United States
Environmental Protection
Agency
Environmental Monitoring Systems
Laboratory
Las Vegas NV 89193
Research and Development
EPA/600/S4-90/034 Apr. 1991
§r EPA Project Summary
Evaluation of Exposure Markers
R. R. Tice and C. H. Nauman
A novel microgel electrophoresis as-
say has been developed for directly
evaluating, in individual cells, the fre-
quency of single strand DNA breaks
and/or alkali-labile sites. This technique,
called the single cell gel (SCG) electro-
phoresis assay, requires processing
only a few hundred to a few thousand
cells. The requirement for an extremely
small number of cells makes it pos-
sible to evaluate the lever and intercel-
lular variability of DNA damage induced
by genotoxic agents in virtually any
eukaryote cell population.
The primary purpose of this research
has been to determine the suitability of
this technique for detecting DNA dam-
age induced by potentially genotoxic
pollutants either in cells sampled from
various organs of rodents or in cells
sampled from humans. In conducting
this work, the focus of the research
has been on: (1) evaluating the speci-
ficity and sensitivity of the technique
by determining the magnitude and ki-
netics of DNA damage induced in cul-
tured mammalian cells (e.g., mouse or
human peripheral blood leukocytes,
Chinese hamster ovary cells, rodent
hepatocytes) by a variety of genotoxic
and nongenotoxic chemicals; (2) de-
veloping appropriate methods for iso-
lating individual cells from organs (e.g.,
blood, brain, liver, spleen, testis, bone
marrow, lung) of rodents; (3) evaluat-
ing the kinetics of DNA damage in-
duced in various organs of male mice
by a representative environmental
genotoxic pollutant; (4) examining the
applicability of the assay to peripheral
blood leukocytes obtained from hu-
mans exposed to genotoxic agents; and
(5) comparing the levels of DNA damage
in the organs of mice collected at an
EPA Superfund site and a concurrent
control site.
In many of these studies the induc-
tion of DNA damage was investigated
using three representative environ-
mental genotoxic pollutants—
acrylamide, trichloroethylene and
dimethylbenzanthracene. Based on the
results obtained, this technique will
provide, with greater sensitivity than
any other method currently available,
data on the induction and persistence
of organ-specific levels of DNA dam-
age resulting from environmental ex-
posure to genotoxic pollutants.
This Project Summary was developed
by EPA's Environmental Monitoring
Systems Laboratory, Las Vegas, NV, to
announce key findings,of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering Information at
back).
Introduction
One approach for assessing the possible
environmental consequences of hazardous
waste pollution involves the assessment
of genotoxic damage, cytotoxic damage
and other adverse health effects in senti-
nel organisms. In marine environments,
sea urchins, mussels, benthic worms, and
various species of fish have been used
(or proposed for use) as organisms with
which to monitor for adverse effects re-
sulting from toxic pollution. In terrestrial
Printed on Recycled Paper
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environments, birds and plants, particu-
larly the Tradescantia stamen hair system,
have long been used to assess toxic lev-
els of environmental pollution. More re-
cently, interest has focused on mamma-
lian species living in close proximity to
man. Data have been published demon-
strating the demographic impact of toxic
wastes at Love Canal, New York, on resi-
dent meadow vole populations. Associa-
tions have been reported between prox-
imity to industrial areas and increased
levels of genotoxic damage in feral house,
mice. Recent research has reported an
increased frequency of genotoxic damage
among rodents collected at a hazardous
waste site in New Jersey.
Techniques that permit the sensitive
detection of DNA damage are useful in
studies of toxicology and carcinogenesis.
Since the effects of toxicants are often
tissue and cell-type specific, it is important
to develop techniques that can detect DNA
damage in a variety of organs or, more
importantly, in individual cells obtained
from various organs. Currently, the three
most commonly used in vivo methods for
ascertaining the ability of chemicals to in-
duce DNA damage involve the scoring of
chromosomal aberrations, micronuclei and/
or sister chromatid exchanges in prolifer-
ating cell populations; the detection of DNA
repair synthesis (so-called unscheduled
DNA synthesis or UDS) in individual cells;
and the detection of single-strand DNA
breaks and/or alkali labile sites in pooled
cell populations.
