r/EPA
United States
Environmental Protection
Agency
Environmental Monitoring
Systems Laboratory
P.O. Box 93478
Las Vegas NV 89193-3478
EPA/600/4-90/034
December 1990
Research and Development
Evaluation of
Exposure Markers
Final Report
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EVALUATION OF EXPOSURE MARKERS
Final Report For September 21, 1988 To September 20, 1990
by
Raymond R. Tice, Ph.D., Principal Investigator
Integrated Laboratory Systems
P. 0. Box 13501
Research Triangle Park, NC 27709
EPA Contract Number 68-C8-0069
Project Officer
Charles H. Nauman, Ph.D., M.P.H.
Exposure Assessment Research Division
Environmental Monitoring Systems Laboratory
Las Vegas, NV 89193-3478
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U. S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS, NEVADA 89193-3478
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NOTICE
The information in this document has been funded wholly or
in part by the United States Environmental Protection Agency
under contract 68-C8-0069 to Integrated Laboratory Systems. It
has been subject to the Agency's peer and administrative review,
and it has been approved for publication as an EPA document.
Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
»
11
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ABSTRACT
A novel microgel electrophoresis assay has been developed
for directly evaluating, in individual cells, the frequency of
single strand DNA breaks and/or alkali-labile sites. This
technique, called the single cell gel electrophoresis (SCG)
assay, requires the processing of only a few hundred to a few
thousand cells. This requirement for an extremely small number
of cells makes it possible to evaluate the level and
intercellular variability of DNA damage induced by genotoxic
agents in virtually any eukaryote cell population.
The primary purpose of this contract has been to determine
the suitability of this technique for detecting DNA damage
induced by potentially genotoxic pollutants either in cells
sampled from various organs of rodents or in cells sampled from
humans. In conducting this project, the focus of the research
has been on: (i) evaluating the specificity and sensitivity of
the technique by determining the magnitude and kinetics of DNA
damage induced in cultured mammalian cells (e.g., mouse or human
peripheral blood leukocytes, Chinese hamster ovary cells, rodent
hepatocytes) by a variety of genotoxic and nongenotoxic
chemicals; (ii) developing appropriate methods for isolating
individual cells from organs (e.g., blood, brain, liver, spleen,
testis, bone marrow, lung) of rodents; (iii) evaluating the
kinetics of DNA damage induced in various organs of male mice by
a representative environmental genotoxic pollutant; (iv)
examining the applicability of the assay to peripheral blood
leukocytes obtained from humans exposed to genotoxic agents; and
(v) 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 induction of DNA damage was
investigated using three representative environmental 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 damage resulting from
environmental exposure to genotoxic pollutants.
This report was submitted in fulfillment of contract number
68-C8-0069 under the sponsorship of the U.S. Environmental
Protection Agency. This report covers a period from September
21, 1988 to September 20, 1990, and work completed as of August
1, 1990.
iii
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TABLE OF CONTENTS
NOTICE ii
ABSTRACT iii
FIGURES Vi
TABLES xi
ACKNOWLEDGEMENTS xii
1. 0 INTRODUCTION 1
2 . 0 THE BASIC SCG TECHNIQUE 4
3 . 0 IMAGE ANALYSIS SYSTEM 4
4 . 0 TECHNICAL PROBLEMS 8
4.1 Fluorescence Exposure Duration 8
4.2 Slide Placement 8
4.3 Cell Position on the Slide 11
4.4 Electrophoresis Duration 11
4.5 Radical Induced DNA Damage 11
4.6 Cell Fixation 14
4.7 Processing Delays 17
4.8 Dead Cells 17
4.9 Cell Selection Criteria 18
4.10 Other Technical Problems 18
5. 0 IN VITRO EXPERIMENTS 19
5.1 S9 Mix and DNA Migration 19
5.2 Chemically Induced DNA Damage 20
5.2.1 Human Leukocytes 20
5.2.2 Mouse Leukocytes 24
5.2.3 Chinese Hamster Ovary Cells 24
5.2.4 In Vitro Rodent Hepatocyte Assay 40
6 . 0 IN VIVO STUDIES 53
6.1 Collagenase Treatment 54
6.2 Calcium Chelators 54
6.3 A Kinetic Study of Acrylamide-Induced Organ
Specific Levels of Damage 54
7 . 0 HUMAN STUDIES 56
7.1 5K Race Study 63
7.2 Smokers vs Nonsmokers 63
iv
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TABLE OF CONTENTS (CONT)
7.3 Duke Cancer Study 65
7.4 Human Longitudinal Study 68
8.0 HAZARDOUS WASTE SITE STUDIES 72
8.1 Study Area 72
8.2 Live-Trapping Schedule 74
8.3 Tissue Collection 76
8.4 Data Analysis 76
9 . 0 CONCLUSION 85
10.0 REFERENCES 86
11.0 SCG PRESENTATIONS 91
12.0 SCG ABSTRACTS/PUBLICATIONS/MANUSCRIPTS IN PREPARATION. 94
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LIST OF FIGURES
Page No,
1. Schematic Presentation of the Technical 5
Steps Involved in the Single Cell Gel
(SCG) Assay.
2. Flow Chart for the SCE Image Analysis 7
Process.
3. Slide Position on Small Gel Box and DNA 9
Migration Pattern.
4. Slide Position on Large Gel Box and DNA 10
Migration Pattern.
5. Cell Position on a Slide and DNA Migration. 12
6. Electrophoresis and DNA Migration for Human 13
and Mouse Leukocytes Processed as Whole Blood.
7. Effect of DMSO (10%) and Phenanthroline on DNA 15
Damage Induced in Mouse Leukocytes during Lysis.
8. Effect of Desferroxamine on DNA Damage Induced 16
During Lysis of Mouse Whole Blood.
9. Effect of Incubation Duration and S9 Mix on the 21
DNA Migration of Isolated Mouse Leukocytes.
10. Mean DNA Migration for Intact Human Leukocytes 22
or Lysed Cells Exposed to Hydrogen Peroxide.
11. Intercellular Distribution of DNA Migration in 23
Intact and Lysed Human Leukocytes Exposed to
Hydrogen Peroxide.
12. Mean DNA Migration for CHO Cells Exposed to 26
Acrylamide as a Function of Sample Time in the
Presence or Absence of Rat Liver S9 Mix.
13. Sample time Dependent Distribution of DNA 28
Migration Patterns for CHO Cells Exposed to
10 mM Acrylamide in the Presence of Rat Liver
S9 Mix.
14. Sample Time Dependent Distribution of DNA 29
Migration Patterns for CHO Cells Exposed to
10 mM Acrylamide in the Absence of Rat Liver
S9 Mix.
VI
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Page No.
15. Mean DNA Migration for CHO Cells Exposed to 30
Acrylamide as a Function of Dose in the
Presence (4 Hour Treatment) or Absence (8
Hour Treatment) of Rat Liver S9 Mix.
16. Dose Dependent Distribution of DNA Migration 31
Lengths for CHO Cells Exposed to Acrylamide
for 4 Hours in the Presence of Rat Liver S9
Mix.
17. Dose Dependent Distribution of DNA Migration 32
Lengths for CHO Cells Exposed to Acrylamide
for 8 Hours in the Absence of Rat Liver S9 Mix.
18. Mean DNA Migration for CHO Cells Exposed to 33
Trichloroethylene as a Function of Sample Time
in the Presence or Absence of Rat Liver S9 Mix.
19. Sample Time Dependent Distribution of DNA 34
Migration Patterns for CHO Cells Exposed to 10
mM Trichloroethylene in the Presence of Rat
Liver S9 Mix.
20. Mean DNA Migration for CHO Cells Exposed to 35
Trichloroethylene as a Function of Dose in
the Presence (4 Hour Treatment) of Rat Liver
S9 Mix.
21. Dose Dependent Distribution of DNA Migration 36
Lengths for CHO Cells Exposed to
Trichloroethylene for 8 Hours in the Presence
of Rat Liver S9 Mix.
22. Mean DNA Migration for CHO Cells Exposed to 37
Dimethylbenzanthracene as a Function of
Sample Time in the Presence or Absence of Rat
Liver S9 Mix.
23. Sample Time Dependent Distribution of DNA 38
Migration Patterns for CHO Cells Exposed to
100 uM Dimethylbenzanthracene in the Presence
of Rat Liver S9 Mix.
24. Mean DNA Migration for CHO Cells Exposed to 39
Dimethylbenzanthracene as a Function of Dose
in the Presence (4 Hour Treatment) of Rat
Liver S9 Mix.
25. Dose Dependent Distribution of DNA Migration 41
Lengths for CHO Cells Exposed to
Dimethylbenzanthracene for 8 Hours in the
Presence of Rat Liver S9 Mix.
vii
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Page No.
26. Time Course for the Induction of DNA Damage 43
in Primary Mouse Parenchymal Cells by
Cyclophosphamide.
27. Distribution of DNA Migration Lengths among 44
Individual Primary Mouse Parenchymal Cells
Exposed to Cyclophosphamide as a Function of
Sample Time.
28. DNA Migration in Parenchymal Cells as a 46
Function of Cyclophosphamide Dose, Sampled
After 6 Hours of Treatment.
29. Distribution of DNA Migration Lengths among 47
Individual Primary Mouse Parenchymal Cells
Exposed to Cyclophosphamide for 6 Hours.
30. Comparative Analysis of the Induction of DNA 48
Damage in Rat and Mouse Primary Parenchymal
Cells by 2-Acetylaminofluorene and 4-
Acetylaminofluorene.
31. DNA Migration in Mouse Primary Parenchymal 49
and Non-parenchymal Cells Exposed to
Dimethylnitrosamine for 6 Hours.
32. Distribution of DNA Migration Lengths among 50
Individual Mouse Primary Parenchymal Cells
Exposed to Dimethylnitrosamine for 6 Hours.
33. Ethylmethanesulphonate-Induced DNA Damage in 51
Mouse Primary Parenchymal and Nonparenchymal
Cells Exposed for 6 Hours.
34. Distribution of DNA Migration Lengths among 52
Individual Mouse Primary Parenchymal Cells
Exposed to Ethylmethanesulphonate for 6 Hours.
35. Effect of EDTA on the DNA Migration for Liver 55
Cells Processed from a Control Mouse.
36. Evaluation of Acrylamide-Induced DNA Damage, 57
as a Function of Sample Time, in Various
Tissue of Male B6C3F1 Mice.
37. Sample Time Dependent Distribution of 58
DNA Migration Lengths for Mouse Blood
Leukocytes Collected from Acrylamide-Treated
Male B6C3F1 Mice.
Vlll
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Page No,
38. Sample Time Dependent Distribution of DNA 59
Migration Lengths for Mouse Liver Parenchymal
Cells Collected from Acrylamide-
Treated Male B6C3F1 Mice.
39. Sample Time Dependent Distribution of DNA 60
Migration Lengths for Mouse Liver
Nonparenchymal Cells Collected from
Acrylamide-Treated Male B6C3F1 Mice.
40. Sample Time Dependent Distribution of DNA 61
Migration Lengths for Mouse Spleen Cells
Collected from Acrylamide-Treated Male B6C3F1
Mice.
41. Sample Time Dependent Distribution of DNA 62
Migration Lengths for Mouse Testis Cells
Collected from Acrylamide-Treated Male
B6C3F1 Mice.
42. DNA Migration Lengths for Blood Leukocytes 64
Sampled from Three Smokers and Three
Nonsmokers (Experiment 1).
43. DNA Migration Lengths for Blood Leukocytes 66
Sampled from Three Smokers and Three
Nonsmokers (Experiment 2).
44. Distribution of DNA Migration Lengths for 67
Blood Leukocytes Sampled from Three
Smokers and Three Nonsmokers (Experiment 2).
45. DNA Migration Lengths for Blood Leukocytes 69
Sampled from 6 Duke Hospital Chemotherapy
Patients.
46. Distribution of DNA Migration Lengths for 70
Blood Leukocytes Sampled from First 3 Duke
Hospital Chemotherapy Patients.
47. Distribution of DNA Migration Lengths for 71
Blood Leukocytes Sampled from Second 3 Duke
Hospital Chemotherapy Patients.
48. Location of the North Carolina State 73
University EPA Superfund Site.
49. An expanded view of the North Carolina 75
State University Superfund Site. The
isopleths indicate concentrations
(ug/1) of trichloroethylene contamination
in the ground water.
ix
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50. Evaluation of DNA Damage in Various Tissue
of O. nuttalli Collected at the North
Carolina State University EPA Superfund
Sites as Compared to Animals Collected
from Concurrent Control Sites.
51. A Plot of Individual Animal Responses,
Ranked in Order from Low to High DNA
Migration Length.
52. A Plot of Individual Animal Responses,
Ranked in Order from Low to High
Dispersion Coefficient.
53. Distribution of DNA Migration Lengths for
Blood, Bone Marrow, Brain and Liver Cells
Sampled from a Representative Animal for
the Hazardous Waste and Control Sites.
54. Correlation Between of DNA Migration
Length and Dispersion Coefficient for
Brain and Liver Cells in Animals Collected
at the Hazardous Waste and Control Sites.
Page No,
80
81
82
83
84
x
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LIST OF TABLES
Page No.
Table 1. Population Demographics of Animals 77
Collected at the North Carolina
State University Hazardous Waste
Site and the Concurrent Control Sites.
Table 2. Frequencies of Micronucleated 78
Polychromatic Erythrocytes and the
Percentage of Polychromatic
Erythrocytes in Bone Marrow of
Animals Collected at the North
Carolina State University Hazardous
Waste Site and the Concurrent Control
Sites.
XI
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ACKNOWLEDGEMENTS
The development and application of the SCG technique to a
variety of in vitro and in vivo (laboratory animal, free-living
animals, human) systems and the wealth of data collected could
not have been obtained without the involvement and technical
expertise of a number of talented individuals whose participation
far exceeded the required scope of the project. These
individuals include: P.W. Andrews, D. Croom, B. Nascimbini, and
M. Phillips at ILS, and Dr. O. Hirai, a visitor in residence for
a year from Fujisawa Pharmaceutical Company, Osaka, Japan. The
application of the SCG technique to the in vitro rodent
hepatocyte culture system is entirely due to Dr. Hirai's
abilities and persistence. The U.S. Army Biomedical Research and
Development Laboratory, Ft. Detrick, Frederick, MD provided
partial support to this research under Interagency Agreement No.
RW21934132-0 (U.S. Army Project Order No. 89PP9951) with the U.S.
Environmental Protection Agency.
Xll
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1.0 INTRODUCTION
The ecological impact and risks to human health (e.g. cancer,
heritable mutations) associated with exposure to environmental
pollutants are exceedingly difficult to evaluate. Adverse
exposure situations resulting from the improper disposal of
hazardous wastes are generally identified by epidemiologic
investigations or by analytical techniques characterizing the
levels of known pollutants. However, because of the generally
limited sample sizes intrinsic to studies involving point sources
of pollution, the inaccurately defined exposure conditions and,
for cancer induction, the long lag time before expression,
epidemiologic studies are of limited value in preventing adverse
health outcomes resulting from localized pollution. Analytical
techniques, while of great value in delineating the kinds of
pollution present, do not provide insight into the biological
hazards associated with complex mixtures as they interact or are
acted upon by various environmental pathways (Vaughan, 1984).
An additional approach for assessing the possible
environmental consequences of hazardous waste pollution involves
the assessment of genotoxic damage, cytotoxic damage and other
ill health effects in sentinel organisms. In marine environ-
ments, sea urchins, mussels, benthic worms, and various species
of fish (Kligerman and Bloom, 1976; Black, 1984) have been used
(or proposed for use) as organisms with which to monitor for
adverse effects resulting from toxic pollution. In terrestrial
environments, birds (Hill and Hoffman, 1984) and plants,
particularly the Tradescantia stamen hair system (Schairer et
al., 1978), have long been used to assess toxic levels of
environmental pollution. More recently, interest has focused on
mammalian species living in close proximity to man. Rowley et
al. (1983) published data demonstrating the demographic impact of
toxic wastes at Love Canal, New York, on resident meadow vole
populations. Nayak and Petras (1985) reported an association
between proximity to industrial areas and increased levels of
genotoxic damage in feral house mice. More recently, Tice et al.
(1988) reported an increased frequency of genotoxic damage among
rodents collected at a hazardous waste site in New Jersey.
Techniques which 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 which 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
induce DNA damage involves the scoring of chromosomal
aberrations, micronuclei and/or sister chromatid exchanges in
proliferating cell populations (Tice and Ivett, 1985; Allen,
1988; Tice, 1988), the detection of DNA repair synthesis (so-
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called unscheduled DNA synthesis or UDS) in individual cells
(Furihata et al., 1984; Mirsalis and Butterworth, 1980; Mirsalis,
1988) and the detection of single-strand DNA breaks and/or alkali
labile sites in pooled cell populations (Petzold and Swenberg,
1976; Cavanna et al., 1980; Barbin et al., 1983; Bermudez, 1988).
