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OCR for page 38
2
DNA-Adduct Technology
The consensus of several recent meetings on DNA-adduct research has
been that new assay systems for detecting and measuring DNA adducts and
protein adducts have the potential to improve markedly the biologic bases
for estimating the risks of human exposure to several important classes of
environmental pollutants (Bartsch et al., 1988; Berlin et al., 1984; de Serres,
1988; de Serres et al., 1985; Farmer et al., 19871. This chapter describes
and evaluates some of the currently used assays and discusses how the
information they provide can lead to the improvement of risk assessment and
epidemiological studies.
New DNA-adduct technology embodies striking technologic improvements
in sensitivity and specificity and permits measurement of mammalian re-
sponse to small, intermittent environmental exposures. For example, molec-
ular dosimetric assays and mutation analysis in mammalian cells in tissue
culture suggest that low exposures to genetic toxicants produce DNA lesions
at approximately 2,000 per cell in the lung after exposure to aromatic amines
and 100,000 per cell in the upper layer of skin after exposure to the ultraviolet
component of sunlight (Lohman et al., 1985~. Those figures correspond to
about 1 adduct per 10-7 bases in the lung and at least 1 adduct per 10-4
bases per day in the upper layer of skin.
The new methods of measuring DNA adducts are in many instances rel-
atively inexpensive, fast, and reproducible. They can be applied to readily
available samples of body fluids, such as blood, semen, and urine, and to
small samples of cells, such as buccal mucosa or skin biopsy specimens.
38
OCR for page 39
DNA-Adduct Technology 39
TECHNIQUES FOR DETECTING DNA ADDUCTS
Chromatographic and Spectrometric Methods
Chromatography has many variations, but all involve the flow of test
material through tubing containing stationary material designed to adsorb the
components of the test mixture selectively, and thus create different flow
rates and a series of bands (chromatograms) by which their identity can be
determined.
In spectrometry, the sample Interacts with light or particles to yield dis-
tinctive spectral signals. One version is mass spectrometry, the most accurate
technique for trace organic-chemical analysis, according to the National Bu-
reau of Standards.
The polarity and size of DNA adducts, or fragments of DNA adducts, can
markedly influence the separation power and thus the sensitivity of these
techniques. Each analytic method possesses potentially good to high resolving
power, but the suitability and sensitivity of any method or combination of
methods commonly depends on the physicochemical properties of the adduct
or the class of adducts to be tested.
. . · . - . . ~
LIQUID CHROMATOGRAPHY/MASS SPECTROMETRY (LC/MS)
In this method, DNA adducts are separated from the test sample by ad-
sorption on an activated surface. They are then put into a mass spectrometer
for final analysis.
HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC)
This method combines speed and high-resolution power to fractionate DNA
adducts by column chromatography of modified DNA bases. It can detect
fluorescence.
ATOMIC-ABSORPTION SPECTROMETRY (AAS)
This quantitative analytic technique detects adducts containing metals by
absorbing light of specific wavelengths from excited atoms.
TANDEM MASS SPECTROMETRY (MS/MS)
Two mass spectrometers are used in sequence to detect fragments (ions)
of specific molecules, such as DNA adducts. An ion from the mixture is
selected and focused through the first mass spectrometer; thereafter, it is
.
OCR for page 40
40 DRINKING WATER AND HEALTH
fragmented into smaller portions for further, high-resolution analysis in the
second mass spectrometer (Farmer et al., 1988~.
FLUORESCENCE LINE-NARROWING SPECTROMETRY (FLNS)
Particularly useful for fluorescent adducts of polycyclic aromatic hydro-
carbons, FENS uses low temperature and laser excitation to improve sen-
sitivity in the quantitative fluorometric analysis of DNA adducts (Jankowiak
et al., 1988).
ULTRAVIOLET RADIATION/HIGH-PERFORMANCE LIQUID
CHROMATOGRAPHY (UV/HPEC)
Because all adducts absorb ultraviolet radiation, testing for UV absorption
is useful for large, bulky adducts, such as those formed by aflatoxin, but is
rarely sensitive enough for human monitoring. Sensitive HPLC can be added
to detect fluorescence after hydrolysis of particular carcinogen-DNA ad-
ducts. For example' synchronous fluorescence spectrometry (SFS) scans a
,
~ · - ^ ^ · . · . . - · · -
sample with a flxecl wavelength oliterence between excitation and emission.
Three-dimensional plots of fluorescence intensity, emission, and excitation-
emission wavelength difference should be able to help identify some unknown
adducts (Farmer et al., 19871.