While providing information about dam-
age in individual cells, the cytogenetic
techniques (chromosome aberrations, etc.)
are of limited value because of the need
for proliferating cell populations and be-
cause the DNA damage must be pro-
cessed into microscopically visible lesions.
The autoradiographic technique (for the
detection of UDS) is based on the excision
repair of DNA lesions, as demonstrated
by the incorporation of tritiated thymidine
into DNA repair sites. While providing in-
formation at the level of the individual cell,
the technique is technically cumbersome
and not all DNA lesions are repaired with
equal facility. Biochemical techniques to
evaluate DNA damage directly (e.g., DNA
strand breaks), such as alkaline elution or
alkaline gel electrophoresis, appear to cir-
cumvent some of the problems associated
with the other two techniques. However,
the use of pooled cells eliminates an
evaluation of damage in small target tis-
sues and ignores the importance of inter-
cellular differences in response.
Biochemical approaches for detecting
DNA damage directly in single cells have
been developed but have not been ap-
plied formally to in vivo research. DNA
damage may now be directly quantitated
in individual cells by lysing cells embedded
in agarose on slides under mild alkaline
conditions to allow the partial unwinding
of DNA. To improve sensitivity for detect-
ing DNA damage in isolated cells, a
microgel electrophoresis technique has
been devebped in which cells are em-
bedded in agarose gel on microscope
slides, lysed by detergents and high salt
and then electrophoresed under neutral
conditions. Cells with increased DNA
damage display increased migration of
DNA from the nucleus towards the anode.
The migrating DNA is quantitated by
staining with ethidium bromide and by
measuring the intensity of fluorescence at
two fixed positions within Jhe migration
pattern using a microscope photometerl
While the neutral conditions for lysis and
electrophoresis permit the detection of
double-strand DNA breaks, they dp not
allow for the detection of either single-
strand breaks or alkali-labile sites. Since
many agents induce from 5 to 2000 fold
more single-strand breaks than double-
strand breaks, neutral conditions are
clearly not as sensitive as alkaline condi-
tions in detecting DNA damage.
Recently, a microgel electrophoretic as-
say has been introduced which is capable
of detecting DNA single-strand breaks and/
or alkali-labile sites in individual cells. The
importance of this assay lies in its ability
to detect intercellular differences in DNA
damage/repair and in the requirement for
extremely small cell samples. Furthermore,
this single cell gel (SCG) technique ap-
pears to be quite sensitive, being capable
of detecting on the order of 250 single-
strand breaks and/or alkali-labile sites in
the DNA of a single cell. While not all
DNA lesions are alkali-labile, nor do all
lesions result in visible cytogenetic dam-
age, many classes of lesions are revealed
by this technique.
The results reported here center on the
evaluation of the SCG assay for use as a
primary approach for detecting the possible
exposure of mammalian organisms to
genotoxic pollutants. This work has in-
cluded experiments to develop and char-
acterize the assay and data obtained from
studies to explore the sensitivity of the
assay for detecting genotoxic damage in-
duced in vitro and in vivo. In many of these
experiments, specific attention has been
paid to the ability of acrylamide,
dimethylbenzanthracene and trichloroeth-
ylene, three representative environmental
pollutants, to induce single-strand DNA
breaks and/or alkali-labile sites in the DNA
of mammalian cells. The principal purpose
of this research has been to expand the
application of the SCG assay to the de-
tection of DNA damage induced by
chemicals in mammalian cells in vitro and
in vivo and ultimately to the assessment
of genotoxic damage in resident free-living
animals or in humans environmentally ex-
posed to hazardous pollutants.