While providing information about damage in individual cells, the
cytogenetic techniques are of limited value because of the
necessity for proliferating cell populations and because the DNA
damage must be processed into microscopically visible lesions.
The autoradiographic technique is based on the excision repair of
DNA lesions, as demonstrated by the incorporation of tritiated
thymidine into DNA repair sites. While providing information at
the level of the individual cell, the technique is technically
cumbersome and not all DNA lesions are repaired with equal
facility. For example, the repair of 0 -methylguanine is largely
independent of excision repair processes (Tice and Setlow, 1984).
Furthermore, based on statistical grounds, the technique is
insensitive to weak mutagens (Margolin and Risko, 1988).
Biochemical techniques to evaluate DNA damage directly such as
alkaline elution or alkaline gel electrophoresis appear to
circumvent some of the problems associated with the other two
techniques (Petzold and Swenberg, 1976; Cavanna et al., 1980;
Larsen et al., 1982; Sina et al., 1983; Barbin et al., 1983;
Bermudez, 1988). However, the use of pooled cells eliminates an
evaluation of damage in small target tissues and ignores the
importance of intercellular differences in response.
Furthermore, possible artifacts associated with the technique
(Taningher et al., 1987) and technical limitations associated
with experiment to experiment variability (Doerjer et al., 1988)
have limited the general use of this approach.
Biochemical approaches for detecting DNA damage directly in
single cells have been developed but have not been applied
formally to in vivo research. Rydberg and Johanson (1978) were
the first to directly quantitate DNA damage in individual cells
by lysing cells embedded in agarose on slides under mild alkali
conditions to allow the partial unwinding of DNA. After
neutralization, the cells are stained with acridine orange and
the extent of DNA damage quantitated by measuring the ratio of
green (indicating double-stranded DNA) to red (indicating single-
stranded DNA) fluorescence using a photometer. To improve the
sensitivity for detecting DNA damage in isolated cells, Ostling
and Johanson (1984) developed a microgel electrophoresis
technique. In this technique, cells are embedded 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 the migration pattern
using a microscope photometer. However, while the neutral
conditions for lysis and electrophoresis permit the detection of
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double-stranded DNA breaks, they do riot allow for the detection
of either single-stranded breaks or alkali-labile sites. Since
many agents induce from 5 to 2000 fold more single-stranded
breaks than double-stranded ones, neutral conditions are clearly
not as sensitive as alkaline conditions in detecting DNA damage.
Recently, Singh et al. (1988) introduced a microgel electro-
phoretic assay 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, the single cell gel
(SCG) technique appears 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 (Singh et al., 1988).
While not all DNA lesions are alkali-labile (neither are all
lesions repaired by a long-patch repair process, nor do all
lesions result in visible cytogenetic damage), many classes of
lesions are labile under alkaline conditions (Swenberg et al.,
1976; Yang et al., 1984). Furthermore, since this technique can
be used to follow the excision repair of alkali-insensitive
lesions, as demonstrated by the presence of single strand breaks
formed during the repair process, the absence of alkali-labile
sites may not be a critical problem.
It is this SCG technique that has been evaluated for use as
a primary approach for detecting the possible exposure of
mammalian organisms to genotoxic pollutants. In this final, two-
year report, the experiments conducted to develop and
characterize the assay and data obtained from studies to explore
the sensitivity of the assay for detecting genotoxic damage
induced in vitro and in vivo are presented. In many of these
experiments, specific attention has been paid to the ability of
acrylamide, dimethylbenzanthracene and trichloroethylene, 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 contract has been to expand
the application of the SCG assay to the detection 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 exposed to
hazardous pollutants. Data obtained should help to better
characterize environments that pose a significant health hazard
to mammalian species, including man.
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2.0 THE BASIC SCG TECHNIQUE
Up to 10,000 cells of a cell suspension are mixed with 75 ul
of 0.5% low melting point agarose at 37°C and then placed on a
precleaned, fully-frosted microscope slide previously coated with
0.5% regular agarose (90 ul). The cell suspension is immediately
covered with a #1 coverglass (40 x 50 mm) 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 (1%
sodium sarcosinate, 2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris, pH
10, 10% DMSO, and 1% Triton X-100, added fresh) at 4 C for l 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 electrophoretic buffer (1 mM
Na^EDTA and 300 mM NaOH; pH >13) to a level 0.25 cm above the
slides. The slides are allowed to set in this high pH buffer for
20 minutes to allow unwinding of the DNA followed by
electrophoresis for 10 to 40 minutes at 25 volts. All of the
steps described above are conducted under yellow light or in the
dark to prevent additional DNA damage. After electrophoresis,
the slides are rinsed gently, to remove alkali and detergents
which 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 ul of a 10 ug/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 nm. This
protocol is schematically presented in Figure 1.
3.0 IMAGE ANALYSIS SYSTEM
In the studies published by Singh et al. (1988), DNA migration
patterns were determined by photographing representative cells on
the gel, developing the negatives and measuring DNA migration
length on a small number of cells using lOx magnifying device
with an inset millimeter ruler. Not only is this method
extremely labor intensive, the resulting data are of limited
accuracy for detecting small but biologically important
differences in migration. These two facts led us to seek an
alternative method for obtaining DNA migration data. After
reviewing a number of direct and indirect systems, a microscope-
based image analyzer was selected as the method offering the
greatest sensitivity and efficiency. After comparing various
image analyzing systems, the Cambridge Instrument's Quantimet 520
image analyzer was selected and placed into operation late in the
winter of 1989. All of the data presented in this report
originate from this imaging device.
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SUMMARY OF EXPERIMENTAL PROTOCOL
IMAGE
ANALYZER
STAIN / / '. ' '.7
elhidium bromide
NEUTRALIZE //''."./
ptl 7.5
10 minutes
WHOLE
BLOOD
ELECTROPHORESIS
20-40 minutes
25V. 300mA
UNWINDING
20 mlnules
pH 10
I hour
Figure l. Schematic Presentation of the Technical Steps
Involved in the Singel Cell Gel (SCG) Assay.
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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
digitablet for editing the image, a dot matrix printer, and a
Zenith 386 PC with a separate graphics monitor for running the
Cambridge software. The Cambridge software allows for the
setting of brightness and contrast levels, saving the image in
memory, setting image intensity detections thresholds, editing
and/or amending the image, calibrating to relative units, and
finally measuring the migration length electronically. This
process, although far better than that employed earlier, was
still time consuming. To streamline the cell measurement
process, a program in QBASIC (a Cambridge modification of the
BASIC programming language) was written. The first version of
the program, which still required manual manipulation of the
software, could only measure the migration length in screen
pixels and give a rough estimate of DNA intensity. The current
program (Figure 2), which is in its ninth version, first sets
optical shading, then asks for a data file name to store results.
From the main menu, the user may input what objective is being
used, the type of cell being scored (e.g., parenchymal, testis,
lymphocyte), how many cells will be scored per slide, the size of
the measuring frame, and pixel detection thresholds. When
finished, calibration factors are automatically set and the
current slide I.D. is entered. Once in the cell to cell loop,
the scorer simply selects a cell, focuses it on the image monitor
and presses the space bar. The program then stores the image in
memory to reduce background and proceeds to first dilate (5
times) and then erode (5 times) the image. This step is neces-
sary since many of the DNA migration tails are long, thin and
spotty, and a continuous image is required for length measure-
ment. An EDIT screen gives the scorer the option of removing
unwanted background objects and/or connecting large tail pieces
to the nucleus. The parameters finally recorded are the cell
count, migration length (now calibrated in microns), a shape
factor (perimeter /area) , the diameter of the nucleus (also in
microns), and the intensity of the migration tail. Tail inten-
sity allows an approximate quantitation of the amount of DNA that
has migrated from the nucleus. Thus, even in the absence of any
DNA movement in the gel system, all cells have a minimal image
length (called here DNA migration), a shape close to 1.0 (i.e.,
round), a minimal diameter and a measurable tail intensity. It
should be noted that while shape is dimensionless and thus inde-
pendent of call type, all of the other measurements have char-
acteristic values for the type of cell being evaluated for DNA
damage (e.g. parenchymal vs. nonparenchymal cells, diploid testes
vs. haploid testes cells). These data are stored in a DOS file
which can later be accessed for adding more information. After
scoring the proper number of cells per slide, the program loops
back to the main menu for the next slide. With this program,
scoring time has been reduced to approximately 30 seconds per
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Image Analysis of SCG Slides
Enter Data File
Calibrate Magnification
Shading Correction
Adjust Measure Frame
Set Detection Thresholds
Select Cell/Gray Store
Amend Cell
Edit Binary Image
Measure Features
Output Results
No
Figure 2. Flow Chart for the SCE Image Analysis Process,
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cell (10-15 minutes per slide if 25 cells are scored). A
spreadsheet template in QUATTRO PRO (software similar to Lotus 1-
2-3) has been prepared which imports the data file, calculates
means and standard errors for each slide, evaluates frequency
distributions, then presents the data in tabular form.
Sigmaplot, a graphics software package, is used to create the
line and bar graphs used for presentations.
4.0 TECHNICAL PROBLEMS
In developing an image analysis-based SCG system and in
applying the technique to different biological systems, a number
of technical parameters had to be evaluated. In addition,
problems throughout the course of the project were encountered
which had to be solved.
4.1 Fluorescence Exposure Duration
The first parameter evaluated was the effect of the duration
of exposure to the incident fluorescent light on the magnitude of
the emitted ethidium bromide fluorescence signal from the image.
In this experiment, the length of DNA migration for a cell with a
short migration pattern and for a cell with an extended migration
pattern was repeatedly measured (25 times over a 15 minute inter-
val) . The cells used in this experiment were human peripheral
blood leukocytes randomly selected on a slide electrophoresed for
either 10 or 20 minutes. The resulting data demonstrated initial
rapid quenching of the fluorescent signal over the first 5 min-
utes, followed by a slower decline in signal strength over the
remaining 10 minutes. This experiment indicated the necessity of
measuring only one cell within the same microscope field of view.
4.2 Slide Placement
The second problem formally evaluated was the effect of slide
placement (on the horizontal gel electrophoresis unit) on the
resulting DNA migration patterns. In the first experiment, human
blood cells were mixed with agarose and placed on several
microscope slides. After lysis, a slide was placed on the
horizontal gel box (18 x 24 cm) at each of the four corners and
then electrophoresed for either 20 or 40 minutes. An analysis of
DNA migration patterns among 25 cells selected in the same area
on each slide revealed that the more distant the slide was from
the anode, the less the average migration (Figure 3). The
differential effect was more pronounced the greater the extent of
DNA migration present among the cell population being scored. A
second experiment resulted in a similar finding, while a third
experiment (Figure 4) using a larger gel box (26 x 37 cm)
revealed less of a position effect for the same average migration
length. These data indicate the need to be aware of the
potential for bias in migration resulting from slide placement
and that more homogeneous data are obtained when slides are
8
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DMA MIGRATION AND GEL BOX POSITION
SMALL GEL BOX
200
0)
X
1
z
LJ
150-
100--
0
UPPER
RIGHT
LOWER
RIGHT
UPPER
LEFT
LOWER
LEFT
Figure 3. Slide Position on Small Gel Box and DNA Migration
Length. Group mean data are presented, error bars indicate
standard error of the mean among cells. The indicated positions
are in relation to the anode, which is attached to the upper
right. Data based on 25 cells scored per slide.
-------
DNA MIGRATION AND GEL BOX POSITION
LARGE GEL BOX
V)
c
2
o
O
z
LJ
Z
O
0
UPPER
RIGHT
LOWER
RIGHT
UPPER
LEFT
LOWER
LEFT
Figure 4. Slide Position on Large Gel Box and DNA Migration
Length. Group mean data are presented, error bars indicate
standard error of the mean among cells. The indicated positions
are in relation to the anode, which is attached to the upper
right. Data based on 25 cells scored per slide. Electrophoresis
duration was 20 minutes.
10
-------
electrophoresed on a larger gel box. Currently, to minimize this
effect, replicate slides are randomly dispersed on the gel box,
with at least one control slide placed closest to the anode.
4.3 Cell Position on the Slide
To evaluate the possible effect on DNA migration of the posi-
tion of a cell on an individual slide, microscope slides were
arbitrarily divided into thirds and 15 cells in each third were
evaluated for DNA migration patterns. Slides electrophoresed for
20 and 40 minutes were used so that we could evaluate the effect
of small or large migration lengths. In each case, cells were
selected for scoring by moving in a preset number of viewing
fields from the top edge to the bottom edge of the slide. This
pattern would also enable an analysis of across slide position
effects. While cell to cell variability in DNA migration lengths
were present, no position-related differences in DNA migration
were detected (Figure 5).
4.4 Electrophoresis Duration
In one of the first series of pilot electrophoresis
experiments, human and mouse peripheral blood leukocytes were
electrophoresed over a range of times (5 to 25 minutes) to
evaluate optimal electrophoresis durations. Representative data
from one experiment is presented in Figure 6. These initial
studies indicated technical problems in conducting the assay
since the DNA migration lengths exhibited by cells electro-
phoresed for 20 minutes were much greater than anticipated from
data collected in N.P. Singh's laboratory (see Singh et al.,
1988). Furthermore, the extent of DNA migration increased
dramatically when the slides were stored in the lysis solution
for longer than a few hours. This observation was also inconsis-
tent with data from N.P. Singh, who observed no change in migra-
tion patterns among cells stored in the lysis solution for at
least a week. Initially, considerable effort was spent in at-
tempting to determine the source of this problem, evaluating such
factors as the source of water used in solution preparation, the
company from which the reagents were purchased, the types of con-
tainers used in the experiments, and other experimental condi-
tions. None of these factors were responsible for the high level
of DNA damage observed among control cells. Ultimately, much of
the difficulty was traced to the formation of iron-mediated free
radicals generated during lysis (see below).
4.5 Radical Induced DNA Damage
As mentioned above, it became apparent in our earlier experi-
ments that, under apparently identical experimental condition,
the processing of whole blood resulted in leukocytes with greater
DNA migration than that observed for cells isolated from blood by
Ficoll-hypaque centrifugation. The observation that the lysing
11
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CELL LOCATION ON SLIDE
250
_cn
-------
200
_g 150 +
Q.
Z
o
100--
§ 50 +
Q
OO human leukocytes
mouse leukocytes
0
5 10 15 20
ELECTROPHORESIS DURATION (min.)
25
Figure 6. Electrophoresis and DNA Migration for Human and Mouse
Leukocytes Processed as Whole Blood. Data are group menas for 25
cells, error bars indicate the standard error of the mean among
cells.
13
-------
solution turned pinkish when whole blood was lysed, suggested the
presence of liberated hemoglobin and/or iron. Additionally,
during this time, considerable inter-experiment variability in
the extent of DNA migration among cell samples from control
animals was occurring. It occurred to us that iron-mediated free
radical generation may be responsible for the damage induced
during lysis and for the excessive interexperiment variability.
To evaluate this possibility, phenanthroline and desferroxamine,
two iron chelators, were tested for their ability to suppress the
increased level of DNA damage in leukocytes lysed as whole blood.
In the first experiment, phenanthroline, dissolved in
dimethylsulfoxide (DMSO) due to its insolubility in water, was
tested over a range of concentrations. While the resulting data
demonstrated the complete lack of a phenanthroline inhibitory
response (in fact the compound appeared to induce DNA damage), it
appeared that DMSO alone was capable of inhibiting the increased
levels of DNA damage induced during the lysis step (Figure 7).
Two additional experiments with DMSO were subsequently conducted.
First, increasing concentrations of DMSO were evaluated for their
ability to inhibit the induction of DNA damage during lysis and
10% DMSO was determined to be the optimal concentration. Second,
it was determined that the presence of DMSO had no effect on DNA
damage induced in mouse leukocytes by hydrogen peroxide prior to
the lysis step. Furthermore, DMSO appears to protect cells in
lysis from damage for at least two weeks. Presently, DMSO is
routinely included in the lysis solution.
Incubation of whole blood slides in desferroxamine also
inhibited the induction of DNA damage in human leukocytes during
lysis (Figure 8). Because the sample of desferroxamine available
was small, a complete range of concentrations could not be
tested. Since this compound is a specific chelator of free iron,
these data suggest the importance of eliminating iron from the
lysis solution.
4.6 Cell Fixation
An extensive series of experiments were conducted to evaluate
various methods of cell fixation prior to SCG analysis. We hoped
that a method could be developed whereby cell samples could be
stored and then processed when convenient for the SCG assay.
