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
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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-
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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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