Procedure
The Basic SCG Technique
Up to 10,000 cells of a cell suspension
are mixed with 75 uJ of 0.5% tow melting-
point agarose at 37°C and then placed on
a precleaned, fully-frosted microscope slide
previously coated with 0.5% regular agar-
ose. The cell suspension is immediately
covered with a #1 coverglass and the
slides kept at 4°C for 5 minutes to allow
solidification of the agarose. After adding
a third layer of low-melting agarose, and
allowing for solidification, the slides are
immersed in a lysing solution at 4°C for 1
hour to lyse the cells. The slides are then
removed from the lysing solution and
placed on a horizontal gel electrophoresis
unit.
The unit is filled with fresh electro-
phoretic buffer to a level 0.25 cm above
the slides. The slides are left in this high
pH buffer for 20 minutes to allow unwind-
ing of the DNA. This is followed by elec-
trophoresis for 10 to 40 minutes at 25
volts. After electrophoresis, the slides are
rinsed gently, to remove alkali and deter-
gents that would interfere with ethidium
bromide staining, by flooding them slowly
with 0.4 M Tris, pH 7.5. After three 5-
minute rinses, the slides are stained by
placing 50-75 u.l of a 10 u.g/ml ethidium
bromide solution in distilled water on each
slide and covering the slide with a
coverglass. Observations are made using
a Zeiss fluorescent microscope equipped
with an excitation filter of 515-560 nm and
a barrier filter of 590 jim.
The Image Analysis System
After compariing various image analyz-
ing systems, the Cambridge Instrument's
Quantimet 520* image analyzer was se-
lected for use. The Quantimet 520 consists
of a gated CCD camera attached to the
fluorescent microscope and wired into the
image analysis hardware. The hardware
is in turn attached to a graphics monitor
for visualization of the digitized image, a
mouse-controlled digrtablet for editing the
image, a dot matrix printer, and a Zenith
386 PC with a separate graphics monitor
for running the Cambridge software.
'Mention of trade names or commercial products does
not constitute endorsement or recommendation for
use.
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The Cambridge software allows for the
setting of brightness and contrast levels,
saving the image in memory, setting im-
age intensity detections thresholds, edit-
ing and/or amending the image, calibrat-
ing to relative units, and finally measuring.
the migration length electronically. This
process, although far better than that used
initially, was still time-consuming. To
streamline the cell measurement process,
a program in QBASIC (a Cambridge
modification of the BASIC programming
language) was written.
With the current version of this program,
scoring time has been reduced to ap-
proximately 30 seconds per cell (10-15
minutes per slide if 25 cells are scored). A
spreadsheet template has been prepared
which imports the data file, calculates
means and "stahdara1 errors for each slide,
evaluates frequency distributions, and
presents the data in tabular form. A
graphics software package is then used
to create the line and bar graphs for pre-
sentations.
Results and Discussion
In Vitro Experiments
A series of in vitro studies was con-
ducted to investigate the applicability of
•the SCG assay to the detection of chemi-
cally induced DNA damage in mammalian
cells treated in vitro with a variety of DNA
damaging agents. In these experiments
human leukocytes, mouse leukocytes,
Chinese hamster ovary cells, and mouse
and rat hepatocytes were exposed to
graded doses of several genotoxic chemi-
cals.
The first series of experiments with hu-
man leukocytes was conducted to exam-
ine the differential ability of hydrogen per-
oxide to induce damage in the DNA of
intact cells vs the DNA of cells after lysis.
At the levels tested, hydrogen peroxide
induced a significant increase in the mi-
gration of DNA, regardless of whether
metabolically active cells or lysed cells
were treated. However, the extent of DNA
migration appeared much greater for lysed
cells than for intact cells.