This step would greatly increase the utility of the assay by
delaying the need for immediate laboratory processing. A number
of fixation methods were attempted, including fixation in
absolute methanol, in 70% methanol, in 3:1 methanol:glacial
acetic acid, in glacial acetic acid, and in various
concentrations of glutaraldehyde. All of these methods resulted
in DNA which, when processed using the usual SCG technique, was
overly condensed when stained and imaged on the gel. This state
of contraction indicated a lack of normal unwinding during lysis
and an inability to undergo normal movement during
14
-------
CO
c
o
L_
u
Sc
50
40-
30-
2Q--
10-
BLOOD
LEUK
ISOL
PBL
DMSO 0.01 0.1 1.0 10.0
PHENANTHROLINE (mM)
Figure 7. Effect of DMSO (10%) and Phenanthroline on DNA Damage
Induced in untreated Mouse Leukocytes during Lysis. Data are group
means for 25 cells, error bars indicate the standard error of the
mean among cells. LEUK = leukocytes; ISOL PBL = Ficoll-isolated
peripheral blood lymphocytes.
15
-------
to
c
O
y
E
Z
o:
o
z
Q
25
20-
15-
10-
5--
XJUCJCJC
BLOOD
LEUK
ISOL
PBL
0.1 1.0 10.0
DESFERROXAMINE (uM)
Figure 8. Effect of Desferroxamine on DNA Damage Induced During
Lysis of Mouse Whole Blood. Data are group means for 25 untreated
control cells, error bars indicate the standard error of the mean
among cells. LEUK = leukocytes; ISOL PBL = Ficoll-isolated
peripheral blood
lymphocytes.
16
-------
electrophoresis. While experiments to evaluate other methods or
these same methods under different experimental conditions have
not been conducted, the potential utility of a proper fixation
method suggests the importance of additional studies at some time
in the future.
4.7 Processing Delays
As more complex experiments were undertaken, it became
necessary to identify steps in the assay which could be delayed
without compromising the integrity of the experiment. As already
discussed, it proved possible to leave the cells in lysis for at
least a few weeks once DMSO was routinely incorporated into the
lysis solution. The only other steps considered to be amenable
to processing delays were at the time of cell collection or after
the cells had been stained with ethidium bromide, but before
image analysis. Since a delay in sample processing prior to
lysis was not considered initially to be a problem, our first
efforts focused on evaluating how long stained slides could be
retained before scoring. In these experiments, the DNA migration
patterns for a randomly selected cell population (generally mouse
or human peripheral blood leukocytes) were determined immediately
after staining or after storage of the slides. Initially, we
determined that slides stored in a cold, humidified, scalable
container could be accurately scored over a period of 3 to 4
days. Longer storage durations resulted in the excessive
diffusion of small pieces of DNA away from the tail. The maximum
length of storage time is dependent on the initial size of the
image being analyzed. DNA diffusion from smaller cells (e.g.,
spermatocytes) created a greater problem over the same period of
time than DNA diffusion from larger cells (e.g. parenchymal
cells). To evaluate the possibility of storing the slides for
more extended periods, some gels were dehydrated in 70% or 100%
methanol and stored cold. Subsequent rehydration, with or
without additional ethidium bromide staining failed to provide
usable fluorescence images. We also evaluated whether freezing
gels at -20°C or -70°C could be used for long term slide storage.
Best results were obtained with quick -70°C freezing and slow
thawing. However, this process also resulted in agarose
dehydration and increased background from the slide frosting.
4.8 Dead Cells
Our major concern about the SCG assay, as would be for any
assay based on the detection of single strand breaks, is the
possible effect of the presence of dead cells on data analysis.
In conducting a number of SCG experiments, it was generally noted
that some cells on the gel exhibited no point of origin (i.e., no
originating cell nucleus remained, but rather a cloud of migrated
DNA). Based on a significant correlation between their frequency
and viability data, we concluded that these objects represented
17
-------
dead cells. To determine the accuracy of this conclusion, human
peripheral blood leukocytes were isolated by Ficoll-hypaque
centrifugation, and then incubated for 60 minutes at 60°C.
Trypan blue exclusion was used to indicate viability. Under
conditions which resulted in 100% dead cells, all cells appeared
as clouds. Our thought was that any dead cell would exhibit this
same pattern of migration. However, data obtained on cells
exposed to 60°C for a shorter time suggested a continuation of
DNA migration patterns. A comparison with vital dye exclusion
frequencies suggested that the cloud pattern depended on how long
the dead cell had been dead (i.e., the autolysis of DNA to
extremely small fragments takes time). These data suggest the
need to routinely obtain viability data and to conduct
experiments at doses that do not result in a significant
depression in viability (i.e., below 80-90%). More recently, we
have been using a dual viability stain consisting of 5-6
carboxyfluorescein diacetate (CFDA) and ethidium bromide. In
this technique, ethidium bromide stains the DNA red in any cell
with a compromised cell membrane, while CFDA stains the cytoplasm
green in any cell metabolically active. Thus, the combination of
the two stains permits an assessment of dead cells (red with no
green), compromised cells (red and green) and live cells (green
but not red). Currently, as a quality assurance check, cells are
routinely monitored for viability prior to SCG analysis.
4.9 Cell Selection Criteria
One of the major potential sources for bias in this assay is
the basis on which individual cells are selected for analysis.
Since the extent of intercellular DNA damage is very
heterogeneous in many cases, it is quite possible that a scorer
may be biased towards selecting cells with more or less damage.
This is especially true if the cell density on the slide is high
(i.e., more than 2-3 cells per field). To reduce this possi-
bility, a set criteria for cell selection was developed. In
scoring a slide, the scorer scans the slide either horizontally
or vertically, limiting image analysis to cells which intersect
the image box on the computer monitor. Using this approach,
sample bias is largely avoided. To demonstrate the success of
this approach, we have determined for a number of control and
treated slides the distribution of cells with no tails, short
tails, long tails, and with no origin (i.e., clouds) among 100
cells and compared the results with the distribution of cells
among the various cell types actually analyzed for DNA migration.
The differences have been insignificant.
4.10 Other Technical Problems
Several factors were found to be critical in maintaining
proper background control levels and gel quality on the slides.
At one point, a defective batch of fully frosted slides was
18
-------
received from the supplier. These slides apparently were not
ground as well as previous batches. This resulted in a
significant proportion of the agarose gels sliding off the slides
during processing (with the complete loss of several experi-
ments) . We determined that this problem could be circumvented by
increasing the agarose concentration of the bottom layer to 0.6%.
Currently, when a new batch of microscope slides is obtained, the
ability of the standard first agarose layer to remain attached to
a number of slides is checked.
Another critical factor was the pH's of the lysing solution
and the electrophoresis buffer. During the normal shelf life of
these solutions, it was discovered that the pH can shift by as
much as one pH point in either direction. An increased pH in the
lysing solution results in increased migration tails, while a
decreased pH in the electrophoresis buffer results in the lack of
DNA migration. The pH of both these solutions are now routinely
checked and adjusted prior to use.
Yet another problem occurred when new Coplin jars were
purchased. Several of these jars contained a residue which
resulted during the lysis step in increased DNA migration tails.
Extensive cleaning of these coplin jars with DMSO ended this
problem.
A fourth problem was the frequent high background fluores-
cence from the slide frosting which interfered with slide
scoring. This problem was corrected by simply increasing the
volume of the bottom agarose layer from 75 to 95 microliters and
by decreasing the volume of ethidium bromide from 75 to 50 ul.
Lastly, it was discovered that stock bottles of Triton X-
100, one of the detergents in the lysing solution, are extremely
prone to bacterial growth when opened frequently. This also
resulted in longer migration tails amongst our controls. This
problem was solved by aliquoting 10 ml samples into smaller
bottles for daily use.
5.0 IN VITRO EXPERIMENTS
A series of in vitro studies were conducted to investigate the
applicability of the SCG assay to the detection of chemically-
induced DNA damage in mammalian cells treated in vitro with a
variety of DNA damaging agents.
5.1 S9 Mix and DNA Migration
To investigate in vitro the genotoxic potential of chemicals,
an exogenous source of metabolic activation is often provided to
metabolize inert parent compounds to reactive species. This is
19
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normally accomplished by adding an S9 mix, a source of mixed
function oxidases obtained from the liver of rats (generally
Aroclor-treated). In this study, isolated mouse blood leukocytes
were incubated in complete medium with and without the addition
of an S9 mix at 37°C for 1 to 4 hours and the extent of DNA
migration determined in cells electrophoresed for 15 minutes
(Figure 9). Although the effect was small, the resulting DNA
migration suggested an increase in DNA damage which depended both
on the incubation time and the presence of rat liver S9.
5.2 Chemically Induced DNA Damage
5.2.1 Human Leukocytes; The first series of experiments
involved the exposure of mammalian cells to hydrogen peroxide
(H202) . The SCG assay is an extremely sensitive technique for
evaluating free-radical induced DNA damage, such as that caused
by this reactive species. We decided to examine the differential
ability of H2O2 to induce damage in the DNA of intact mammalian
cells as compared to the DNA of cells after lysis. We considered
the ability to test the DNA-damaging activity of chemicals
against purified double-stranded DNA as one of the important
aspects of the SCG technique. In this way, chemicals requiring
intercellular metabolic processing could be discriminated from
pure, direct acting chemicals. Five ul samples of whole blood
were obtained from a single male donor, and added to 1 ml of
RPMI-1640 containing 10% fetal calf serum. After centrifugation,
the pellet was either suspended in Hanks calcium-magnesium free,
balanced salt solution containing H2O2 (44 and 176 uM) for 1 hour
at 4°C or mixed, as described, with 0.5% LMA and layered onto
microscope slides. For the former method, the cells were
collected by centrifugation, and processed for SCG analysis as
described. For the latter, after 1 hour in the lysis solution,
the slides were rinsed twice in cold PBS and then placed for 1
hour in a coplin jar containing H202 (44 and 176 uM) at 4°C. All
control and treated slides were electrophoresed for 20 minutes,
and 50 cells were scored per treatment.
At these concentrations, H202 induced a significant increase
in the migration of DNA, regardless of whether metabolically
active cells or lysed cells were treated (Figure 10). However,
the mean extent of DNA migration appeared much greater for lysed
cells than for intact cells. This apparent difference was due to
the differences in the intercellular distribution of DNA damage
between these two experimental conditions. When intact human
leukocytes were exposed to H202, the intercellular distribution
of DNA migration was much more heterogenous than that detected
for lysed cells (Figure 11). Based on a dispersion analysis
(where H, the dispersion coefficient, is equal to the variance/
mean), the DNA migration lengths among the lysed cells exposed to
H202 was distributed as a Poisson (i.e., randomly). Among the
intact cell population, the majority of the cells
20
-------
15
w
c
o
u
t««»
* 10-
O
<
Q
0
0.0
1.0 2.0 3.0
S9 INCUBATION PERIOD (hours)
4.0
Figure 9. Effect of Incubation Duration and S9 Mix on the DNA
Migration of Isolated Mouse Leukocytes. Data are group means for
25 cells, error bars indicate standard error of the mean among
cells. Open symbols indicate cultures without 59, solid symbols
indicate cultures with S9.
21
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H202-INDUCED DAMAGE IN HUMAN PEL
30
en
c
O
o
'E
O
z
LJ
_l
z
O
£
C£
O
25--
20-
OO whole blood
O lysed cells
-O
50
100
CONCENTRATION (uM)
150
200
Figure 10. Mean DNA Migration for Intact Human Leukocytes or
Lysed Cells Exposed to Hydrogen Peroxide. Data are group means
for 50 cells, error bars indicate standard error of the mean
among cells.
22
-------
00 20-
LJ
O
o 60+
LJ
O
I
LJ
O
LJ
D_
40-
20--
0
intact cells
lysed cells
44 uM
176 uM
10 20 30 40
DMA MIGRATION (microns)
Figure 11. Intercellular Distribution of DNA Migration in Intact
and Lysed Human Leukocytes Exposed to Hydrogen Peroxide. Data are
for 50 cells per sample. The width of each bar represents 2 micron
intervals.
23
-------
exhibited no damage, with a subpopulation of cells expressing DNA
migration patterns consistent with that detected among the lysed
cells (Figure 11). These increased migration patterns could not
be explained by the presence of dead cells among the exposed
population since viability measurements routinely resulted in 97%
to 100% viability. Several explanations are possible for this
extensive heterogeneity in DNA damage among intact, metabolically
active cells. For instance, individual cells may vary in their
permeability to H202, their radical scavenging capabilities, the
accessibility of DNA to the damaging species and other mechanisms
which either enhance or diminish the effects of this potent
radical.
5.2.2 Mouse Leukocytes; In the second series of in vitro
experiments to formally evaluate the induction of DNA damage by a
test chemical, ficoll-hypaque isolated mouse leukocytes were
incubated with dimethylbenzanthracene (DMBA) at 1 to 50 ug/ml, in
the presence of a standard rat liver S9 mix for 4 hours. DNA
migration in treated cells appeared to be increased, but the
increase did not depend on the dose of DMBA. Because the
possibility existed that the S9 mix was not properly active, the
next series of experiments used acrylamide (ACR), a direct acting
chemical as the test agent. Isolated mouse leukocytes were
incubated for four hours in the presence of ACR (5 to 1000 uM),
including and excluding a rat liver S9 mix. DNA damage was not
detected in either set of cultures. Subsequently, an experiment
was conducted in which mouse blood leukocytes were incubated in
complete medium at 37°C in the presence of ACR at 1000 uM for 5,
15, 30, 60 and 120 minutes. Exposures of short duration (30
minutes or less) resulted in a significant increase in DNA
damage. By 1 to 2 hours, the extent of DNA migration was
returning to control distances. These data indicate that the
previous experiments were negative because the sample time was
inappropriate, permitting sufficient time for DNA repair to
remove the damage. Because DNA repair processes effectively
repair the damage, we determined that the addition of cytosine
arabinoside (ARA-C), a DNA synthesis chain terminator, could be
used to prevent ligation of the repair sites during unscheduled
DNA synthesis. Since, under these conditions, any DNA damage
would either directly result in strand breaks and/or alkali-
labile sites or indirectly result in single strand breaks formed
as a consequence of DNA repair, this step makes it unnecessary to
evaluate DNA damage induction kinetically. It would be of
interest to more completely characterize this approach as it
applies to routine testing.
5.2.3 Chinese Hamster Ovary Cells; To better standardize the in
vitro SCG assay, we decided to focus additional research on
Chinese hamster ovary (CHO) cells, a transformed fibroblast cell
line commonly used in genetic toxicology. CHO cells were
obtained from American Type Culture Collection, Rockville, MD and
stored in liquid nitrogen. Stock cultures were maintained in
24
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Ham's F-12 medium supplemented with 10% fetal bovine serum, 2 mM
L-glutamine, 100 units/ml penicillin and 100 ug/ml streptomycin.
All cultures were maintained at 37 + 1°C in an atmosphere of 5 +
1% C02 and 95% relative humidity. For the experiments, cultures
were seeded 16-20 hours prior to treatment in 35 mm petri dishes
at a density of 2 x 10 cells/dish. Dilutions of the 3 test
chemicals were prepared in sterile phosphate buffered saline (pH
7.4) for ACR and DMSO for trichloroethylene (TCE) and DMBA.
Based on data from experiments using primary rat hepatocytes
(Hirai et. al., 1990), cyclophosphamide (CP), an alkylating agent
requiring metabolic activation, was selected as a positive
control for the S9 activated portions. Mitomycin C (MMC) and
hydrogen peroxide, two direct acting compounds not requiring
metabolic activation, were used as a positive controls for the
nonactivated experiments. Exogenous metabolic action was
provided by using a liver homogenate fraction (S9) prepared from
Aroclor 1254-induced male Sprague Dawley rats. The final
concentrations within the S9-cofactor pool were 4.5 mg
isocitrate, 2.4 mg NADP and 0.02 ml S9 per milliliter of culture
medium.
A preliminary toxicity test was performed to determine the
optimum doses for testing in the SCG assay. Duplicate cultures
seeded 16-20 hours earlier were exposed to five doses of each
chemical, both with and without metabolic activation, in half log
intervals starting with high doses of 10 mM for ACR and TCE, and
1 mM for DMBA. After 4 hours of exposure for the S9 activated
cultures and 8 hours for the nonactivated cultures, cells were
harvested by trypsinization and the percentage of viable cells
calculated using the trypan blue dye exclusion method. Only
doses with approximately 80% or greater viability were used in
the SCG assay. For the DNA damage assay, overnight growth media
was removed from the cultures and replaced with 2.0 ml of media
with 5% FBS for the nonactivated cultures or with 1.6 ml media
and 0.4 ml S9 mix for the S9 activated cultures. Twenty
microliters of the test chemical dilutions were then added to the
appropriate dishes in duplicate and all cultures placed at 37°C
incubation. After 1-8 hour exposure periods for the nonactivated
cultures and 0.5-4 hours for the S9 activated cultures, the test
medium was removed, the cultures rinsed once in 1.0 ml cold
Hank's Balanced Salt Solution (BBSS) and 0.5 ml more HBSS added.
