United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
Las Vegas NV 89114
Research and Development
EPA/600/S4-87/005 June 1987
SERA Project Summary
Carcinogen-DNA Adducts:
Introduction, Literature
Summary, and
Recommendations
S. D. Soileau
This report summarizes the literature
concerning adducts formed by xenobi-
otics with DNA and protein in order to
determine their feasibility as a moni-
toring tool for use in exposure and risk
assessment and to propose compounds
and methods that may be appropriate
for preliminary field studies. This report
is divided into three segments.
The first segment provides an intro-
duction to DNA damage and its relation
to carcinogenesis. This segment also
discusses available methodology for
the measurement of macromolecular
(DNA, protein) adducts. The tech-
niques were evaluated according to
their sensitivity, selectivity, limitations,
and future possibilities. The next seg-
ment provides a summary of the cur-
rent literature on the individual chem-
icals found to form adducts in both man
and in experimental animals. The
information in this segment and addi-
tional information was tabulated and is
presented in the appendix. Finally, the
conclusion and recommendation sec-
tion discusses the overall potential for
the use of macromolecular adducts as
a measure of dose, given the current
technology. Recommendations on the
analytical detection methodologies,
applicable chemicals, and populations
to be used for a human monitoring pilot
study were offered.
This Project Summary was devel-
oped by EPA's Environmental Monitor-
ing Systems Laboratory, Las Vegas,
NV, to announce key findings of the
research project that is fully docu-
mented in a separate report of the same
title (see Project Report ordering
information at back).
Introduction
The Environmental Protection Agency
(EPA) is charged to protect human health
and the environment, and it has acted
by placing restrictions and regulations on
chemicals that have been shown to be
detrimental to human health or to the
environment. Accurate dose measure-
ments 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. Therefore, there is an
intense interest in devising techniques
that can be used as monitoring tools to
quantitate exposure to xenobiotics and
that can eventually be used in risk
assessment. Consequently, the EPA has
developed an initiative designed to
develop, refine, and apply appropriate
biomarkers that can be used in conjunc-
tion with other environmental monitor-
ing data to provide a better estimate of
risk to individuals and populations. By
linking biological measurements to
environmental monitoring measure-
ments, it will be possible to determine
relationships that exist between total
exposure, dose, and disease.
The first stage of the EPA initiative is
to evaluate the feasibility of using
biomarkers as a monitoring tool for use
in exposure and risk assessment. This
will include a compilation of available
biomonitoring methods for assessing
environmental exposures and methods
for predicting associated health risks.
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One biomomtonng method is the
measurement of the reaction products of
possible carcinogens with DNA and
protein Structural identification and
quantitation of carcmogen-DNA and
protein add ucts that result from exposure
to these xenobiotics could provide a more
accurate measure of exposure
This report discusses the feasibility of
the use of DNA and protein adducts as
a valid biological dosimeter to monitor
the integral dose of genotoxic environ-
mental chemicals The report is divided
into three segments The first segment
provides an introduction to DNA damage
and its relation to carcmogenesis This
segment also discusses available metho-
dology for the measurement of macro-
molecular (DNA, protein) adducts The
analytical techniques were evaluated
according to their sensitivity, selectivity,
limitations, and future possibilities The
next segment provides a summary of the
current literature on the individual
chemicals found to form adducts m both
man and in experimental animals The
information in this segment and addi-
tional information was tabulated and is
presented m the Appendix Finally, the
conclusion and recommendation section
discusses the overall potential for the use
of macromolecular adducts as a measure
of dose, given the current technology
Recommendations on the analytical
detection methodologies, applicable
chemicals, and populations to be used
for a human monitoring pilot study were
offered
Procedure
Two data bases were searched, Can-
cerlme and Medlme from 1981 to June
1986 The search terms used were
environmental mutagens and carcino-
gens, Pharmaceuticals and DNA or
protein adducts In addition to the
computer search, two separate manual
searches were conducted The articles m
volume 62 of Environmental Health
Perspectives were searched for pertinent
citations Also, Chemical Abstracts was
searched back to 1 976 for review articles
on DNA adducts A total of 335 citations
were located and 112 compounds were
found to form DNA and''or protein
adducts The 1 1 2 compounds were
cross-referenced with the HEALS priority
compound list and only one match was
found, styrene The literature was sum-
marized and the pertinent information
was summarized in tabular form An
introduction on carcmogenesis, DNA
adduct formation, and the methods used
to measure adduct format ion was written
and recommendations were made for
compounds to study and methodology to
use
Discussion
DNA or protein adduct formation
occurs when electrophilic molecules
enter or are created within the body
These electron-poor molecules attack