A second set of experiments examined
the effect of exposure duration on DNA
migration length in ficoll-hypaque isolated
mouse leukocytes exposed to
dimethylbenzanthracene (DMBA) and
acrylamide (ACR). Exposure for 4-hour
periods to DMBA resulted in increases in
DNA migration that were not dose depen-
dent, while similar exposures to ACR pro-
duced negative results. However, when
mouse leukocytes were incubated in com-
plete medium at 37°C in the presence of
1000 u.M ACR for 30 min or less, a signifi-
cant increase in DNA damage resulted;
by 1 to 2 hours the extent of DNA migra-
tion was returning to control levels. Thus,
initial experiments were negative because
long sample times permitted sufficient time
for DNA repair to remove the damage. It
was determined that adding cytosine ar-
abinoside (ARA-C), a DNA synthesis chain
terminator, could be used to prevent liga-
tion of repair sites during unscheduled
DNA synthesis.
Chinese hamster ovary cells were used
in a series of experiments to evaluate the
response to ACR (a direct acting agent),
and to trichloroethylene (TCE) and DMBA
(agents requiring metabolic activation). The
presence of S9 was required before an
increase in DNA migration could be dem-
onstrated following exposure to DMBA and
TCE, arid a positive dose response was
seen with S9 present. Response to ACR
was generally greater when 89 was
present, and a positive dose response
was demonstrated both with and without
S9, the response being steeper with 89.
Migration patterns were more heteroge-
neous for cells exposed to TCE and DMBA
than for those exposed to ACR. One ex-
planation for this may be that individual
cells vary in their permeability to the 39-
dependent active metabolite(s) of TCE and
DMBA. The overall results of these ex-
periments are consistent with ACR and a
metabolite of ACR having genotoxic activ-
ity, and with TCE and DMBA requiring
metabolic activation to reactive forms.
The final set of in vitro experiments
adapted the rodent hepatocyte assay to
SCG procedures. Mouse hepatocytes were
freshly isolated for each test. Resulting
cultures were exposed to two doses of
cyclophosphamide (CP), a well-known al-
kylating agent requiring metabolic activa-
tion. Parenchyma! cells (which possess
the capability for metabolic activation)
demonstrated significant increases in the
lengths of DNA migration; furthermore, the
intercellular distribution of DNA migration
patterns was more homogeneous with in-
creasing doses of CP.
Additional studies were conducted with
mouse and/or rat liver parenchyma! cells
which were exposed to a variety of com-
pounds, including diethylnitrosamine
(DEN), ethylmethanesulphonate (EMS),
and 2- and 4-acetylaminofluorene.
In Vivo Experiments
A series of experiments was conducted
to evaluate the ability of ACR, DMBA and
TCE to induce DNA damage in mice in
four different tissues (brain, liver, spleen,
blood). Male B6C3F1 mice were exposed
acutely by gavage to 100 mg/kg ACR or
DMBA, or to 1000 mg/kg TCE.
Four hours after treatment with ACR,
cells from all four organs/tissues exhibited
a significant increase in DNA migration,
with liver cells showing the greatest per-
centage increase in response. By 24 hours
after treatment, only blood leukocytes still
exhibited an increased level of damage.
None of the cells sampled four hours after
treatment with DMBA exhibited an increase
in DNA migration. However, at 24 hours
after treatment, cells from all but brain
exhibited a significant increase in DNA
migration, with spleen cells showing the
greatest response. Four hours after treat-
ment with TCE, cells from all four tissues
exhibited a significant increase in DNA
migration, with spleen cells showing the
greatest response.
These pilot studies demonstrated that
the level of DNA damage induced by these
chemicals was agent-, organ-, and sample
time-dependent. They also showed the
utility of the approach and the feasibility of
detecting DNA damage in individual cells
isolated from different organs of mice.
However, the range of variation among
cell samples from control mice (about 2-3
fold) was disappointing and led to an ex-
amination of factors involved in the pro-
cessing of in vivo tissues.
Three factors were investigated. The
collagenase treatment used to isolate
single cells from brain and liver resulted
in a significant increase (about 50%) in
DNA migration. Mincing alone, without
collagenase, was found to be sufficient for
ensuring an adequate sample of single
cells from every tissue tested. Secondly, it
was found that the addition of calcium
chelators, EDTA or EGTA, to the media
solutions resulted in a very significant re-
duction in DNA migration in control cells.