The cells were dislodged into the HBSS using a teflon scraper.
Ten microliters of this cell suspension were mixed with 75 ul of
LMA and layered onto microscope slides.
Acrylamide; Initial kinetic studies were performed with 10,
1.0 and 0 mM doses of ACR for 0.5, 1, 2 and 4 hours in the
presence of S9 and for 2, 4, 6 and 8 hours in the absence of S9
activation. Increased exposure times resulted in an increase in
the length of DNA migration (Figure 12). An earlier, significant
increase over controls for ACR with S9 (2 hours vs 4 for the
nonactivated cultures) was observed for both the 10 and 1.0 mM
25
-------
ACRYLAMIDE IN CHO CELLS
-
en
c
o
.H
O
c:
o
z
Q
30
25-
20-
15-
10-
0-
O O 10 mM
- 1 mM
A-A P2S
25--
20-
15-
10-
5-
0-
0
WITHOUT S9
O O 10 mM
- 1 mM
A- A PBS
246
EXPOSURE TIME (hours)
8
Figure 12 Mean DNA Migration for CHO Cells Exposed to Acrylamide
as a Function of Sample Time in the Presence or Absence of Ra?
thrL.n M/X* /a?^ P2int rePresent^ the mean and standard error of
scorSI duplicate cultures. For each culture 25 cells were
26
-------
doses. Control values (6-8 urn) represent cell diameters after
DNA unwinding only, with little to no DNA migration. Electrophor-
etic migration profiles for the DNA of cells after treatment with
10 mM ACR over time are shown in Figure 13 with S9 and Figure 14
without S9. These fragments represent a combination of frank
aerylamide-induced breaks and breaks resulting from the hydro-
lysis of alkali-labile lesions under the conditions of the lysing
and electrophoresis experiments. Based on these results, dose
response experiments were conducted over a broader range of con-
centrations (0, 0.1, 0.5, 1.0, 5.0 and 10 mM) at a single expo-
sure time (4 hours with S9 and 8 hours without S9). Increasing
the concentration of ACR results in a progressive increase in the
DNA migration length, indicating a dose-dependent generation of
DNA strand breaks (Figure 15). At equal exposure times, ACR (10
mM) with S9 induced longer migration lengths than the same dose
without S9 and twice the exposure time. Migration length fre-
quency histograms for all doses are shown in Figure 16 with S9
and Figure 17 without S9.
Trichloroethylene; Identical kinetic studies were performed
with 10, 1.0 and OmM doses of TCE both with and without S9 acti-
vation. Increased exposure times resulted in an increase in the
length of DNA migration for the S9 activated cultures only (Fig-
ure 18). In the absence of metabolic activation, a small but
non-significant increase in the length of DNA migration was ob-
served in cells exposed to 10 mM TCE after 6 hours of exposure.
DNA migration profiles for CHO cells after treatment with the 10
mM dose over time with S9 are shown in Figure 19. Based on these
results, a dose response experiment was conducted in the presence
of S9 over a broader range of concentration (0.1-10 mM) at a
single exposure time of 4 hours. Increasing the concentration of
TCE likewise results in a progressive increase in the DNA migra-
tion length (Figure 20). A significant increase in the length of
DNA migration over controls was observed in cells exposed to TCE
at doses as low as 0.5 mM. The DNA migration histograms for the
six doses are shown in Figure 21.
Dimethylbenz(a)anthracene; Exposure to 100, 10 and 0 uM of
DMBA induced a time-dependent increase in DNA damage in the pre-
sence of metabolic activation only (Figure 22). After 2 hours of
exposure, the level of DNA damage was significantly increased at
the 100 uM dose relative to DMSO control levels. An analysis of
the distribution of migration patterns among individual cells in-
dicated a heterogenous response among cells in the top dose (Fig-
ure 23). No significant increase in the length of DNA migration
was observed in cells exposed in the absence of metabolic activa-
tion even at the top dose and longest exposure time. A dose re-
sponse experiment was conducted in the presence of S9 over a
broader range of concentrations (1-lOOuM) at a single 4 hour ex-
posure time. Increasing the concentration of DMBA resulted in a
progressive increase in the DNA migration length (Figure 24). A
significant increase in the length of DNA migration was observed
27
-------
10 mM ACRYLAMIDE (+S9)
PERCENTAGE OF CELLS
ro^cn N>-J^O> ho-^c
3000000 OoOOC
' . 1 ' ........ ^ v-' *-
I _ 30 min
I II-
D 10 20 30
I,
3 10 20 30
ll.l.llll-
^0 10 20 30
ou -
40-
20-
n.
.lllll
40 5
1 HR.
x = 7.7
40 5
2 HRS.
x = 14.6
40 5(
4 HRS.
x = 23.7
1 1
0
10 20 30 40
DMA MIGRATION (microns)
50
Figure 13. Sample Time Dependent Distribution of DNA Migration
Patterns for CHO Cells Exposed to 10 mM Acrylamide in the Presence
of Rat Liver S9 Mix. Data are for 50 cells per sample.
The width of each bar represents 2 micron intervals. X = group
mean DNA migration.
28
-------
LJ
O
LL.
O
LJ
O
LJ
O
£E
LJ
Q_
60-
40-
20--
60
40-
20-
60
0
40-
20-
60
40-
20--
0
10 mM ACRYLAMIDE (-S9)
10
20
30
III..!-...
10
20
30
-I
10
20
30
2 MRS
x = 7.8
40
50
4 MRS.
x = 13.0
40
50
6 MRS.
x = 15.8
40
50
8 MRS.
x = 19.1
10 20 30 40
DNA MIGRATION (microns)
50
Figure 14. Sample Time Dependent Distribution of DNA Migration
Patterns for CHO Cells Exposed to 10 mM Acrylamide in the Absence
of Rat Liver S9 Mix. Data are for 50 cells per sample.
The width of each bar represents 2 micron intervals. X = group
mean DNA migration.
29
-------
ACRYLAMIDE IN CHO CELLS
8 hr. exposure (-S9)
4 hr. exposure (+S9)
I
LJ
2
g
o
10
Figure 15. Mean DNA Migration for CHO Cells Exposed to
Acrylamide as a Function of Dose in the Presence (4 Hour
Treatment) or Absence (8 Hour Treatment) of Rat Liver S9 Mix.
Each point represents the mean and standard error of the mean for
duplicatie cultures. For each culture 25 cells were scored.
30
-------
ACRYLAMIDE (+59) 4 HOUR EXPOSURE
u
u
L.
O
LJ
LU
U
c:
u
Q.
40--
20--
40-
20-
40-
20-
40-
20-
40-
20-
40-
20--
JL
Jill
I
PBS
x - £.3
0.1 mM
x = 8.6
1.0 mM
x - 11.7
5.0 mM
x = 11.0
5.0 mM
x = 19.1
10 mM
x = 26.3
10 20 30 40
DMA MIGRATION (microns)
50
Figure 16. Dose Dependent Distribution of DNA Migration Lengths
for CHO Cells Exposed to Acrylamide for 4 Hours in the Presence of
Rat Liver S9 Mix. Data are for 50 cells per sample. The width of
each bar represents 2 micron intervals. X = group mean DNA
migration.
31
-------
ACRYLAMIDE (-S9) 8 Hour Exposure
(A
_J
U
U
Lu
O
U
0
p
LJ
o
tr
u
Q_
60
40-
20-
fl-
SC
40-
20-
0
60-
40-
20-
0-
60-
40-
20-
0-
60-
40-
20-
0-
60-
40-
20-
0-
C
1
ll.
Ill _
Hi.. .
-III........
' t ' i ' i '
Illi^i !!
> 10 20 30
PBS
0.1 mM
x = 9.0
0.5 mM
x - 10.5
1.0 mM
x = 12.1
5.0 mM
x = 16.6
i
10 mM
x = 17.9
,
40 5C
DNA MIGRATION (microns)
Figure 17. Dose Dependent Distribution of DNA Migration Lengths
for CHO Cells Exposed to Acrylamide for 8 hours in the Absence of
Rat Liver S9 Mix. Data are for 50 cells per sample. The width
of each bar represents 2 micron intervals. X = group mean DNA
migration.
32
-------
c
o
l_
y
'E
c:
o
<
Q
30-r
25;-
20-
is-
le-
s'-
0-
TRICHLCROETHYLENE IN CHO CELLS
WITH S9
OO 10 mM
- 1 mM
A---A DMSO
A
w
z
o
i
o
20
15-L
10-
0
WITHOUT S9
O-
A-
-O 10 mM
- 1 mM
A DMSO
8
EXPOSURE TIME (hours)
Figure 18. Mean DNA Migration for CHO Cells Exposed to
Trichloroethylene as a function of Sample Time in the Presence or
Absence of Rat Liver S9 Mix. Each point represents the mean and
standard error of the mean for duplicative cultures. For each
culture 25 cells were scored.
33
-------
UJ
o
u_
o
UJ
o
P
UJ
u
ce:
UJ
£L
10 mM TRICHLOROETHYLENE (+S9)
60
40--
20-
0
60-
40-
20-
60
40-
20-
0
60'
40-
20-
0
-T»*
20
30
I
_-,-_
10
20
30
lilli
10
20
30
30 min
x = 11.5
40
50
1 HR.
x = 10.7
40
50
2 MRS
x = 14.9
40
50
4 HRS
x = 22.6
10 20 30 40
DNA MIGRATION (microns)
50
Figure 19. Sample Time Dependent Distribution of DNA Migration
Patterns for CHO Cells Exposed to lOmM Trichloroethylene in the
Presence of Rat Liver S9 Mix. Data are for 50 cells per sample,
The width of each bar represents 2 micron intervals. X = group
mean DNA migration.
34
-------
TRICHLOROETHYLENE (+S9)
O
LJ
z
O
h;
O
4 hr. exposure
4 6
DOSE (mM)
8
10
Figure 20. Mean DNA Migration for CHO Cells Exposed to
Trichloroethylene as a Function of Dose in the Presence (4 Hour
Treatment) of Rat Liver S9 Mix. Each point represents the mean
and standard error of the mean for duplicate cultures. For each
culture 25 cells were scored.
35
-------
TRICHLORCETHYLENE (4S9) 4 HOUR EXPOSURE
CO
_i
u
u
L.
O
LJ
LJ
O
c:
LJ
D.
601
40 T
20 4-
DMSO
II,
8.6
U ' ! ' 1 1
60*
T I
40
20
0-
60-
40-
20-
0-
60-
40
20-
60-
40-
20-
A
60-
40-
20-
n.
1.
«K
'
-|l
1 1 - I 1
Jill ... .
1 I ' I ' 1
.III.. ..
|MH||||
0.1 mM
x = B.1
0.5 mM
x = 14.0
1 1 |ii
1.0 mM
x = 14.3
5.0 mM
x = 14.6
10 mM
x = 22.5
_
10 20 30 40
DNA MIGRATION (microns)
50
Figure 21. Dose Dependent Distribution of DNA Migration Lengths
for CHO Cells Exposed to Trichloroethylene for 8 Hours in the
Presence of Rat Liver 59 Mix. Data are for 50 cells per sample.
The width of each bar represents 2 micron intervals. X = group
mean DNA migration.
36
-------
C
o
u
2
g
c;
o
<
Q
DIMETHYLBENZANTHRACENE IN CHO CELLS
WITH S9
30
25-
15-
10
5
OO 100 uM
- 10 uM
A- A DMSO
A
0-
I
o
z.
o
c:
<
Q
20
15T
WITHOUT S9
OO 100 uM
- 10 uM
A- A DMSO
0-
EXPOSURE TIME (hours)
Figure 22. Mean DNA Migration for CHO Cells Exposed to
Dimethylbenzanthracene as a Function of Sample Time in the
Presence or Absence of Rat Liver S9 Mix. Each point represents
the mean and standard error of the mean for duplicate cultures.
For each culture 25 cells were scored.
37
-------
100 uM DMBA (-fS9)
u
u
u
1
u
CJ
tr
LJ
CL
O^J
40
20
n
u
rn
cu
40-
20-
0"
U i
(
40-
20-
t
60 C
DU
40-
20-
n.
u ~
0
30 MIN
i ll
D 10 20 30 40 5
1 HR.
x = 9.8
lln
D 10 20 30 40 5
2 MRS.
x = 13.3
..III .
) 10 20 30 40 5
4 MRS.
x = 20.0
10 20 30 40 5(
0
0
0
D
DMA MIGRATION (microns)
Figure 23. Sample Time Dependent Distribution of DNA Migration
Patterns for CHO Cells Exposed to 100 uM Dimethylbenzanthracene
in the Presence of Rat Liver S9 Mix. Data are for 50 cells per
sample. The width of each bar represents 2 micron intervals.
X = group mean DNA migration.
38
-------
DMBA (+S9)
^
D
~j-
O
2*
U
0
£
o:
o
uu
25-
20-
15-
10-
4 hr. exposure
T__ ~~*
/T
b*
0
20
40 60
DOSE (uM)
80
100
Figure 24. Mean DNA Migration for CHO Cells Exposed to
Dimethylbenzanthracene as a Function of Dose in the Presence (4
Hour Treatment) of Rat Liver S9 Mix. Each point represents the
mean and standard error of the mean for duplicate cultures. For
each culture 25 cells were scored.
39
-------
in cells exposed to DMBA beginning at the 10 mM dose. The
migration histograms for the six doses are shown in Figure 25.
In contrast to the more homogeneous DNA migration patterns
observed for cells exposed to ACR, exposure to TCE and DMBA
resulted in a more heterogenous intercellular response, with the
majority of the cells widely distributed in terms of migration
length. Several explanations are possible for this heterogeneity
in DNA damage: individual cells may vary in their permeability to
the S9-dependent active metabolite(s) of TCE and DMBA, in the
accessibility of DNA to the damaging species and/or in repair
capacity or in other mechanisms which either enhance or diminish
the effects of the compound. These results are consistent with
ACR and a metabolite ACR having genotoxic activity and with TCE
and DMBA requiring metabolic activation to reactive forms.
5.2.4 In Vitro rodent Hepatocvte Assay: Currently, one of the
primary in vitro methods for ascertaining the ability of
chemicals to induce DNA damage involves an evaluation of DNA
repair synthesis (so-called unscheduled DNA synthesis or UDS) in
rodent hepatocytes (Butterworth et al., 1987). This technique is
based on an autoradiographic determination of the incorporation
of tritiated thymidine into DNA repair sites. Rodent hepatocytes
are used because the target cells themselves are metabolically
competent, eliminating the need for an exogenous source of
metabolic activity. The primary rat hepatocyte UDS assay is
considered to be an excellent method for detecting the genotoxic
activity and potential carcinogenicity of chemicals (Probst et
al., 1980; Williams et al., 1982; Mitchell et al., 1983).
However, the very nature of the technique limits its detection
generally to those lesions requiring long patch repair, involves
the use of radioactive precursors to DNA and requires an extended
processing time. Furthermore, based on statistical grounds, the
assay is reported as insensitive to weak mutagens (Margolin and
Risko, 1988) . Based on these considerations, we have begun
studies to evaluate the applicability of the SCG assay to a
detection of DNA damaging agents using the in vitro rodent
hepatocyte cell system. The data presented here comes from an
initial experiment involving cyclophosphamide (CP), a well-known
alkylating agent requiring metabolic activation.
Mouse hepatocytes were freshly isolated for each test by a two
step in situ perfusion of the liver of B6C3F1 male mice (10 - 12
weeks old age, Taconic Farms, Germantown, NY). The procedure
used has been previously described for obtaining hepatocytes from
rats (Williams, 1977; Butterworth et al., 1987), as modified for
the smaller animals (McQueen et al., 1981; Maslansky and
Williams, 1982). The liver was initially perfused with 0.5 mM
EGTA in Ca2+, Mg2+ free Hanks balanced salt solution for 3
minutes at 6 ml/min. This was followed by collagenase (type IV,
40
-------
DIMETHYLBENZANTHRACENE ( + S9) 4 HOUR EXPOSURE
LJ
u
U_
O
LJ
O
Z
U
u
Cf
LU
CL
60 T
40-
20-
60-f
40
T
20 r
JL
DWSO
x - 8.6
1.0 uM
x « 8.1
60-
40-
20-
0-
60-
40-
20-
0-
60-
40-
20-
0
60-
40-
20-
nJ
1 . ' i i 1 1
III..