GAS CHROMATOGRAPHY/ELECTRON CAPTURE NEGATIVE TON MASS
SPECTROMETRY (GC/ECNIMS)
In this technique, the test material is derivatized and volatilized into a
carrier gas stream whose components pass through a chromatography column
at different rates, where the adducts are separated. The sample then enters
the mass spectrometer, where distinctive ions are formed and detected with
high sensitivity and specificity. A related, less specific method is GC with
electron capture detection (GC/ECD).
Quantitative Immunoassays
Monoclonal or polyclonal antisera specific for carcinogen-DNA adducts
or carcinogen-modified DNA are used in immunoassays to quantify the bind-
ing of known carcinogens with DNA in biologic samples of nucleic acid
(Poirier, 19841. These immunoassays are simple to perform, inexpensive,
and thus appropriate for human samples; but the antisera are chemical-spe-
cific, and thus different antisera must be developed for each adduct of interest
(Santella, 19881.
The most sensitive immunoassays are run in a competitive mode, in which
OCR for page 41
DNA-Adduct Technology 41
two chemically identical haptens (in this case, DNA adducts) compete for
an antibody binding site. The concentration of 1 hapten (usually an assay
standard) is always kept constant, and that hapten is radioactively labeled
(in radioimmunoassays) or bound to the bottom of microtiter wells (in en-
zyme-linked immunosorbent assays). The other hapten is used in increasing
concentrations to compete with the constant hapten for binding to the anti-
body. The variable hapten can be either a standard immunogen or an unknown
sample. Quantitation is based on comparison of unknowns with the inhibition
curve generated by the standard immunogen.
RADIOIMMUNOASSAY (RIA)
In conventional RIA, a competitive technique, the antigen-antibody com-
plex is separated from the whole mixture in tubes by a variety of physical
or chemical methods (Poirier, 1981), and the standard hapten is radioactively
labeled.
ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA)
In ELISA a solid-phase competitive assay that uses microtiter plates, the
antibody bound after competition can be measured by a second enzyme-
linked antibody used to cleave a specific substrate. One of the most commonly
used enzyme conjugates is alkaline phosphatase, which cleaves a variety of
phosphorylated substrates into products that can be detected by spectropho-
tometry or fluorescence (ELISA) (Poirier, 19811. In general, microtiter-plate
assays can be more sensitive than RIAs, but they are also more often in-
consistent and variable.
ULTRASENSITIVE ENZYMATIC RADIOIMMUNOASSAY (USERIA)
The procedure for USERIA is similar to that of ELISA, except that the
substrate obtained after application of the first antibody is labeled with ra-
dioactive isotopes for radiochemical measurement of enzyme interaction.
Counting requires that samples be manually removed from each well (Farmer
et al., 1987; Santella, 1988~.
m mu noh istochem ice ~ Tech n iq ues
Enzyme-staining with a fluorescence or peroxidase end point can identify
the specific cell types in which DNA adducts occur (and thus the cells that
are targets for carcinogens) within a complex tissue sample. Monoclonal or
polyclonal antibodies are used in conjunction with other antibodies that con-
tain peroxidase enzyme or a fluorescent probe. With the fluorescent probe,
OCR for page 42
42 DRINKING WATER AND H"LTH
images: of the adducts can be enhanced by computer for analysis. Polyclonal
antibodies are less easily identified by the most sensitive image analysis
systems (Adamkiewicz et al., 1985), but they have been used with micro-
fluorometry for semiquantitative comparison between samples (Huitfeldt et
al., 1987) and to detect, but not quantify, binding in human tissues (den
Engelse et al., 1988~.
32P-PostIabeling Technique
DNA is enzymatically hydrolyzed and the digest is labeled with phosphor-
ylating enzyme to incorporate radioactivity. Thin-layer chromatography (TLC)
is used to separate the adducts, which can be detected by autoradiography,
and a portion of the chromatogram is excised for estimation of the total count
(Gupta et al., 19821. Less useful for low-molecular-weight compounds, 32p_
postlabeling is more sensitive for aromatic or bulky hydrophobic adducts and
has been able to show the extent and persistence of adduct formation in
animals by more than 70 compounds, including aromatic hydrocarbons, ar-
omatic amines, estrogens, and methylating agents (Gupta and Randerath,
1988~. With 32P-postlabeling, it is possible to monitor human carcinogen
exposure (Randerath et al., 1988~. This technique is also capable of analyzing
DNA adducts formed by unknown hydrophobic compounds (Farmer et al.,
19871; tandem technology might someday be developed for identifying the
structure of such adducts.
