlorine over
boiling toluene until it has increased 38% in weight (HSDB,
1989r). Benzyl chloride is used in the manufacture of benzyl
compounds, perfumes, pharmaceutical products, dyes, synthetic
tannins and artificial resins (Merck, 1983).
23.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Benzyl chloride has been identified in surface water, oil
refinery and industrial effluents, pharmaceutical factories and
spray painting booths in a U.S. automobile manufacturing factory
(Abdel-Rahman and Saxena, 1988). The TLV for benzyl chloride is
1 ppm (5 mg/cu m) in air (ACGIH, 1986h).
23.3 KNOWN HEALTH EFFECTS
Benzyl chloride, a direct alkylating agent, is intensely
irritating to the skin, eyes and mucous membranes; large doses
cause CNS depression (Merck, 1983). It is mutagenic in
Salmonella typhimurium without metabolic activation and has
induced local sarcomas with lung metastases in rats after
subcutaneous injection (Walles, 1981) . In some workers exposed
to 2 ppm benzyl chloride, abnormal liver function and mild
leukopenia have been observed (Abdel-Rahman and Saxena, 1988).
23.4 METABOLISM
23.4.1 Detoxification
In exposed animals, the highest concentrations of the
chemical are found in the liver where benzyl chloride is
metabolized by conjugation with GSH to form benzyl mercapturic
acid (Abdel-Rahman and Saxena, 1988).
23.4.2 Activation
No relevant information was found in the literature.
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TABLE 15. U.S. PRODUCTION OF BENZYL CHLORIDE
MANUFACTURERS
SITES OF PRODUCTION
Monsanto Chemical Co.
AKZO, Chemical Division
Chicago, IL
Spectrum Chemical Mfg.
Gardena, CA
Bridgeport, NJ
Edison, NJ
Gardena, CA
YEAR
PRODUCTION
SOURCE
1977
1982
1984
4.30 X 10E10 G
(9.55 X 10E7 Ibs)
3.30 X 10E10 G
(7.33 X 10E7 Ibs)
4.99 X 10E10 G
(1.10 X 10E8 Ibs)
HSDB, 1989r
HSDB, 1989r
HSDB, 1989r
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23.4.3 Host Factors
No relevant information was found in the literature.
23.5 PROTEIN ADDUCTS
No relevant information was found in the literature.
23.5.1 Characterization
No relevant information was found in the literature.
23.5.2 Rate of Formation
Only one report on adduct formation by benzyl chloride was
found in the literature. Walles (1981) determined the in vitro
rate constant, k^,, for the reaction between benzyl chloride and
Hb to be 1.6 X 10"3/l/g Hb/hr. The degree of alkylation of Hb
was measured in terms of radioactivity bound. No data on
specific benzyl chloride-protein adducts are available in the
literature.
23.5.3 Dose-Response
No relevant information was found in the literature.
23.5.4 Background Levels
No relevant information was found in the literature.
23.5.5 Methods of Detection
No relevant information was found in the literature.
23.6 RESEARCH NEEDS
More research is needed in all areas from adduct
identification to method development. Of particular interest for
the purpose of exposure monitoring would be any stable adduct
with either the N-terminal valine of Hb or the N-terminal
aspartic acid of serum albumin.
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24.GROUP V CHEMICALS:
PENTACHLOROPHENOL
24.1 MANUFACTURE AND USE
Pentachlorophenol (PCP) (CAS f 87-86-5) is produced in the U.S.
by the chlorination of phenol for use as a fungicide,
molluscicide, herbicide, and wood preservative (Sax, 1987). PCP
was the second most heavily used pesticide in 1980, but its use
has declined recently in response to federal regulatory measures
(Zeighami, 1987).
24.2 SOURCES AND LEVELS OF HUMAN EXPOSURE
Due to the persistence and the extent and mode of use of
PCP, this compound is ubiquitous in the environment, and may be
detected in the bodily fluids and fat of non-occupationally-
exposed people. In the general population, chronic PCP may occur
by contact with or ingestion of materials contaminated with the
pesticide (Uhl et al., 1986). Production workers remain the
single largest exposed population, but exposures are likely to be
kept at a minimum (Zeighami, 1987). The TLV for PCP is 0.5 mg/cu
m of air or approximately 0.05 ppm (Sax, 1987).
24.3 KNOWN HEALTH EFFECTS
PCP is toxic by inhalation, ingestion and skin absorption,
and abuse of the compound can be fatal (U.S. EPA, 1984g). Acute
poisoning is marked by weakness, dermatitis, respiratory, blood
pressure and urinary output changes, convulsions and collapse.
Chronic exposure to PCP can cause liver and kidney damage (Sax,
1984) . Phenolic substances such as PCP tend to precipitate
cellular proteins. Evidence to date suggests that PCP is neither
mutagenic, teratogenic nor carcinogenic (EPA, 1984g).
24.4 METABOLISM
24.4.1 Detoxification
The bulk of ingested PCP is eliminated in the urine either
unchanged or conjugated to glucuronide (Uhl et al., 1986). PCP
has an unusually long elimination half-life of 20 days. Strong
noncovalent binding to plasma protein and a high rate of tubular
resorption allow the steady state human body burden to be a
factor of 10-20 times higher than the value extrapolated from
animal pharmacokinetic data. The attainment of steady state
urinary levels of PCP may require 3 months, and will reflect
exposure over several weeks rather than the usual 24-48 hours.