.III...
Illllll
-.III ...
5.0 uM
x - 9.9
10 uM
x = 12.8
50 uM
x = 14.4
100 uM
x = 17.0
10 20 30 40
DNA MIGRATION (microns)
50
Figure 25. Dose Dependent Distribution of DNA Migration Lengths
for CHO Cells Exposed to Dimethylbenzanthracene for 8 Hours in
the Presence of Rat Liver S9 Mix. Data are for 50 cells per
sample. The width of each bar represents 2 micron intervals.
X = group mean DNA migration.
41
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Sigma, 540 U/mg, Lot No. 78F-6814: 100 U/ml of Williams Medium E)
for 5 -7 minutes at 5 ml/min. The viability of the cells was
determined by trypan blue dye exclusion and only preparations
with a cell viability greater than 80% were used. Approximately
1.5 X 10 viable cells were plated in a tissue culture plate (6
wells/plate, 3.5 X 1.0 cm, Limbro; Flow Laboratories). The cells
were allowed to attach for 1.5 - 2 hr in 2 ml Williams Medium E
(WME, GIBCO) supplemented with 10% fetal calf serum (GIBCO) and 2
mM glutamine (GIBCO) at 37°C in a 95% air/5% C02 incubator. At
the end of this attachment interval, the cultures were washed
once with WME.
In the time course study, cultures were exposed to 25 or 250
ug/ml CP for 1 to 8 hr. The selection of these doses was based
on published UDS results and on initial toxicity studies
indicating that hepatocyte viability was not decreased below 80%.
For a dose response evaluation, cultures were exposed to 0.8,
2.5, 8.0, and 25 ug/ml CP for 6 hours. Control and treated cells
were washed once with PBS (pH 7.4) and harvested by gentle
scrapping with a polyethylene scrapper into cold PBS containing 4
mM EDTA. Viability of the cell suspension was determined by the
Trypan blue exclusion test and doses which resulted in more than
a 20% decrease in viability were not evaluated. The suspensions
were kept on ice until mixed with low melting agarose for the SCG
technique.
Identification of Parenchymal versus Nonparenchymal Cells; In
pilot experiments to evaluate the applicability of the SCG
technique to the in vitro hepatocyte cell culture system, cells
with a wide range of diameters were noted. A determination of
cell diameters (i.e., the diameter of the nuclear mass), under
conditions where DNA unwinding but not DNA migration was
permitted, indicated that cells with two discrete size
distributions were present. In general, after the unwinding
period, approximately 94% of the cells had a nuclear diameter of
6 to 10 um, while the remaining 10% had a diameter of 3 to 5 urn.
About a third of the larger sized nuclei appeared as doublets,
indicating that they had originated from binucleate cells. Based
on their size and morphology, nuclei with a diameter above 6 um
were classified as parenchymal cells while nuclei smaller than 5
um were classified as nonparenchymal cells.
Time Course Study: Beginning at the two hour sample time, the
incubation of mouse hepatocytes with CP at 25 and 250 ug/ml
resulted in a significant increase in the length of DNA migration
in parenchymal cells (Figure 26). After 4 hours of exposure, the
level of DNA damage was not significantly different between the
two doses and appeared to have saturated. An analysis of the
distribution of migration patterns among individual parenchymal
cells indicated an increasingly homogeneous response with
increasing sample time (Figure 27). Viability in this experiment
remained above 90%.
42
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V)
O
o
30
25
20
15
10
5--
0.0
OO control
- 25 ug/ml T
A- A 250 ug/ml .J.T
A
-o
2.0 4.0 6.0
EXPOSURE DURATION (hr)
8.0
Figure 26. Time Course for the Induction of DNA Damage in
Primary Mouse Parenchymal Cells by Cyclophosphamide. Data are
presented as the mean and standard error of the mean for
triplicate cultures. 25 cells were scored in each culture.
43
-------
_
u
o
L_
O
U
O
LJ
U
o:
LU
D.
CZH control
25 ug/ml
250 ug/ml
10 20 30 40
DNA MIGRATION (microns)
Figure 27. Distribution of DNA Migration Lengths among
Individual Primary Mouse Parenchymal Cells Exposed to Cyclophos-
phamide as a Function of Sample time. Data for each sample based
on 75 cells. The width of each bar represents 2 microns.
44
-------
Dose Response Study: Based on this initial kinetic study, a
dose-response experiment was conducted using a 6 hour exposure
period and CP at doses between 0.8 and 25 ug/ml. At the highest
dose tested, viability was not decreased below 93%. Under these
conditions, CP induced a progressive increase in DNA migration
length, indicating a dose-dependent generation of DNA strand
breaks (Figure 28). An analysis of the distribution of migration
patterns among individual cells indicated that the intercellular
distribution of DNA migration patterns was more homogenous with
increasing CP dose (Figure 29).
Subsequent to these initial in vitro mouse hepatocyte studies,
several additional chemicals, including 2-acetylaminofluorene, 4-
acetylaminfluorene, benz(a)pyrene and mitomycin C, have been
examined for their ability to induce DNA damage in mouse and/or
rat liver cells. Data on 2-acetylaminofluorene and 4
acetylaminofluorene for both mouse and rat hepatocytes are
presented in Figure 30.
One of the more interesting aspects of this research was the
observation that parenchymal cells, the hepatocytes responsible
for the metabolic activation of procarcinogens to carcinogens,
could be readily distinguished on the basis of size from
nonparenchymal support cells. This suggested that, by analyzing
the induction of DNA damage in both cell types concurrently,
direct-acting genotoxic chemicals (i.e., those capable of
inducing damage without metabolic activation) could be
discriminated from chemicals requiring metabolic activation. For
procarcinogens, it would be anticipated that DNA damage would
preferentially occur in parenchymal cells, with DNA damage in
nonparenchymal cells occurring as a result of the release of
reactive intermediates into the culture media. For direct-acting
chemicals, the level of damage in both cell types should be
approximately equal, assuming equal transport of the chemical
into the cell and equal repair capacity. Several experiments
were conducted to verify this hypothesis. For example, the
induction of DNA damage by dimethylnitrosamine (DMN) was highly
cell-type specific. A significant increase in DNA migration was
detected in parenchymal cells at lower doses and a earlier times
than in nonparenchymal cells (Figure 31, 32). Since this
chemical is not genotoxic unless metabolically activated, the
data support the initial hypothesis. An experiment involving the
treatment of rat hepatocytes with ethylmethanesulphonate (EMS), a
direct-acting chemical, demonstrates equal levels of DNA
migration among the two cell types (Figure 33, 34).
While not exhaustive, these experiments strongly suggest the
potential wide-spread applicability of the SCG technique to the
in vitro rodent hepatocyte culture system. Not only due the data
demonstrate the sensitivity and reproducibility of the assay, but
indicate also the ability to distinguish between direct-acting
and metabolically-requiring genotoxic agents.
45
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IN VITRO MOUSE HEPATOCYTE ASSAY
CYCLOPHOSPHAMIDE
Q
0
0
10 15 20
DOSE (ug/ml)
25
30
Figure 28. DNA Migration in Parenchyma! Cells as a Function of
Cyclophosphamide Dose, Sampled after 6 Hours of Treatment. Data
are presented as the mean and standard error of the mean for
triplicate cultures. 25 cells were scored in each culture.
46
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10 20 30 40
DMA MIGRATION (microns)
50
Figure 29. Distribution of DNA Migration Lengths among Individual
Primary Mouse Parenchymal Cells Exposed to Cyclophosphamide for 6
Hours. Data for each sample based on 75 cells. The width of each
bar represents 2 microns.
47
-------
0.4 0.6 0.8
CONCENTRATION (mM)
1.0 1.2
2AAF
-D-
mouse
0.2 0.4 0.6 0.8 1.0
MOUSE CONCENTRATION (mM)
RAT CONCENTRATION (uM x 10)
1.2
Figure 30. Comparative Analysis of the Induction of DNA Damage in
Rat and Mouse Primary Parenchymal Cells by 2-Acetylaminofluorene
and 4-Acetylaminofluorene. Data are presented as the mean and
standard error of the mean for triplicate cultures. 25 cells were
scored in each culture.
48
-------
microns
<
tz
o
2
<
Q
DIMETHYLNITROSAMINE
40
30--
o 20--
10 +
T
?
o-
A-
-O o
- 30 uM
A 1 mM
-_ I
-
4 6
TIME (hours)
8
OO parenchymal
nonparenchymal
10 15 20
DOSE (uM)
25
Figure 31. DNA Migration in Mouse Primary Parenchymal and
Nonparenchymal Cells Exposed to Dimethylnitrosamine for 6 Hours.
Data are presented as the mean and standard error of the mean for
triplicate cultures. 25 cells were scored in each culture.
49
-------
fl PARENCHYMAL
NONPARENCHYMAL
60-
40-
_l
LU 0-
^60-
Lu
O '
LJ40-
O
b;20-
LJ
QX 0
LiJ 60
Q_
40
20
n
_i
0.0 uM "
r-|
3.0 uM.
n
-yl nftln
Ib-^rdL Minn
' 30 uM "
.
_ i M ut 1 Jl UWUl
0
10 20 30 40
DMA MIGRATION (microns)
50
Figure 32. Distribution of DNA Migration Lengths among Individual
Mouse Primary Parenchymal Cells Exposed to Dimethylnitrosamine for
6 Hours. Data for each sample based on 75 cells. The width of
each bar represents 2 microns.
50
-------
ETHYLMETHANESULPHONATE-INDUCED DNA DAMAGE
60
LJ
4--
* PARENCHYMAL
NONPARENCHYMAL
z
g
en
e
u
o.
en
a
6--
0
9
01
I 6 +
3--
0.00
0.25 0.50 0.75
CONCENTRATION (uM)
1.00
Figure 33. Ethylmethanesulphonate-Induced DNA Damage in Mouse
Primary Parenchymal and Nonparenchymal Cells Exposed for 6 Hours.
The presence of damage is measured by changes in DNA migration,
shape (perimeter squared/area), and dispersion (variance/mean).
Data on image diameter are presented to indicate the difference in
size between parenchymal and nonparenchymal cells. Data are
presented as the mean and standard error of the mean for triplicate
cultures. 25 cells were scored in each culture.
51
-------
LJ
O
u_
o
LJ
O
Ld
O
o:
LJ
CL
60--
40--
20
0
60
40-
20-
60-
40-
20-
60-
40-
20-
0
PARENCHYMAL
NONPARENCHYMAL
0.0 uM
0.1 uM
0.3 uM
1.0 uM
10 20 30 40
DNA MIGRATION (microns)
50
Figure 34. Distribution of DNA Migration Lengths among
Individual Mouse Primary Parenchymal Cells Exposed to
Ethylmethanesulphonate for 6 Hours. Data for each sample based
on 75 cells. The width of each bar represents 2 microns.
52
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6.0 IN VIVO STUDIES
Initially, a series of experiments were conducted to evaluate
the ability of ACR, DMBA and trichloroethylene (TCE) to induce
DNA damage in mice in four different tissues (e.g., brain, liver,
spleen and blood). In these experiments, male B6C3F1 mice were
exposed acutely by gavage to 100 mg/kg ACR in PBS, 100 mg/kg DMBA
in corn oil or to 1000 mg/kg TCE in corn oil. Groups of mice (4
per group) were killed by etherization at 4 and 24 hours after
treatment and peripheral blood, liver, spleen and brain samples
obtained from each mouse. These samples were processed with
(brain, liver) or without (blood, spleen) collagenase treatment
and evaluated for DNA damage using the SCG technique. The doses
and the sample times used in this study are based on previous
experience with these chemicals in this and other laboratories.
Mortality among the treated animals was not anticipated under
these experimental conditions and did not occur. Tissue
selection was based on technical ease of processing (blood,
spleen), on metabolic (liver) and neurological (brain) importance
and on the expectations that DNA damage in these tissue would
accumulate under multiple exposure conditions. Because of fairly
extensive tissue and experiment to experiment differences in
control migration patterns, the percentage of increase is
discussed rather than the actual raw data.
Four hours after treatment with ACR, cells from all four
organs/tissues exhibited a significant increase in DNA migration,
with liver cells exhibiting the greatest percentage increase in
response. By 24 hours after treatment, only blood leukocytes
(PBL) still appeared to exhibit an increased level of damage.
None of the cells obtained from organs/tissues sampled four hours
after treatment with DMBA (100 mg/kg) 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 exhibiting the greatest response. Four hours
after treatment with TCE (1000 mg/kg), cells from all four
organs/ tissues exhibited a significant increase in DNA migra-
tion, with spleen cells exhibiting the greatest response. By 24
hours after treatment, all tissues exhibited DNA migration
patterns indistinguishable from that observed for control mice.
These pilot studies demonstrated, not surprisingly, that the
level of DNA damage induced by these chemicals was agent, organ
and sample time dependent. They also demonstrate the utility of
the approach and the feasibility of detecting DNA damage in
individual cells isolated from different organs of mice. How-
ever, the range of variation among cell samples from control mice
(approximately 2 to 3 fold) was extremely disappointing and
suggested the need to characterize the processing of in vivo
tissues in greater depth. Based on conversations with other
scientists while at the International Environmental Mutagen
Society meeting in Cleveland, the first technical factor
53
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evaluated was the effect of the collagenase treatment used to
isolate single cells from the brain and liver. The second factor
was the presence of calcium chelators in the mincing and lysing
solutions. Finally, the third factor considered was the influ-
ence of blood contamination on the resulting DNA migration
patterns.
6.1 Collagenase Treatment
In this experiment, brain and liver tissue were removed from a
single mouse and minced, either in the presence or absence of
collagenase prior to SCG analysis. The resulting data indicated
that collagenase treatment resulted in a significant increase in
DNA migration (approximately 50% greater) and that mincing alone
was sufficient for ensuring an adequate sample of single cells
from every tissue tested (including spleen, liver, brain,
testis).
6.2 Calcium Chelators
In the original protocol developed by Singh and his colleagues
(Singh et al., 1988), calcium chelators such as EDTA or EGTA were
omitted from the media solutions because it did not appear as if
DNA damage resulting from the presence of nucleases occurred at
any time during the processing of human leukocytes. However,
liberation of such nucleases is more likely during the mincing
and handling of in vivo tissues, resulting in increased levels of
DNA damage. In several experiments, this possibility was
evaluated by adding various concentration of EDTA or EGTA, two
calcium chelators, to the mincing solutions used during the
processing of liver tissue. In Figure 35, group mean migration
lengths from a representative experiment involving EDTA are
presented. These data demonstrate the importance of the addition
of a calcium chelator to the mincing solutions.
The adverse impact of blood in the lysing solution and its
correction by the addition of DMSO has already been discussed.
Based on the results of these experiments, the protocol has been
modified by the addition of 20 mM EDTA (or EGTA) to the mincing
solutions, by the addition of 10% DMSO to the lysing solution,
and by the omission of collagenase from the mincing solution.
6.3 A Kinetic Study of Acrylamide-Induced Organ Specific Levels
of Damage
After the several experiments discussed above to improve our
in vivo tissue sampling procedures, we decided to return to an
evaluation of organ-specific levels of DNA damage induced in vivo
in mice by an environmentally important pollutant with genotoxic
activity. The chemical we selected to evaluate first was
acrylamide (ACR). Studies with TCE were started but not
54
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en
c
o
u
X
I
o
2
LJ
IN VIVO TISSUE ISOLATION
LIVER CELLS
CONTROL
5.0 10.0
DOSE (mM)
20.0
Figure 35. Effect of EDTA on the DNA Migration for Liver Cells
Processed from a Control Mouse. Bars indicate group means and
the standard error of the mean for 25 cells per sample.
55
-------
completed due to technical difficulties already discussed (e.g.,
problems with slide batches, coplin jars, electrophoresis buffer
pH, etc.) and a decision to focus additional resources on the
collection of human data and on animals inhabiting hazardous
waste sites.
Briefly, male B6C3F1 mice (10 - 13 weeks of age, 25 to 32 gm
in body weight, 4 mice per group) were gavaged with 10 and 100
mg/kg ACR in PBS. At 3, 6, 12, 24 and 48 hours after treatment,
groups of mice were killed by C02 asphyxiation, and samples of
liver, spleen and testis were removed from each mouse and stored
on ice in Hank's buffer containing 20 mM EDTA. Blood samples
were collected by adding 5 uL to RPMI-1640. The samples of
liver, spleen and testis were minced, while the blood sample was
centrifuged to recover the leukocytes. An aliquot of cells were
mixed with LMA and placed on microscope slides as described
earlier. The cells were electrophoresed for 20 minutes, stained
with ethidium bromide and 25 cells per cell type per sample were
evaluated for DNA migration using the Quantimet 520 image
analyzer. Total leukocytes were scored from blood, splenocytes
from spleen, parenchymal and nonparenchymal cells from liver,
while the analysis of damage in testis was limited to diploid
cells. Cell viability measurements, as described earlier, were
also conducted on each tissue and viability was routinely above
90%.