TECHNIQUES FOR DETECTING PROTEIN ADDUCTS
Some of the same methods for detecting DNA adducts are applied to the
determination of protein adducts (GC, GC-MS, immunoassay, and fluores-
cence detection with HPLC). New techniques for detecting protein adducts,
especially those using hemoglobin as a target molecule (Bailey et al., 1987;
Osterman-Golkar, 1988; Neumann, 1984, 1988), offer high sensitivity and
specificity in detecting exposure of animals to alkylating agents. Several
studies have found a direct correlation between hemoglobin-adduct and DNA-
adduct concentrations in exposed experimental animals (Adriaenssens et al.,
1983; Pereira, et al., 1981; Shugart, 1985; Wild et al., 19861. An additional
practical advantage of measuring protein adducts is that large amounts of
some proteins (especially hemoglobin) can be obtained from human subjects.
Thus, protein adducts might provide more reliable measurements than DNA
adducts for evaluating both normal background concentrations of adducts
and deviations from the normal.
OCR for page 43
DNA-Adduct Technology 43
SENSITIVITY AND SPECIFICITY
The intrinsic sensitivity of an assay to detect the molecular effect of a
given hypothetical chemical is usually expressed as femtomoles (fmol; 10- Is
moles) of adduct per milligram of DNA or protein. Tables 2-1 and 2-2 give
estimates of the intrinsic sensitivities of various DNA and protein binding
assays. The estimates are approximations and sensitivity might vary by sev-
eral orders of magnitude, depending on the physicochemical nature of the
chemical or adduct.
Tables 2-1 and 2-2 show that some of the immunochemical assays and
the postlabeling assay have good sensitivity and do not require invasive
techniques or large tissue samples. The MS/MS method, a physicochemical
method, has the same advantages as the immunochemical and postiabeling
assays, but its dependence on expensive and sophisticated equipment could
severely limit widespread application. Other techniques, such as immuno-
chemical methods for detecting DNA adducts at the single-cell level (Bean
et al., 1988; Perera et al., 1988; Van Benthem et al., 1988) and the recently
introduced laser-scan immunofluorescence microscopy (Bean et al., 1986),
are also limited by unique instrumentation requirements. However, the MS/
MS (Farmer et al., 1988), RIA (Umbenhauer et al., 1985), and 32P-postla-
beling (Randerath et al., 1988) methods can detect interactions with small
amounts of unidentified alkylating agents associated with occupational and
low-level environmental exposures.
Both physicochemical and immunochemical methods for detecting adducts
or metabolites of genetic toxicants in urine have high sensitivity and speci-
f~city (Oshima and Bartsch, 1988; Shuker and Farmer, 1988; Vanderlaan et
al., 1988), but still require validation as indicators of internal exposure. A
high adduct concentration in urine is often assumed to indicate high internal
exposure, but this assumption is not necessarily correct. Proper mass-balance
evaluations are needed to measure the intake and excretion of genetically
toxic agents and their metabolites. Without such determinations, it is equally
justifiable to relate the presence of a high concentration of adducts in urine
to detoxif~cation or to low internal exposure (Lohman et al., 1984; van Sittert,
19841.
Although technologic improvements make feasible the sensitive measure-
ment of exposure to genetic toxicants in animal models and humans, no
generally applicable methods have been developed for estimating genetic risk
(Wogan, 19881. Attempts are under way to relate target dose in humans to
biologically adverse effects of small exposures to genetically toxic agents
(Ehrenberg, 1988~.
The use of these new, ultrasensitive analytical techniques in risk assessment
will depend on an understanding of the mechanistic relationships between
DNA alterations and the ultimate expression of toxic effects. Recent devel
OCR for page 44
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DNA-Adduct Technology 47
opments in the study of DNA binding and protein binding provide a useful
tool for beginning to acquire that understanding. However, additional infor-
mation, such as clarification of the role of background or baseline adducts
that are always present in animals and humans, will be needed to make full
use of the advanced technology that is currently available.
APPLICATIONS OF DNA ADDUCT TECHNOLOGY
General Utility in Risk Assessment
DNA-adduct and protein-adduct technology is potentially useful in the
processes of hazard identification and risk assessment. The NRC has per-
formed comprehensive toxicological assessments for the Environmental Pro-
tection Agency on chemicals found in drinking water (NRC 1977, 1980a,b,
1982, 1983, 1986, 19871. In these assessments, information on acute, sub-
chronic, and chronic effects is assembled and evaluated. Considerations of
exposure and pharmacokinetics play an important role in risk assessment for
chemicals with identified toxicity (NRC, 19871. It appears that adduct tech-
nology could be extremely valuable in estimating dosimetry and systemic
distribution, in establishing possible target tissues or organs, and in deter-
mining the potential for irreversible toxicity such as cancer, mutation, or
developmental effects.
