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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