Based on an analysis of mean DNA migration patterns, treatment
with ACR induced a significant increase in DNA damage in all
organs at all sample times (Figure 36), the magnitude of which
was dose (100 « 10 mg/kg), tissue blood leukocytes > spleen =
liver > testis) and sample time (maximum response at 12 hours)
dependent. In liver, both parenchymal and nonparenchymal cells
exhibited a similar increase in DNA damage. The corresponding
distributional data are presented, by tissue, in Figures 37-41.
This analysis indicates that even at 10 mg/kg, a small but
significant subpopulation of cells in each tissue expressed some
damage. The analysis also indicates that the extent of DNA
migration shifted in almost all cells of the liver, spleen and
testis toward control levels by 48 hours. At this delayed sample
time, blood leukocytes, however, exhibited greater intercellular
heterogeneity in DNA migration patterns. This suggests that the
leukocytes in the blood are more variable in repair than cells in
the other tissues. These data also demonstrate, under the
modified sampling protocol, reproducible control data for each
tissue between sample times, and reproducible data among animals
at a specific dose of ACR.
7.0 HUMAN STUDIES
Because one of the ultimate goals of this project is to be
able to evaluate and compare, where feasible, data obtained on
both animal and human populations, several pilot studies were
56
-------
30
15--
C 15
O
s§ °
z:
o 15
h-
co o
0
T
A-
BLOOD
A
A
SPLEEN
-A-
I 1 1 1
LIVER-PARENCHYMAL
LIVER-NONPARENCHYMAL
TESTIS
o
10 20 30 40
TIME (hours)
50
Figure 36. Evaluation of Acrylamide-Induced DNA Damage, as a
Function of Sample Time, in Various Tissue of Male B6C3F1 Mice.
Data are presented as group mean and the standard error of the mean
among 4 animals. 25 cells were scored per tissue. Treatment was
by gavage. Open triangles = 100 mg/kg; solid circles = 10 mg/kg;
open circles = phosphate buffered saline only.
57
-------
BLOOD
80
40
0
40
en
UJ Q
0 U
o
0 40'
PERCENT
o
40-
0
40-
n
n 0 mg/kfl BB 10 mg/kc M 100 ma/*
f
1
n
[
r
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n 3 Hrs
1
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6 Hrs
L.
12 Hrs
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. 24 Hrs
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J-,n ,
[I
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rt
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n 3 Hrs
h
n 6 Hrs
prli.llinipl^l-r
. 12 Hrs
jlllll-r
24 Hrs
1 r
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J..H. ,
10 20 30 40 0
DNA MIGRATION (microns)
10 20 30 40
DNA MIGRATION (microns)
50
Figure 37. Sample Time Dependent Distribution of DNA Migration
Lengths for Mouse Blood Leukocytes Collected from Acrylamide-
Treated Male B6C3F1 Mice. Data based on 100 cells per sample.
The width of each bar represents 2 microns.
58
-------
80
40-
40-
LJ
o
40
O 0
cc.
u
Q_
40|
40-
0
LIVER-PARENCHYMAL
0 ms/kg BB 10 ma/kg
3Hra
6Hrs
12Hre
Mrs
48 Mrs
0 10 20 30 40 0
DNA MIGRATION (microns)
6 Mrs
12 Mrs
24 Mrs
48 Hrs
10 20 30 40 50
DNA MIGRATION (microns)
Figure 38. Sample Time Dependent Distribution of DNA Migration
Lengths for Mouse Liver Parenchymal Cells Collected from
Acrylamide-Treated Male B6C3F1 Mice. Data based on 100 cells per
sample. The width of each bar represents 2 microns.
59
-------
LIVER-NONPARENCHYMAL
ou
40
40
(n
UJ n
0 U'
o
:ENTAGE
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mill.
6Hrs
^,lli|..,r
12Hrs
frH*"M-i--i i
24 Mrs
I,..
48 Mrs
I
yii
H11^
DMA MIGRATION (microns) DNA MIGRATION (microns)
Figure 39. Sample Time Dependent Distribution of DNA Migration
Lengths for Mouse Liver Nonparenchymal Cells Collected from
Acrylamide-Treated Male B6C3F1 Mice. Data based on 100 cells per
sample. The width of each bar presents 2 microns.
60
-------
90
45
0
45'
CO
LJ n
0 U
O
LJ 4 =
O 4i3
PERCENT
0
45'
0-
45-
n,
SPLEEN
O 0 mg/k; BB 10 mg/kfl § 100 mg/kq
1
fl
fl
[I
. 3 Hrs
I
i
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if
JL
r
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lllli.I... ..
6 Hrs
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24 Hrs
Lilm.
48 Hrs
1
DL,,.
10 20 30 40 0
DNA MIGRATION (microns)
10 20 30 40
DNA MIGRATION (microns)
50
Figure 40. Sample Time Dependent Distribution of DNA Migration
Lengths for Mouse Spleen Cells Collected from Acrylamide-Treated
Male B6C3F1 Mice. Data based on 100 cells per sample. The width
of each bar represents 2 microns.
61
-------
60
30
0
30
to
o
L.
O
ft 30
Ld
O 0
0-
LJ
CL
30
0
30'
0
TESTIS-DIPLOID
C3 Omg/kg SB 10 mfl/kg
100 mg/Rf
3 Hrs
6 Mrs
12Hrs
.«.
24Hrs
48Hrs
I,
i
L,
6 Hrs
12 Hrs
24 Hrs
48 Hrs
0 10 20 30 40
DNA MIGRATION (microns)
0 10 20 30 40
DNA MIGRATION (microns)
50
Figure 41. Sample Time Dependent Distribution of DNA Migration
Lengths for Mouse Testis Diploid Cells Collected from Acrylamide-
Treated Male B6C3F1 Mice. Data based on 100 cells per sample. The
width of each bar represents 2 microns.
62
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conducted and/or are in progress to examine the utility of the
SCG assay in human biomarker studies.
7.1 5K Race Study
In a very early study, peripheral blood was sampled from three
individuals participating in a 5K road race. While this study
may be perceived as being unusual, it was felt that obtaining
samples under these conditions would demonstrate the feasibility
of conducting similar studies outside the laboratory. Three
runners were involved and samples were obtained by finger prick
prior to the start of the race (within 5 to 15 minutes), within 5
minutes after completion of the course, and at one-half and one
hour later. The samples were kept cold, transported back to the
laboratory approximately 2 hours after the initial sample and
processed in the SCG assay. Two of the runners (runners 1 and 3)
exhibited no increase in DNA migration immediately after the
completion of the race, while one runner (runner 2) exhibited a
large increase in DNA migration. While conducted under less than
rigorous conditions and before the affect of various
subpopulations of leukocytes in the peripheral blood on DNA
migration patterns was fully appreciated, the study does indicate
the feasibility of utilizing this technique for conducting human
studies outside the laboratory.
7.2 Smokers vs Nonsmokers
In another, early study, peripheral blood was obtained from
three heavy smokers and three, age and sex-matched control
nonsmokers. A blood sample, obtained by finger prick, was
obtained early in the morning after each smoker had completed one
cigarette. After being processed in the SCG assay, with samples
being electrophoresed for either 20 or 30 minutes, DNA migration
in 50 cells for each individual was determined. At 20 minutes of
electrophoresis, the extent of DNA migration was not different
between the smokers and nonsmokers (Figure 42). At 30 minutes of
electrophoresis, the DNA migration patterns among the smokers was
less than that observed for the nonsmokers (Figure 42). This was
due to the presence of a larger population of leukocytes among
the blood from the nonsmokers with extended DNA migration
patterns. Several hypothesis for this surprising difference were
considered. First, we considered that the differences was due to
increased levels of DNA crosslinking among the cells from the
smokers. Based on hypothetical grounds, increased crosslinking
would result in decreased migration. Second, we considered the
possibility that the difference in migration may reflect
differences in lymphocyte population dynamics/turnover between
the two groups. Finally, it was suggested that smokers may have
induced levels of DNA repair enzymes; thus lower levels of
background damage.
63
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to
c
o
CJ
1,
z
o
tc
o:
o
z
Q
*JU ~
25-
20-
15-
10-
5-
n.
«
T
'^^^
1 1
r 0
; 8 o ° o ° "
1 1 1 1 1 1
1 2 3
SMOKERS
1 2 3
NONSMOKERS
Figure 42. DNA Migration Lengths for Blood Leukocytes Sampled from
Three Smokers and Three Nonsmokers (Experiment 1) . Electrophoresis
was for either 20 (open symbols) or 30 (closed symbols) minutes.
Each symbol represents the mean and standard error of the mean for
50 cells.
64
-------
Based on observations made on blood samples stored in the
laboratory for several days, a second smokers study was
subsequently conducted in which DNA migration patterns were
compared for fresh blood or for blood stored for 48 hours. In
both cases, the DNA was electrophoresed for 20 or 40 minutes.
The results of this experiment are presented in Figure 43.
Again, no difference in migration was detected in fresh blood
electrophoresed for 20 min. Again, except for one smoker with
elevated migration lengths, the DNA from the nonsmokers appeared
to migrate more at 40 minutes of electrophoresis than the DNA
from the smokers. However, when the blood was stored for 48
hours and then run on the SCG assay, DNA migration patterns were
not different between smokers and nonsmokers at either electro-
phoresis duration. In fact, it appeared as if the cells with the
extended migration patterns had disappeared during this
incubation period from each individual's blood (Figure 44). This
resulted in a population of cells with almost a complete lack of
DNA migration. We subsequently determined, using hydrogen
peroxide treated cells, that damage, in and of itself, did not
lead to a disappearance of damaged cells from whole blood over
this period of time. Rather, we speculate, that a subpopulation
of cells, probably neutrophils, are either depressed in number or
in capability, in the blood from smokers. Experiments to
determine if this hypothesis is correct are planned.
7.3 Duke Cancer Study
In this study we attempt to ascertain the utility of the (SCG
assay in evaluating DNA damage in peripheral blood leukocytes
from individuals being treated with high-dose alkylating agents.
Patients studied were under the care of the Bone Marrow Trans-
plant Program at Duke University Medical Center. Twelve patients
with metastatic breast tumors were sampled before, during and
after the intravenous (IV) administration of antineoplastic
alkylating agents. The patients received indwelling, triple
lumen, right atrial catheters for venous access prior to
treatment. The transplant preparative regimen was as follows:
cyclophosphamide (CTX) was administered as a 1 h infusion on days
-6,-5,-4 at a dose of 1,875 mg/m/d; cisplatin (cis-DDP) (165
mg/m ) was given with hydration by continuous infusion over 72 h
from day -6 to -3; carmustine (600 mg/m) was infused on day -3
at a rate of 5 mg/m /min. On day 1 (3 days following completion
of chemotherapy) autologous bone marrow, harvested and cryo-
preserved prior to drug treatments, was thawed and rapidly
infused. Blood specimens for the SCG study were taken as
follows: on day -6, before drug treatment; on day -4, following 2
days administration of CTX and cis-DDP and upon hematopoietic
recovery (when peripheral blood white cell counts increase to at
least 1,000/ m.
PBLs were isolated from heparinized blood using Lymphocyte
Separation Medium (LSM) (Organon Teknika-Cappel, Durham, NC).
65
-------
n
c
8
.0
j,
2
O
i
o
^
2
Q
O 20 MIN ELECTRO. 40 MIN ELECTRO.
3D
25-
20-
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n.
A. FRESH BLOOD
-
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-
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o ° o o o
1 1 1 1 1 !
1 2 3
SMOKERS
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XCON
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n.
B. STORED BLOOD
T
* ? 9
o o o o o o
1 1 1 1 1 1
1 2 3
SMOKERS
1 2 3
NONSMOKERS
Figure 43. DNA Migration Lengths for Blood Leukocytes Sampled from
Three Smokers and Three Nonsmokers (Experiment 2) . Electrophoresis
was for either 20 or 40 minutes. Electrophoresis was for either 20
(open symbols) or 40 (closed symbols) minutes. Each symbol
represents the mean and standard error of the mean for 50 cells.
66
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20 MINUTE ELECTROPHORESIS
40 MINUTE ELECTROPHORESIS
100
CO
UJ
75
50-
CJ 25 f
O
Ld
O
75
O
cr
LJ
D. 50
25-
-f-
FB
i i smokers
H nonsmokern
FB
J
SB
10 20 30 40
DNA MIGRATION (microns)
10 20 30 40
DNA MIGRATION (microns)
50
Figure 44. Distribution of DNA Migration Lengths for Blood
Leukocytes Sampled from Three Smokers and Three Nonsmokers
(Experiment 2). Data based on 50 cells per sample. The
width of each bar represents 2 microns. FB = fresh blood;
SB = stored blood.
67
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Briefly, 6 - 7 ml blood were carefully layered over 4 ml LSM and
centrifuged at 400 x g for 20 minutes. The buffy layer was
removed, and the viable lymphocytes and monocytes counted using
1.0% trypan blue (Sigma). The plasma was saved for use in
further cell handling. The cells were washed with TC-199 medium
(Gibco) by centrifugation at 400 x g for 10 minutes and
aspiration of the supernatant. Cells to be assayed fresh were
resuspended at approximately 10 /ml in medium with 20% plasma.
Media TC-199 and plasma, at final proportions of 70% and 20%
respectively, were added to resuspend the cell pellet. DMSO
(Sigma) was then added at 10% (v/v) and the cell suspensions were
transferred to plastic freezing vials (NUNC) in 1 ml aliquots
containing approximately 10 cells each. Vials were placed
directly into a -70°C freezer. As needed, vials were retrieved
and placed in a 37°C water bath and shaken gently until melting
of the last ice crystal.
For SCG analysis, 5-10 ul samples were mixed with low melting
agarose and processed as described (electrophoresis was for 20
minutes). For each sample, 25 cells were scored on each of two
slides (50 cells total) for DNA migration. The mean DNA
migration length for each sample analyzed to date are presented
in Figure 45, with the corresponding histogram data presented in
Figure 46 and 47. Clearly, there are differences among the
individuals in regard to either the level of DNA damage present
in the admission sample, the sample obtained after two days of
chemotherapy, and in the sample obtained after hematopoietic
recovery. The significance of these differences in terms of
various pretreatment regimens, the chemotherapy outcome, and or
various in vitro bone marrow treatments requires additional
information, both about the patients and about the relative
ability of the various chemotherapy drugs to induce DNA damage,
as measured by the SCG assay. It is unfortunate that samples are
not available after the last day of treatment. The lack of this
sample makes any correlation with therapy outcome more difficult
to interpret. However, the data collected demonstrate the
potential utility of the SCG assay in human studies. Additional
samples, including both fresh and frozen blood samples from the
same patient continue to be analyzed for DNA damage.
7.4 Human Longitudinal Study
Very early in the course of this project we decided to conduct
a longitudinal study to evaluate the extent of interindividual
variability in DNA migration in human leukocytes. While we
recognized that such a study would be labor-intensive, we
believed that the resulting data would be important in any human
monitoring study. A blood sample was obtained by fingerprick
from a group of volunteers consisting of three males and three
females. One male smoker and one female smoker was included in
the group. Blood samples were obtained generally each week (in
some cases every other week) over a period of approximately 6
68
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DUKE CANCER STUDY
25
CO
c
o
L_
o
E
o
o
2
<
Q
20--
15--
10--
0
pre
EZ3 dy 2
I post
02 03 04 05 06
BREAST CANCER PATIENT
Figure 45. DNA Migration Lengths for Blood Leukocytes Sampled
from 6 Duke Hospital Chemotherapy Patients. Samples were
obtained upon admission (pre), after 2 days of a 3-day
treatment protocol (dy 2), or after hematopoietic recovery
(post). Electrophoresis was for 20 minutes. Each bar
indicates the mean and standard error of the mean for 50 cells
per sample.
69
-------
100
PATENT 01
H 1 1-
PATIENT 02
i ii t
PATIENT 03
75
50
25
C/)
d o
UJ
o
U_ 75
O
UJ
O
50
I"
O
e °
CL
75
50
25
0
10 20 30 40 0 10 20 30 40 0 10 20 30 40 50
DNA MIGRATION (microns) DMA MIGRATION (micron*) DMA MIGRATION (microns)
Figure 46. Distribution of DNA Migration Lengths for Blood
Leukocytes Sampled from First 3 Duke Hospital Chemotherapy
Patients. Samples were obtained upon admission (A), after
2 days of a 3-day treatment protocol (B), or after hemato-
poietic recovery (C). Electrophoresis was for 20 minutes.