electron-rich sites wit hi n the body These
sites are primarily found m DNA and
proteins It is believed that adduct
formation with DNA results m an alter
ation of the molecule that eventually
results in the creation of a mutation
within the genome Through poorly-
understood steps, the DNA mutation
transforms the normal cell into a can-
cerous one Although mutations cannot
be easily measured, adduct formation
with both DNA and protein can be
measured
After DNA adduct formation occurs,
their levels can be modulated by the rate
of cell division or by DNA repair There-
fore, DNA adducts may be a good
indicator of risk but may not be an
accurate measure of exposure However,
if animal studies indicate that a linear
exposure-dose relationship exists, then
DNA adducts would be a good indicator
of exposure In general, protein adducts
would give a better indication of exposure
because little if any protein adduct repair
occurs
Several methods exist for the detection
of carcmogen-macromolecule adducts,
but only a couple are suitable for human
studies The use of radiolabelled carcin-
ogens provides a very sensitive measure
of adduct formation, but it is only of use
in animal studies because human pop-
ulations are normally not exposed to
radiolabelled compounds
Spectrophotometric and fluorometric
methods can be used to measure DNA
to protein adducts that are formed with
compounds that absorb strongly or that
fluoresce The disadvantage of these
methods are that they are either not
sensitive enough or that they are useful
only for certain compound classes
Chemical denvatization methods are
sensitive, but the methodology is rela-
tively new and unproven It may also
prove to be too expensive for use in
studies involving many subjects
Conclusions and
Recommendations
Two procedures appear to be ideally
suited for human monitoring studies The
first is the use of antibody techniques
Antibodies are selective for a particular
three dimensional structure and can be
very sensitive Both polyclonal and
monoclonal antibodies can be used, but
monoclonal antibodies have the potential
for being the most selective The antib
odies are used in competitive assays
where the sample adduct competes with
a known amount of added adduct The
sample adduct levels are calculated by
using a standard curve
Another method has been developed
that has the sensitivity advantage of
radiolabelled compounds, and it does not
require that the carcinogen be radiola
belled The method is referred to as ' P
postlabeling, and the method is summar
ized as follows Adducted DNA is isolated
from a tissue source and is digested to
form 3'-mononucleotides <-'P is incorpo
rated on the 5' end of the neucleotides,
and the adducts are separated by using
multidimensional thin layer chromato-
graphy The separated adducts are
quantified by using autoradiography
Because of the high specific activity of
the '-'P, this method can detect adducts
at about one adduct per 10'1 bases from
a 1 /jg sample of DNA This makes '-'P
postlabeling one of the most sensitive
methods available This level of sensi
tivity may be required when one is
looking for DNA adducts induced by
environmental carcinogens m the
general population because of the low
exposure situations Both immunoassay
and '-'P-postlabelmg techniques should
be adaptable to nearly any DNA adduct
that is characterized It is suggested that
a possible method to monitor DNA
adducts would include a rapid screening
with the immunoassay method followed
by the !-'P-postlabelmg method for those
samples that show up negative on the
immunoassay screening This procedure
would be the most cost-effective as only
those samples with very low levels of
DNA adducts would be analyzed by the
more expensive !-'P-postlabelmg method
Currently, protein adducts can only be
monitored by using immunoassay tech-
niques because the 3;'P-postlabeimg
technique is specific for DNA adducts
However, protein adducts should occur
at higher concentrations than the DNA
adducts because of the lack of protein
adduct repair systems and because
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greater amounts of sample are available.
Therefore, sensitivity should not be an
insurmountable problem.
Two main points were considered
when the tissues or fluids to detect
adduct formation were chosen. The first
point was one of invasiveness. If the
sample collection technique is too inva-
sive, it will be impossible to obtain
enough volunteers for the studies. The
second point involved the usefulness of
the sample. The further away the sample
is from the target organ, the less repre-
sentative the measured adduct levels will
be of the adduct levels at the target site,
unless animal studies indicate other-
wise. Therefore, tissue and fluid selec-
tion was a compromise between these
two points.
The best compromise fluid is blood.
Sample collection is relatively non-
invasive, and it contains several mole-
cules that can contain covalent adducts.
White blood cells contain DNA; therefore,
DNA adduct formation can be monitored.
However, one must remember that the
DNA adduct level may not correlate with
the DNA adduct level in the target
organ(s) or with the dose received. In
addition, protein adducts can be moni-
tored. The two main proteins to be
monitored would be hemoglobin and
human serum albumin. Each protein
would give different levels of integration
data as the half-lives of the proteins are
120 and 20 days, respectively. Also,
human serum albumin is extra-cellular
and is not protected by a cellular mem-
brane. Therefore, it might show a higher
level of protein adduct formation. In
summary, both integration and equilibra-
tion data can be obtained from blood.