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24.4.2 Activation
The primary microsomal oxidative metabolites of PCP in man,
i.e., the benzoguinone or the semiguinone forms of 1,4- and 1,2-
tetrachlorohydroquinone (TCHQ), can bind covalently to proteins
and, to a much lesser extent, to DNA (Van Ommen et al., 1986).
in vitro ascorbic acid inhibits covalent binding to a protein by
reducing the benzo- and semiguinone forms to the hydroguinone
form. Glutathione also inhibits protein binding in vitro by
forming conjugates with the reactive metabolites of PCP.
24.4.3 Host Factors
As an inhibitor of cytosolic sulfotransferase, PCP will
reduce the toxicity of compounds such as N-hydroxy-2-
acetylaminofluorene and I'-hydroxysafrole (U.S. EPA, 1984g). PCP
itself is best activated by cytochrome P-450d, the isosafrole-
induced isozyme (Van Ommen et al., 1986).
24.5 PROTEIN ADDDCTS
24.5.1 Characterization
No information on specific PCP-protein adducts was found in
the literature. In the in vitro studies of Van Ommen et al.
(1986), binding of PCP to protein was measured as the total
radioactivity bound to the proteinaceous extract of the
microsomal incubation medium. It seems likely, however, that at
least some of the observed binding was the result of reaction
between cysteine residues in proteins and the benzo- or
semiguinone forms of 1,4- and 1,2-tetrachlorohydro-guinone. The
extent to which such binding may occur in vivo will be limited by
reduction and conjugation mechanisms.
24.5.2 Rate of Formation
PCP (100 /iM) bound to rat liver microsomal proteins in vitro
at a the rate of about 70 pmol/mg/min (Van Ommen et al., 1986).
No value for the rate constant of reaction between PCP
metabolites and protein was reported.
24.5.3 Dose-Response
No relevant data were found in the literature.
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24.5.4 Background Levels
No data relevant to the formation of PCP-protein adducts,
background or otherwise, were found in the literature. However,
background levels of the parent compound have been reported in
human fat and blood plasma (Uhl et al., 1986).
24.5.5 Methods of Detection
To date, the only method used to detect binding of PCP to
proteins is liquid scintillation counting of microsomal extracts
from in vitro incubations (Van Ommen et al., 1986). No other
relevant data were found in the literature.
24.6 RESEARCH NEEDS
More protein-adduct research is needed for PCP in all areas
from adduct identification to methods development. A first
priority of such research should be to determine the in vivo
formation of N-terminal valine adducts in Hb. However,
considering the unusually long elimination half-life of PCP (20
days—the same as the half-life of human serum albumin) and the
apparent absence of any carcinogenic potential, urinalysis may be
adequate for the purpose of monitoring exposure to PCP in humans.
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Abdel-Rahman, M.S., and S. Saxena (1988). Effects of benzyl
chloride on rat hepatocytes. J. Toxicol. Environ. Health, 25:
453-459
ACGIH (1986a). American Conference of Governmental Industrial
Hygienists. Ethylene Oxide, pp. 256-257 in Documentation of the
threshold limit values and biological exposure indices. 5th Ed.,
Cincinnatti, Ohio.
ACGIH (1986b). American Conference of Governmental Industrial
Hygienists. Propylene Oxide, pp. 504 in Documentation of the
threshold limit values and biological exposure indices. 5th Ed.,
Cincinnatti, Ohio.
ACGIH (1986c). American Conference of Governmental Industrial
Hygienists. Benzidine, pp. 53 in Documentation of the threshold
limit values and biological exposure indices. 5th Ed.,
Cincinnatti, Ohio.
ACGIH (1986d). American Conference of Governmental Industrial
Hygienists. 4,4'-Methylenebis(2-chloroaniline), p. 392 in
Documentation of the threshold limit values and biological
exposure indices. 5th Ed., Cincinnatti, Ohio.
ACGIH (1986e). American Conference of Governmental Industrial
Hygienists. Vinyl Chloride, pp. 623-626 in Documentation of the
threshold limit values and biological exposure indices. 5th Ed.,
Cincinnatti, Ohio.
ACGIH (1986f). American Conference of Governmental Industrial
Hygienists. Acrylonitrile, pp. 15-16 in Documentation of the
threshold limit values and biological exposure indices. 5th Ed.,
Cincinnatti, Ohio.
ACGIH (1986g). American Conference of Governmental Industrial
Hygienists. Epichlorhydrin, pp. 233 in Documentation of the
threshold limit values and biological exposure indices. 5th Ed.,
Cincinnatti, Ohio.
ACGIH (1986h). American Conference of Governmental Industrial
Hygienists. Benzyl chloride pp. 55 in Documentation of the
threshold limit values and biological exposure indices. 5th Ed.,
Cincinnatti, Ohio.
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ATSDR (1987a). lexicological profile for chloroform (DRAFT).
Agency for Toxic Substances and Disease Registry.
ATSDR (1987b). Toxicological profile for benzene (DRAFT). Agency
for Toxic Substances and Disease Registry.
ATSDR (1988a). Toxicological profile for benzidine (DRAFT).
Agency for Toxic Substances and Disease Registry.
ATSDR (1988b). Toxicological profile for vinyl chloride.
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