Data based on 50 cells scored per sample.
70
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PATIENT 04
-H 1 1 1 »
PATIENT 05
PATIENT 06
0 10 20 30 40 0
DMA MIGRATION (microns)
10 20 30 40
DMA MIGRATION (microns)
0 10 20 30 40 50
DNA MIGRATION (microns)
Figure 47. Distribution of DNA Migration Lengths for Blood
Leukocytes Sampled from Second 3 Duke Hospital Chemotherapy
Patients. Samples were obtained upon admission (A), after
2 days of a 3-day treatment protocol (B)/ or after hemato-
poietic recovery (C). Electrophoresis was for 20 minutes.
Data based on 50 cells scored per sample.
71
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months. Duplicate slides were prepared, with one slide being
electrophoresed for 20 minutes and the other for 40 minutes.
Fifty cells were scored per slide. Many of the technical
problems already discussed were detected in this on-going study,
with the loss of a significant number of data points.
Furthermore, when the results of the smoker and blood storage
experiments indicated that significant variability in DNA
migration in fresh whole blood samples could be largely due to
leukocyte subpopulation shifts, we decided to stop this study and
discard the data. We still recognize the need of such a study,
but several kinds of experiments must be conducted before the
results could be interpreted correctly.
8.0 HAZARDOUS WASTE SITE STUDIES
The ultimate goal of this research project was to evaluate the
potential of the SCG assay for detecting DNA damage in free-
living rodents inhabitating a hazardous waste site. It was
hypothesized that such animals would express increased levels of
DNA damage and that information on the organ-distribution,
persistence and magnitude of such damage could be used in
evaluating the potential for ill-health effects in near-by human
communities. Clearly, at the present time, while the
extrapolation of such data to human health effects is
speculative, information on the presence or absence of increased
levels of DNA damage in such animal populations would be useful
in categorizing different sites and in indicating the need for
appropriate human studies. Techniques, such as the SCG assay, to
evaluate global levels of DNA damage, would play an important
role in such studies, especially if they could be applied to
similar tissues in human populations.
8.1 Study Area
Because of difficulties in obtaining permission to gain access
to EPA superfund sites, we decided to collect rodents at the
perimeter of a near-by site. This study was conducted at the
North Carolina State University Hazardous Waste (HW) Facility
(NCSU Lot #86, Farm Unit #1) located approximately 0.5 miles west
of the intersection of Blue Ridge Rd. and West Chase Dr.,
Raleigh, NC (Fig. 48). The site is situated in the Lower
Piedmont, a plateau of gently rolling hills (Hurst, 1963), with
an elevation of 450 ft. The study site is occupied by the
Appling soil association, which is characterized by moderately
sloping to strongly sloping acidic soils of Piedmont uplands.
The surface layer is composed of either gravelly sandy loam,
sandy loam, or fine sandy loam with a subsoil of firm clay loam
to clay (Cawthorn, 1970). The site has been classified as a
Superfund site, with the predominant pollutants including TCE,
chloroform, carbon tetrachloride, various pesticides, laboratory
solvents, and other chemicals. Although the selected site is
72
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1 mile
LEGEND
Hazardous Waste Facility
Trapping Area
Forested Area
Figure 48. Location of the North Carolina State University
EPA Superfund Site.
73
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quite small, the level of exposure anticipated to be quite
limited, and the numbers of animals few, we believed that this
study would be useful in determining the potential applicability
of the SCG assay to such field studies.
The HW site is a fenced 1.5 acre grassy area surrounded on
three sides by a pine-hardwood forest and on one side by a dirt
road (Fig. 49). Three control sites were used during this study,
and the reason for each will be addressed below. Control sites
#1 and #2 (Fig. 48) were located approximately 600 ft southwest
and southeast, respectively, of the HW site. Control site #3 was
located approximately 1400 ft southwest of the HW site. The HW
site and control site #2 were located within the same pine-
hardwood forest. Dominant vegetation included Pinus taeda
(loblolly pine), P. virqiniana (Virginia pine), Quercus alba
(white oak), Q. marilandica (blackjack oak), Liquidambar
stvraciflua (sweetgum), Acer rubrum (red maple), Cornus florida
(flowering dogwood), Rubus allegheniensis (blackberry), Rosa spp.
(roses), Lonicera iaponica (Japanese honeysuckle), and Rhus
toxicodendron (poison ivy). Pine needles covered the forest
floor, and Pueraria spp. (Kudzu-vine) formed the ground cover on
the first half of the HW study area along the northern fence
line. Control site #1 was a narrow band of P. taeda, Q. alba, L.
styraciflua, C. florida. R. allegheniensis, and Rosa spp.
surrounded by an unmowed field. Control site #3 was a pine-
hardwood forest surrounded by an unmowed field. Dominant
vegetation included P. taeda, £>. alba. Liriodendron tulipifera
(tulip poplar), L. styraciflua. A. rubrum. R. allegheniensis.
Rosa spp., L. japonica, R. toxicodendron. Smilax rotundifolia
(common greenbrier), and S. walteri (redberry greenbrier). Pine
needles covered the forest floor.
8.2 Live-Trapping Schedule
Ochrotomys nuttalli (Golden mouse) were live-trapped during 6
trapping sessions from 21 May, 1990, to 13 June, 1990. Thirty-
five Sherman traps each were placed along the outside perimeter
of 2 fenced sides of the HW site and on a control site (Fig. 49).
Traps were set and baited (rolled oats) in late afternoon and
checked at sunrise the following morning. Captured animals were
identified by species, and their sex and reproductive condition
were recorded. All 0. nuttalli, excluding lactating females, were
then returned to their respective traps while other species
caught (Blarina carolinensis (Southern short-tailed shrew),
Tamias striatus (Eastern chipmunk), and Sigmodon hispidus (hispid
cotton rat)) were released. Animals were transported to the lab
in traps.
The control site was moved after the first trapping session
because of a difference in species predominance: S. hispidus on
the control site and 0. nuttalli on the HW site. The second
control site capture results were similar to those of the HW Fig
74
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GATE G
FORMER DRUM
STORAGE AREA
Hozardo
hernical
rial
Ar
200 (--
^-FORMER CHEMICAL
STORAGE DUMPSTER AREA
NORTH
NCSU HAZARDOUS
CHEMICAL STORAGE
Figure 49. An expanded view of the North Carolina State
University Superfund Site. The isopleths indicate concen-
trationz (ug/1) of tetrachloroethene, chloroform and carbon
tetrachloride contamination in the ground water.
75
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site. After the second trapping session, traps on the HW site
were placed along a different fenced side in an area of
tetrachloroethene, chloroform, and carbon tetrachloride
concentration plumes (Fig. 49). After the third trapping
session, 15 traps were moved to the control site to increase
capture success. Twenty traps from the control site were stolen
after the fourth trapping session. As a result, a new control
site with 60 traps was established for the sixth trapping
session, and trapping on the HW site was terminated.
8.3 Tissue Collection
0. nuttalli were sacrificed via C02 asphyxiation, and their
body weight, body length, and tail length were recorded. Sub-
sequently, peripheral blood, liver, brain and bone marrow samples
were obtained from each animal and evaluated for DNA damage using
the SCG technique. In addition, bone marrow and peripheral blood
smears were prepared on each animal to evaluate micronuclei (MN)
frequencies in polychromatic erythrocytes (PCE) and the
percentage of PCE among total erythrocytes (a measure of the rate
of erythropoiesis).
8.4 Data Analysis
A total of 13 mice each were collected at the HW site and
among the various control sites. Population demographics were
comparable between the two populations of animals (Table 1). An
initial inspection of MN frequencies in PCE in peripheral blood
indicated that the efficiency of this rodent's spleen in removing
micronucleated PCE from the circulating blood eliminated the
possibility of evaluating PCE in these smears. In bone marrow
smears, 1000 PCE per animal were scored for the presence of MN
and the number of PCE among 200 erythrocytes was determined using
an acridine orange staining technique. No difference between the
HW and control groups in either the frequency of MN-PCE or the
percentage of PCE were observed (Table 2).
The selection of the various tissues for an evaluation of DNA
damage was based on the following premises. First, liver was
selected as the organ largely responsible for the metabolic
activation and detoxification of xenobiotics. Furthermore, since
the cells in this organ are largely terminally-differentiated it
might be anticipated that DNA damage would accumulate throughout
the life-time of the animal. Brain was selected because the
cells of this organ are also terminally differentiated, but lack
many of the protective mechanism inherent to liver cells. Thus,
DNA damage may be expected to accumulate at a greater rate.
Blood was selected based on our limited in vivo laboratory animal
experience indicating that blood leukocytes appear relatively
more deficient in repair than liver, spleen, or testis and
because blood is the tissue most readily obtainable from human
populations. Finally, bone marrow was selected because of the
76
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TABLE 1. POPULATION DEMOGRAPHICS OP Ochrotomvs nuttalli TRAPPED
ON THE CONTROL AND HW SITES OF THE NCSU HW
ANIMAL
NUMBER
01
02
03
04
05
06
07
08
09
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
TRAP
I.D.
H13
H26
H25
HO 2
H29
HO 6
C04
COS
C16
C31
H08
CIO
C13
H08
Hll
H17
C27
Hll
C36
C48
HO 3
H20
C52
C51
C50
C20
AGE
JUV
JUV
SA
SA
SA
AD
SA
JUV
SA
SA
JUV
JUV
SA
JUV
JUV
JUV
JUV
JUV
JUV
SA
JUV
AD
JUV
JUV
JUV
AD
SEX
M
P
M
M
F
M
M
F
F
F
M
M
F
M
M
F
F
F
M
F
F
M
M
M
M
M
REPROD. BODY TOTAL
COND. WT. (g) LENGTH(mm)
SCROTAL
NON-EST
NON-SCR
NON-SCR
ESTROUS
NON-SCR
SCROTAL
NON-EST
NON-EST
NON-EST
NON-SCR
NON-SCR
NON-EST
NON-SCR
NON-SCR
NON-EST
NON-EST
NON-EST
NON-SCR
NON-EST
NON-EST
SCROTAL
NON-SCR
NON-SCR
NON-SCR
NON-SCR
14.5
12.2
16.3
15.2
15.6
18.1
18.2
13.0
16.6
14.7
14.0
13.7
18.7
14.2
16.9
14.7
11.0
12.7
14.6
17.6
11.8
21.4
11.5
11.9
14.3
21.2
133.350
136.520
146.050
158.750
155.575
158.750
139.700
133.350
139.700
142.875
139.700
136.525
152.400
139.700
130.175
139.700
120.650
139.700
136.525
149.225
114.300
152.400
123.825
130.175
139.700
158.750
FACILITY.
TAIL
LENGTH (mm)
57.150
60.325
63.500
76.200
69.850
69.850
57.150
57.150
57.150
63.500
63.500
63.500
69.850
57.150
50.800
60.325
50.800
63.500
57.150
66.675
50.800
63.500
57.150
57.150
63.500
73.025
Codes: H = HW site
C = Control Site
AD = Adult
JUV = Juvenile
SA = Subadult
NON-EST = Non-estrous
NON-SCR = Non-scrotal
77
-------
Table 2. Frequencies of Micronucleated Polychromatic Erythrocytes and
Percentage of Polychromatic Erythrocytes in Bone Marrown of Anir
Collected at the North Carolina State Univeristy Hazardous Waste Site aTTcl
the Concurrent Control Sites.
Hazardous Waste
Concurrent Control
Animal
Number
Sex
MN-PCE/
1000 PCE
%PCE
Animal
Number
Sex
MN-PCE/
1000 PCE
%PCE
01
02
03
04
05
06
11
14
15
16
18
21
22
m
f
m
m
f
m
m
m
m
f
f
f
m
3
3
2
3
2
4
2
10
3
4
5
4
8
54.0
47.0
51.0
53.0
35.0
53.0
37.0
66.0
74.0
61.0
55.0
62.0
26.0
07
08
09
10
12
13
17
19
20
23
24
25
26
m
f
f
f
m
f
f
m
f
m
m
m
m
4
3
3
2
10
4
5
5
1
4
4
6
5
44.0
82.0
71.0
57.0
51.0
48.0
35.0
70.0
40.0
60.0
67.0
52.0
48.0
Group Mean 4.08 51.8
sem 0.66 3.7
4.31
0.60
55.8
3.8
P Value
MN-PCE = 0.6133
%PCE = 0.2382
P-value from one-tailed pairwise comparison based on liklihood ratio using
pooled data.
78
-------
opportunity to compare SCG data with another endpoint (i.e., MN)
and because DNA damage in this tissue would be more indicative of
a recent exposure to genotoxicants, either through increased body
burdens of reactive pollutants or through concurrent
environmental exposures.
The group mean DNA migration length and dispersion data are
presented in Figure 50. In addition, for a better inspection of
individual animal responses, the data for animals within each
group, by tissue, were ranked from low to high and plotted
individually (Figure 51, 52). Not surprisingly, the extent of
interanimal variability was much greater than that observed
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 the HW site, but only signifi-
cantly in brain (P = <0.05). However, a dispersion analysis
revealed that the bone marrow cells from the HW mice exhibited a
significantly increased dispersion coefficient over that
calculated for the control mice (P = <0.05). This increased
dispersion was due to small numbers of cells with extended DNA
migration patterns among a majority of cells with no or little
DNA migration. This result suggests the presence of low levels
of genotoxic species in this organ and/or differential
sensitivity among the various subpopulations of cells that
comprise this tissue. An example of the distributional data for
DNA migration obtained on a representative control and HW mouse
is presented in Figure 53. An analysis was conducted to evaluate
the degree of correlation among tissues within each animal in the
extent of DNA migration or dispersion. The only significant
correlation occurred for the dispersion of DNA migration in brain
and liver cells (Figure 54). The absence of a significant tissue
correlation suggests that the increases in DNA damage result from
independent events. Trap location, sex or age-dependent differ-
ences in response were not present, due most likely to the
limited number of animals collected.
The results of this small, pilot study indicate the potential
usefulness of the SCG technique in evaluating DNA damage in free-
living rodents. Without knowing the numerically prevalent mammal
on this site, enough individuals were collected that a reasonable
and informative analysis could be conducted. Clearly, consider-
able more work is needed to fully characterize the limitations
and capabilities of the SCG technique in this system. In any
study, the possible influences of animal health, food resources,
etc, on SCG data must be considered when evaluating the resulting
data. For example, increased levels of brain cell damage may
result from food practices or from parasitic infections. Also,
it would be extremely useful if body burdens of various
pollutants are evaluated concurrently in the same
animals.
79
-------
30
'£ 25
I
V
E
g 15
I
a 10
o 5'
0'
*
HW C HW C HW C HW C
BLOOD BONE MAR. BRAIN LIVER
12
o
V
o
c
0
O
CO
c:
u
a.
to
Q
8-
4--
o
in o
HW C
BLOOD
HW C
BONE MAR.
HW C
BRAIN
HW C
LIVER
Figure 50. Evaluation of DNA Damage in Various Tissue of 0..
nuttalli Collected at the North Carolina State University EPA
Superfund Sites as Compared to Animals Collected from Concurrent
Control Sites. Bar heights represent group means, error bars
indicate the standard error of the mean among animals (13 for each
group except for blood which involved 11 hazardous waste mice and
9 control mice). Each symbols represents an individual animal
response. 25 cells were scored per tissue.
80
-------
to
C
o
u.
o
'E
*>» s
-7
^
o
£
C£
O
^
<
z:
Q
ia -
ID-
S'
1 fi
12-
8-
4-
»()
20-
10-
*n
20-
10-
n.
1 i i 1 i ii i : 1 1 \ 1
. A. BLOOD
T
o ?
. °*
1 1 1 1 1 1 1 ' ' III
B. BONE MARROW T T T T
T T A A A A .
T T A-A.A^^AAIA-LA A 4
AAAAjT^x^*- ~
.
1 1 1 1 1 1 1 1 1 1 1 1 1
1 1 1 1 1 1 1 1 1 II'
C. BRAIN D n D T 9
9 9 "
$**
n nn*
i i i i i i i i i i i i i
1 ! 1 1 1 1 1 I j I 1 1 C
D. LIVER ^
O
C
o
0^0*0+0+0+°*°*°* * * .