Table 2-3 lists some potential applications of DNA-adduct analysis to the
toxicologic evaluation of drinking water contaminants for risk assessment.
The table is arranged as a matrix with the components of toxicity assessment
on one axis and the potential contribution of DNA-adduct analysis on the
other. The second column identifies when the specific method chosen to
detect adducts is important. Toxic effects can be adduct-specific, and three
toxicologic components relationship of adducts to toxic response, muta-
genicity, and species extrapolation might require methods that identify the
adducts detected. Some analytic methods (e.g., 32P-postlabeling) do not
identify the DNA adduct detected. The third column specifies whether or
not it is desirable to identify the DNA adducts in using the technology for
toxicologic assessments. Specific adduct identification is needed to correlate
toxicity with adducts and is desirable in studies of mutagenic activity. Some
DNA adducts, such as N7 alkylguanines, appear to have only a small role
in mutation induction. Different components of toxicologic assessment have
different requirements for quantitative or qualitative test results. These are
identified in the table, as is the need for other biologic data. For example,
information on chronicity of exposure is important in establishing a carcin-
ogenicity hazard associated with a substance that induces formation of DNA
adducts. The last column of the table identifies the extent to which DNA-
adduct technology can now be used routinely in toxicologic assessments;
OCR for page 48
48
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DNA-Adduct Technology 49
most of the technology is still~in the research and development stage, and
none can yet be considered routine. The methods listed in the table are
expected to evolve, so the conditions identified will also change. However,
the table should facilitate an understanding of which aspects of toxicologic
testing can be aided by DNA-adduct technology, which methods to consider
first, and what data one should expect from a specific approach.
Features of the four categories of methods now available for use in toxicity
testing are summarized in Table 2-4. Immunochemical and~physical methods
require considerable expertise in chemical synthesis, antibody production and
radiolabeling, and analytic instrumentation. Some methods, such as 32p_
postlabeling, have been developed for use with high-molecular-weight (bulky)
adducts, especially polycyclic aromatic hydrocarbons and aromatic amines.
Use of this assay with low-molecularweight alkylating agents is not now
feasible.
The published literature has been searched for specific information re-
garding the reactivity of 16 compounds that have been identified in drinking
water and are known to be carcinogenic, mutagenic, teratogenic, or genet-
ically toxic in experimental animals. Thirteen were recently reviewed by the
NRC (1986) for EPA. Chemical structure, use, occurrence or source of
exposure, association with DNA adduct formation, tissue distribution, car-
cinogenicity, mutagenicity, and other health effects were the data elements
sought. The results are summarized in Appendix A, which can be referred
to for some evidence for the theoretical assessments. Table 2-5 summarizes
speculations about the ability of the 16 compounds to form DNA adducts.
With the exception of benzoLalpyrene, for which considerable data exists
concerning adduct analysis, the chemicals have not been the subject of ex-
tensive DNA-binding studies.
Epidemiology and Human Monitoring
Proper investigation of relationships between disease in humans and ex-
posure to drinking water contaminants has been hampered by the difficulty
of assessing exposure to contaminants appropriately and by the limitations
inherent in the use of traditional end points, such as the development of
cancer, which are both rare and characterized by long latency. Interest in
the incorporation of biologic markers into studies of human exposure to
xenobiotic substances has increased, with the hope that use of such markers
will enable scientists to characterize the empirical associations between ex-
posures and outcomes, improve the accuracy of exposure assessment, en-
hance understanding of toxic mechanisms, increase the ability to detect early
subclinical effects of exposure, and make better use of data from laboratory
animals in predicting the effects of exposures of humans (NRC, 19871.
Protein and DNA adducts in humans have been proposed as markers both
OCR for page 50
50 DRINKING WATER AND HEALTH
TABLE 2-4 Evaluation of New Molecular Methods in DNA- or Protein-
Adduct Technology
-
Method of Biologic Monitoringa
for DNA Adducts
Physical Immunochemical 32P-Post- For Protein
Characteristic Methods Methods Labeling Adducts
. .
Appropriateness for measuring
exposure
Qualitative ( + ) + + +
Recent (1-week) internal dose ? + + +
Long-term body burden ? + + (+)
Dose at target site ? + +
Appropriateness for assessing
health effects
Reversible ? ( - ) ( - ) ( - )
Irreversible ( - ) ( - ) ( - ) ( - )
Interpretation of results
On individual basis + + + +
On group basis + + + +
Precision of method
Technical reproducibility ? ( + ) ( + ) +
Stability (+) (+) + (+)
Interlaboratory reproducibility ? ( + ) ( + ) +
Sensitivity
For some environmental exposures
For occupational exposures
For acute exposures
Chemical specificity
Absence of confounding factors
Absence of background adducts
Simplicity
Ease of sample storage
Current applicability
In research
In routine use
?