Urine is also an excellent choice for
monitoring DNA adducts. When DNA
repair occurs, the removed adducts are
excreted in the urine. However, not many
carcinogen adducts have been monitored
in the urine, so additional animal studies
would have to be conducted. In one
animal study, the levels of aflatoxin-DNA
adducts found in the urine correlated
with the administered dose of the
carcinogen. Theoretically, any adduct
that is repaired could be monitored by
using this fluid.
Tissues obtained during a biopsy or
autopsy would provide the most accurate
information concerning the level of
da mage that occurs at the target site. The
problem with biopsy samples is that they
are very difficult to obtain, and it would
be difficult to obtain volunteers for such
a study. The question that needs to be
asked is if target DNA adduct level
information is really needed. If the main
purpose of the planned studies is to
obtain dose information, that is, levels
of the carcinogen that have entered the
body, then the information obtained from
adducts measurable in blood may be
sufficient.
In summary, the best approach to
monitoring human subpopulations
would entail collection of both blood and
urine samples. This approach would
allow the collection of the most data with
a minimum of discomfort to the
individuals.
Several compounds and compound
classes were suggested. The primary
consideration used was to select com-
pounds that have the highest expectation
of producing a usable exposure-dose
relationship. Cisplatin, a cancer che-
motherapeutic agent, was suggested as
an ideal candidate compound for a study
to prove feasibility because the exact
amount of cisplatin administered is
known. One could also determine if it is
possible to correct for mediating factors
such as drug metabolizing enzyme levels,
glutathione levels, membrane transport,
etc.
Other cancer chemotherapeutic
agents might also be useful. For example,
the Health Effects Research Laboratory
(HERL), Research Triangle Park, NC, is
conducting an exposure-dose relation-
ship study with 2,5-diazeridinyl-3,6-
bicarboethoxyamino-1,4-benzoquinone
(AZQ). AZQ is used in the treatment of
brain tumors. Unfortunately, long term
studies are difficult because of the
extremely poor prognosis for patients
with brain tumors.
Psoralens are therapeutic agents used
to treat various skin diseases. If adducts
are found in the blood or urine after
treatment, it may be that an exposure-
dose correlation can be established. At
present, EPA-HERL is conducting an
exposure-dose feasibility study with the
compound Psoraben in a collaborative
study with Dr. Regina Santelli of Colum-
bus University. They are looking at the
dose-response of adducts formed with
hemoglobin and white blood cell DNA.
Alkylating agents found in tobacco
products, specifically 4-(methyl-
nitrosoamino)-1 -(3-pyridyl)-1 -butanone
(NNK) and N-nitrosonornicotine (NNN)
would be good compounds to study.
Again, the amount of carcinogen
received can be accurately estimated.
Snuff was suggested as the tobacco.
product to monitor because it contains
high levels of NNN and NNK and does
not contain polynuclear aromatic hydro-
carbons that are derived from smoking.
Other compounds that were suggested
as possible candidates were aflatoxin 81,
o-toluidine, benzidine, 2-naphthylamine,
vinyl chloride, acrylonitrile, styrene, and
dimethylcarbamyl chloride.
In addition, several compounds have
been identified that are on the EPA
priority list and that may form adducts.
Although little literature has been
located on these compounds, it may be
useful to carry out some preliminary
experiments to determine if these com-
pounds form adducts. The compounds
are:
1. Dichloromethane
2. Carbon tetrachloride
3. Polychlorinated biphenyls
4. Chloroform
5. Toluene
6. Formaldehyde
An initial study with these compounds
might include a simple feeding study to
determine if the compounds do form
adducts. If so, several log dose-response
experiments should be conducted to
check linearity with Hb, serum albumin,
and DNA.
Although benzo(a)pyrene (BaP) does
form DNA adducts, and although many
animal studies have been conducted, it
probably would not make a good test
compound. Because BaP is ubiquitous,
high background levels of BaP-DNA
adducts are present and make the
establishment of and exposure-dose
relationship difficult.
This is not meant to be an all inclusive
list as much as it is meant to comprise
suggestions for compounds to be con-
sidered for environmental monitoring.
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S. D. Soileau is with Lockheed-EMSCO, Inc.. Las Vegas, NV 89114.
Tamar G. Gen is the EPA Project Officer (see below).
The complete report, entitled "Carcinogen-DNA Adducts: Introduction, Literature
Summary, and Recommendations." (Order No. PB 87-145 678/AS; Cost:
$18.95, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield. VA22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas, NV89114
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
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