1 1 1 1 1 1 1 1 1 1 1 1 1
0 1 2 3 4 5 6 7 8 9 10 11 12 13 14
ANIMAL SEQUENCE (low to high)
Figure 51. A Plot of Individual Animal Responses for DNA
Migration. The data are ranked in order from low to high DNA
migration length, alternating between hazardous waste and control
mice. Each symbol represents the mean and standard error of the
mean among 25 cells. Open symbols indicate hazardous waste mice,
solid symbols represent control mice.
81
-------
c
D
A
-------
HW-01
C-12
75
50
25
0
a 75
Ed 5°
O
U. 25
O
LU o
%
g 75
LU
O 50
ft*?
LU
DL 25
0
75
50
25
n
A. BLOOD H = 0.05
_J
-
B. BONE MARROW H = 1.35
J
;
.
a tm n B . . . . .
C. BRAIN H - 4.83
. »i .. "n n,--B^rnn B,
D. LIVER H = 1.49
.III
A. BLOOD H - 0.06
1
B. BONE
MARROW H - 0.11
C. BRAIN H - 0.64
l|l« -, . ,
D. UVER H 1 .85
ill... -
10 20 30 40
DMA MIGRATION (microns)
10 20 30 40
DNA MIGRATION (microns)
50
Figure 53. Distribution of DNA Migration Lengths for Blood,
Bone Marrow, Brain and Liver Cells Sampled from a Represen-
tative Animal for the Hazardous Waste and Control Sites. Data
based on 25 cells per tissue. The width of each bar represents
2 microns.
83
-------
m
c
.8 20-
i 15+
i-
I 10+
5--
' I
R -0.1926
5 10 15 20
BRAIN DNA MIGRATION (microns)
25
o
CO
o:
LJ
D_
CO
Q
o:
5--
4--
3--
2"
1--
0-H
R = 0.5375
468
BRAIN DISPERSION
10
12
Figure 54. Correlation Between of DNA Migration Length and
Dispersion Coefficient for Brain and Liver Cells in each Animal
Collected at the Hazardous Waste and Control Sites. Each symbol
represents the response in an individual animal.
84
-------
9.0 CONCLUSION
While technical difficulties were encountered during the
development and application of the SCG technique to in vitro and
in vivo studies, the results presented in this technical summary
indicate that many of the problems have been surmounted and that
the approach should be of considerable value to scientists
attempting to evaluate animal and human populations for DNA
damage induced by genotoxic agents as a consequence of
environmental pollution.
85
-------
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Andrews, P.W., Tice, R.R., and Nauman, C.H. (1989) The single
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Andrews, P.W., Tice, R.R., and Nauman, C.H. (1990) In vitro DNA
damage in peripheral blood leukocytes and CHO cells as measured
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Ashby, J. et al (1985) Concomitant observations of UDS in the
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Butterworth, B.E., Ashby, J., Bermudez, E., Casciano, D.,
Mirsalis, J., Probst, G. and Williams, G. (1987) A protocol and
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Res. 189:113.
Cavanna, M., Parodi, S., Robbiano, L., Pino, A., Sciaba, L., and
Brambilla, G. (1980) alkaline elution assay as a potentially
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Cawthorn, J. W. (1970) Soil survey of Wake County, North
Carolina. U.S. Government Printing Office, Washington, DC., 118
pp. plus maps.
Doerjer, G., Buchholz, U., Kreuzer, K., and Oesch, F. (1988)
Biomonitoring of DNA damage by alkaline filter elution. Int.
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Furihata, C., Yamawaki, Y., Jin, S., Moriya, H., Kodama, K.,
Matsushima, T., Ishikawa, T., Takayama, S., and Nakadate, M.
(1984) Induction of unscheduled DNA synthesis in rat stomach
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72: 1327.
Hill, E.R., and Hoffman, D.J. (1984) Avian models for toxicity
testing. J. Amer. College Toxicol. 3: 357.
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Technique to the Rodent In Vitro Hepatocyte Culture System: 1.
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Parenchymal Cells.
Hurst, A. G. (1963) A phenological study of the herpetofauna of
William B. Umstead and Reedy Creek State Parks, Wake County,
North Carolina. M.S. Thesis, North Carolina State University,
Raleigh, NC, 66 pp.
Kligerman, A.D., and S.E. Bloom (1976) Sister chromatid
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Larsen, K.H., Brash, D., Cleaver, J.E., Hart, R.W., Maher, V.M.,
Painter, R.B., Sega, G.A. (1982) DNA repair assays as tests for
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Program. Mutat. Res. 98: 287.
Margolin, B.H. and Risko, K.J. (1988) The statistical analysis of
in vivo genotoxicity data. Case studies of the rat hepatocyte UDS
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Programme on Chemical Safety's Collaborative Study on In Vivo
Assays", J. Ashby, F.J. deSerres, M.D. Shelby, B.H. Margolin, M.
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Cambridge, U.K., Vol I, 29.
Maslansky, C.J. and Williams, G.M. (1982) Primary cultures and
the levels of cytochrome P-450 in hepatocytes from mice, rat,
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McQueen, C.A., Maslansky, C.J., Crescenzi, S.B., and Williams,
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U.K., Vol I, 345.
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M.O. (1983) Evaluation of the alkaline elution/rat hepatocyte
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simple technique for quantitation of low levels of DNA damage in
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Parodi, S. (1987) Major discrepancies between results obtained
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Tice, R.R. (1988) The cytogenetic evaluation of in vivo genotoxic
and cytotoxic activity using rodent somatic cells. Cell Biology
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marrow damage, in: "Toxicology of the Blood and Bone Marrow",
R.D. Irons, ed., Raven Press, New York, NY, 119.
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aging organisms and cells, in: "The Biology of Aging", E.L.
Schneider, and C. Finch (eds.), Von Nostrand Reinhold Co., Inc.,
New York, NY, 173.
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D.E. (1988) Environmental biomonitoring with feral rodent
species, in: "Application of Short-Term Bioassays in the Analysis
of Complex Mixtures", S. Sandhu, D.M. DeMarini, M.J. Mass, M.M.
Moore and J.L. Mumford (eds), Plenum Publishing Co., New York,
NY, 175.
Tice, R.R., Andrews, P.W., and Singh, N.P. (1989) The single cell
gel assay: a sensitive technique for evaluating intercellular
differences in DNA damage and repair. Presented at the Fifth
International Conference on Environmental Mutagens, Cleveland,
OH, July 10-15.
Tice, R.R, Andrews, P.W., Singh, N.P. (1990) The single cell
assay: A sensitive technique for evaluating intercellular
differences in DNA damage and repair, in: Methods for the
Detection of DNA Damage in Human Cells (B. Sutherland et al.,
eds), Plenum Press, New York, in press.
89
-------
Tice, R.R., Hirai, O., Andrews, P.W., and Nauman, C.H. (1990) The
single cell gel (SCG) assay: a sensitive technique for human
biomonitoring. Presented at the Twenty-First Annual Meeting of
the Environmental Mutagen Society, Albuquerque, NM, March 25-29.
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Williams, G.M. (1977) Detection of chemical carcinogens by
unscheduled DNA synthesis in rat liver primary cell cultures.
Cancer Res. 37: 1845.
Williams, G.M., Laspia, M.F. and Dunkel, V.C. (1982) Reliability
of the hepatocyte primary culture/DNA repair test in testing of
coded carcinogens and non-carcinogens. Mutat. Res. 97: 359.
Yang, K.H., Shin, C.G., Choe, S.Y., Kim, D.H. (1984) Detection of
DNA single strand breaks induced by procarcinogens in Chinese
hamster ovary cells cocultured with rat hepatocytes. J. Toxicol.
Environ. Hlth 13: 133.
90
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11.0 SCG PRESENTATIONS
1. The Single Cell Gel (SCG) Assay: A Sensitive Technique for
Evaluating Intercellular Differences in DNA Damage and Repair,
invited platform presentation by R.R. Tice, 5th International
Conference on Environmental Mutagens (July 10-15, 1989;
Cleveland, OH).
2. The Single Cell Gel (SCG) Assay: A New Tool for Detecting
Organ-Specific Levels of DNA Damage Induced By Genotoxic Agents,
poster presentation by P.W. Andrews, R.R. Tice, and C.H. Nauman,
5th International Conference on Environmental Mutagens (July 10-
15, 1989; Cleveland, OH).
3. The Single Cell Gel Assay: A Sensitive Technique For
Evaluating Intercellular Differences in DNA Damage and Repair,
invited platform presentation by R.R. Tice, P.W. Andrews, and
N.P. Singh, Conference on Methods for the Detection of DNA Damage
in Human Cells (October 1-4, 1989; Brookhaven National
Laboratory, Upton, NY).
4. The Single Cell Gel Technique: A New Method for Analyzing
DNA Damage and Repair, invited platform presentation by R.R.
Tice, Genotoxicity and Environmental Mutagen Society Seventh
Annual Meeting (October 19, 1989; Raleigh, NC).
5. Detection of Chemically-Induced DNA Damage in Individual
Cells, invited seminar by R.R. Tice, R.J. Reynolds Tobacco
Company (November 15, 1990; Winston-Salem, NC).
6. The Single Cell Gel Assay: A New Technique For Evaluating
Intercellular Differences in DNA Damage and Repair, invited
seminar by R.R. Tice (December 20, 1989; Brookhaven National
Laboratory, Upton, NY).
7. The Single Cell Gel (SCG) Assay: An Electrophoretic Technique
for the Detection of DNA Damage in Individual Cells, invited
platform presentation by R.R. Tice, P.W. Andrews, and N.P. Singh,
4th International Conference on Biological Reactive Intermediates
(January 14-17, 1990; Tucson, AZ).
8. The Single Cell Gel Assay: A New Method for the Detection of
DNA Damage in Human Populations, invited seminar by R.R. Tice,
University of North Carolina- US Environmental Protection Agency
Joint Epidemiology Seminar Series (February 6, 1990; Chapel Hill,
NC) .
9. The Single Cell Gel Technique: A New Method for Evaluating
Intercellular Differences in DNA Damage and Repair, invited
seminar by R.R. Tice, U.S. Food and Drug Administration (March 8,
1990; Washington, DC).
91
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10. Detection of Acrylamide-Induced Organ-Specific Levels of DNA
Damage Using A New Single Cell Electrophoretic Technique, invited
seminar by R.R. Tice, U.S. Environmental Protection Agency (March
9, 1990; Washington, DC).
11. The Single Cell Gel (SCG) Assay: A Sensitive Technique for
Human Biomonitoring, platform presentation by R.R. Tice, 0.
Hirai, P.W. Andrews, and C.H. Nauman, Twenty-First Annual Meeting
of the Environmental Mutagen Society (March 25-29, 1990;
Albuquerque, NM).
12. In Vitro DNA Damage in Peripheral Blood Leukocytes as
Measured by the Single Cell Gel (SCG) Assay, platform
presentation by P.W. Andrews R.R. Tice, and C.H. Nauman, Twenty-
First Annual Meeting of the Environmental Mutagen Society (March
25-29, 1990; Albuquerque, NM).
13. A New Method for Evaluating Intercellular Variability in DNA
Damage, invited seminar by R.R. Tice, University of New Mexico
Toxicology Program (March 30, 1990; Albuquerque, NM).
14. The Single Cell Gel (SCG) Assay: An Electrophoretic
Technique for the Detection of DNA Damage in Individual Cells,
invited seminar by R.R. Tice (June 26, 1990; Merck, Sharp and
Dome Pharmaceutical Co., West Point, PA).
15. The Single Cell Gel (SCG) Assay: A New Method for the
Detection of DNA Damage Resulting From Hazardous Pollutants,
invited presentation by R.R. Tice (August 14-15, 1990; U.S Army
Research Methods Branch Workshop, Frederick, MD).
16. The Single Cell Gel (SCG) Assay: A New Method for Evaluating
Intercellular Differences in DNA Damage and Repair, invited
seminar by R.R. Tice (August 30, 1990; Argonne National
Laboratory, Chicago, IL).
17. Detecting DNA Damage in Single Cells Using a Microgel
Electrophoresis Technique, invited seminar (September 7, 1990;
Bristol-Myers Pharmaceutical Co., Syracuse, NY).
18. DNA Damage Evaluation Using the Rodent In Vitro Hepatocyte
Culture System and the Single Cell Gel (SCG) Electrophoretic
Assay. Presented at the Eight Annual Meeting of the Genotoxicity
and Environmental Mutagen Society, October 25, 1990, Raleigh, NC.
19. Evaluation of DNA Damage in Golden Mice (Ochrotomys
Nuttalli) Inhabiting a Hazardous Waste Site Using the Single Cell
Gel (SCG) Assay. Presented at the Eight Annual Meeting of the
Genotoxicity and Environmental Mutagen Society, October 25, 1990,
Raleigh, NC.
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20. Evaluation of Chemically-Induced DNA Damage in Germ Cells of
Male Mice Using the Single Cell Gel (SCG) Assay. Presented at
the Eight Annual Meeting of the Genotoxicity and Environmental
Mutagen Society, October 25, 1990, Raleigh, NC.
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12.0 SCG ABSTRACTS/PUBLICATIONS/MANUSCRIPTS IN PREPARATION
1. Andrews, P.W., Tice, R.R., and Nauman, C.H., (1989) The
Single Cell Gel (SCG) Assay: A New Tool for Detecting Organ-
Specific Levels of DNA Damage Induced By Genotoxic Agents,
Environ. Molec. Mutagenesis 14, S15, 10.
2. Tice, R.R., Hirai, O., Andrews, P.W., and Nauman, C.H. (1990)
The Single Cell Gel (SCG) Assay: A Sensitive Technique for Human
Biomonitoring, Environ. Molec. Mutagenesis 15, S17, 60.
3. Andrews, P.W., Tice, R.R., and Nauman, C.H. (1990) In Vitro
DNA Damage in Peripheral Blood Leukocytes as Measured by the
Single Cell Gel (SCG) Assay, Environ. Molec. Mutagenesis 15, S17,
6.
4. Tice, R.R., Andrews, P.W., and Singh, N.P. (1990) The Single
Cell Gel Assay: A Sensitive Technique For Evaluating
Intercellular Differences in DNA Damage and Repair, In: Methods
for the Detection of DNA Damage in Human Cells (B. Sutherland, J.
Sutherland, R.B. Setlow, and A. Woodhead, eds.), Plenum
Publishing Co., New York. In Press.
5. Tice, R.R., Andrews, P.W., Hirai, O., and Singh, N.P. (1990)
The Single Cell Gel (SCG) Assay: An Electrophoretic Technique for
the Detection of DNA Damage in Individual Cells.
In: Biological Reactive Intermediates IV (R. Snyder, ed.). In
Press.
6. Tice, R.R., Andrews, P.W., Groom, O.K., and Nascimbeni, B.
(In Preparation) The Single Cell Gel Technique: I. Monitoring
of Organ-Specific Levels of DNA Damage Induced by Acrylamide in
Mice.
7. Andrews, P.W., and Tice, R.R. (In Preparation) The Single
Cell Gel Technique: II. Detection of DNA Damage Induced by
Acrylamide, Trichloroethylene and Dimethylbenzanthracene in CHO
cells in vitro.
8. Hirai, 0., Noguchi, H., and Tice R.R. (in preparation)
Application of the Single Cell Gel Electrophoresis (SCG)
Technique to the Rodent In Vitro Hepatocyte Culture System: 1.
Kinetics of Cyclophosphamide-Induced DNA Damage in Mouse
Parenchymal Cells.
9. Hirai, 0., Noguchi, H., and Tice R.R. (in preparation)
Application of the Single Cell Gel Electrophoresis (SCG)
Technique to the Rodent In Vitro Hepatocyte Culture System: 2.
Comparison of DNA Damage Induced in Parenchymal and
Nonparenchymal Cells by Direct and Indirect Acting Genotoxic
Chemicals.
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10. Hirai, 0., Noguchi, H., and Tice R.R. (in preparation)
Application of the Single Cell Gel Electrophoresis (SCG)
Technique to the Rodent In Vitro Hepatocyte Culture System: 3.
Comparison of DNA Damage Induced in Mouse and Rat Hepatocytes by
2-Acetylaminofluorene, 4-Acetylaminofluorene, Cyclophosphamide,
Dimethylnitrosamine, Benzo(a)pyrene, Ethylmethanesulphonate, and
Mitomycin C.
11. Tice, R.R., Phillips, M., Andrews, P.W., and Groom, D.K. (In
Preparation) Increased Levels of DNA Damage in Free-Living
Rodents Inhabiting a Hazardous Waste Site.
12. Tice, R.R, Strauss, G.S.H., , Everson, R., and Peters, W.H.
(In preparation): High-dose Combination Alkylating Agents with
Autologous Bone Marrow Support in Patients with Breast Cancer:
Preliminary SCG Assessment of DNA Damage in Individual Peripheral
Blood Lymphocytes.
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