(+)
b
+ + + +
?
?
+
+
(+)
(-)
b
(+)
( - )
b
(+)
(-)
b
+
(+)
( - )
+
+ +
(+) (+)
aSymbols: +, applicable or true; (+), probably applicable or true; - , not applicable or not true;
( - ), not now applicable or not now true; ?, unknown.
bCannot be generalized.
OCR for page 51
DNA-Adduct Technology 51
TABLE 2-5 Classification of 16 Dnnking Water Contaminants According to
Their Presumed Ability to Form DNA Adducts
Definite Probable Possible
Ability Ability Ability Insufficient Data
Acrylamide Tnchlorfon Diallate Arsensic
Chromium Sulfallate Nitrofen
Benzo[a]pyrene 1,2-Dichloropropane Pentachlorophenol
Dibromochloro- ~ (1,2-DCP)
propane (DBCP) 1 ,2,3-Trichloro
Ethylene di- propane (1 ,2,3-TCP)
bromide (EDB) 1,3-Dichloropropene
(1,3-DCP)
Di(2-ethylhexyl)
phthalate (DEHP)
Mono(2-ethylhexyl)
phthalate (MEHP)
for use in epidemiologic studies to assess the risks associated with exposure
to potential genetic toxicants and for use in monitoring exposed populations.
However, all the studies that have measured protein or DNA adducts have
focused on humans exposed to carcinogens occupationally, environmen-
tally, or otherwise.
In principle, incorporation of measurements of carcinogen-DNA adduct
formation into epidemiologic studies could offer at least two kinds of benef~ts:
· The use of sensitive methods, such as immunoassays and 32P-postIa-
beling, might afford an opportunity to detect early, subtle effects of small
exposures.
· Human studies incorporating DNA-adduct assays might provide infor-
mation on target-molecule dose that reflects exposure, absorption, metabo-
lism, and DNA-adduct formation and repair rates.
Although the measurement of DNA-adduct formation in humans holds
substantial promise for epidemiologic and monitoring studies, interpretation
of data derived from DNA-adduct measurements is extremely complex, par-
ticularly in humans. In general, further experimental work is required before
measurements of DNA adducts can be successfully incorporated into studies
that assess toxicity from drinking water contaminants in humans. The fol-
lowing issues are of particular concern in considering the potential appli-
cations of DNA-adduct technology in human studies to evaluate potential
toxicity of drinking water contaminants:
· The paucity of information about the kinetics, dose-response relation-
ships, and interindividual and intraindividual variability of DNA-adduct for-
mation in humans renders the proper design and interpretation of human
OCR for page 52
52 DRINKING WATER AND HEALTH
studies using DNA-adduct technology very difficult. Studies that characterize
the variability of adduct levels in humans due to such factors as age, sex,
ethnicity, diet, tobacco use, and such medical conditions as liver disease are
needed. Additionally, studies are needed for proper characterization of base-
line adduct levels in the general population.
· Because numbers of DNA adducts reflect not only exposure, but also
rates of metabolism (in the case of indirect carcinogens) and DNA-adduct
formation and repair, DNA-adduct concentrations are likely to involve com-
plex dynamics. When technically feasible, the use of protein adducts might
prove more appropriate for exposure assessment; such adducts in the he-
moglobin of red blood cells have demonstrated chemical stability and linear
dose-response relationships for a variety of compounds and thus can provide
integrated exposure information. To date, the use of protamine adducts in
germ cells has been limited to studies of small alkylating agents in mice.
Further studies are needed to evaluate the use of human protamines in do-
simetry. Protamine dosimetry might help to identify the exposures that pose
germinal risks.
· In animal studies, it is possible to study DNA-adduct concentrations in
target tissue, but the target tissue of interest in humans is often inaccessible.
Circulating white blood cells or lymphocytes are used as surrogates for
determination of DNA-adduct concentrations. However, the validity of using
surrogate tissue, particularly for human risk assessment, has not been ade-
quately evaluated.
· Some of the better-character~zed chemicals that produce DNA adducts,
such as BaP, are ubiquitous in the environment. That presents difficulties in
epidemiologic studies, because, even with proper selection of controls, back-
ground DNA-adduct concentrations might mask slight differences in con-
centrations between "exposed" and "unexposed" populations.
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Representative terms from entire chapter:
cal cal cal