An adverse impact of blood in the lysing
solution was corrected by adding DMSO.
Experiments with ACR subsequent to
isolating these confounding factors dem-
onstrated, under the modified sampling
protocol, reproducible control data for each
tissue between sample times, and repro-
ducible data among animals at a specific
dose.
Human Studies
One of the goals of this research was to
be able to evaluate and compare data
obtained on both animal and human
populations. Thus, several pilot studies
were conducted to examine the utility of
the SCG assay in human biomarker in-
vestigations.
Blood from runners just completing a
race, and from smoking vs nonsmoking
populations was analyzed through the
SCG assay. These preliminary pilot stud-
.S. GOVERNMENT PRINTING OFFICE: 1991/548-028/20203
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las provided equivocal results, with nu-
merous possible explanations for these
results.
In a more definitive study, the blood
from patients undergoing chemotherapy
at the Duke University Medical Center was
analyzed. Patients with metastatic breast
tumors were sampled before, during, and
after Intravenous administration of
antineoplastic alkylating agents. The pa-
tients were exposed over a several-day
period in a complex regimen to cyclo-
phosphamide, cisplatin, and carmustine.
Results of the SCG assay are consis-
tent across patients, with levels of DMA
migration pre- and post-treatment being
similar. DNA migration levels were el-
evated for samples taken during treatment.
Thus, the data collected demonstrate, the
potential utility of the SCG assay in future
human biomonitoring studies.
Hazardous Waste Site Studies
Feral rodents, Ochrotomys nuttalli
(golden mouse), were live-trapped during
May and June 1990 in this pilot study.
Potentially exposed animals were taken
from an area bordering the fenced North
Carolina State University Superfund site.
Predominant pollutants on the site include
TCE, chloroform, carbon tetrachloride,
various pesticides, laboratory solvents, and
other chemicals. Control animals were
trapped in nearby areas of similar ecology.
Blood, bone marrow, brain and liver tis-
sues from 13 exposed and 13 control ani-
mals were examined via the SCG assay.
Not surprisingly, the extent of interanimal
variability was much greater than that ob-
served normally for laboratory animals.
The level of DNA damage, as measured
by mean migration length, was increased
in all four tissues of animals trapped near
thei Superfjjndjiazardpus^jvaste.site, but
significantly only in brain (P=<0.05). How-
ever, a dispersion analysis revealed that
the bone marrow ceils from the mice living
near or on the hazardous waste site ex-
hibited a significantly increased dispersion
coefficient over that calculated for the
control mice (P=<0.05).
In any study, especially with wild-caught
animals, the possible influence of animal
health, food resources, etc., on the data
collected must be recognized when ana-
lyzing those data. However, the results of
this small pilot study indicate the potential
usefulness of the SCG technique in
evaluating DNA damage in free-living ro-
dents.
Conclusion
Significant technical difficulties were
encountered during the development and
application of the SCG technique to in vitro
and in vivo studies. The results of this re-
search document that many of the prob-
lems Jiaye been| sy,rrnqunted^nd that the_
approach should be of considerable value
to scientists attempting to evaluate animal
and human populations for DNA damage
induced by genotoxic agents acting as
environmental pollutants.
H. R. TTce is with Integrated Laboratory Systems, Research Triangle Park, NC 27709;
and the EPA author, C. H. Nauman (also the EPA Project Officer, see below), is
with the Environmental Monitoring Systems Laboratory, Las Vegas, NV 89193.
The complete report, entitled ""Evaluation of Exposure Markers," (Order No. PB91-
144 675/AS; Cost: $23.00, subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Environmental Monitoring Systems Laboratory
U.S. Environmental Protection Agency
Las Vegas, NV 89193
United States
Environmental Protection
Agency
Center for Environmental
Research Information
Cincinnati, OH 45268
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