United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA 600 1 79-021
May 1979
Research and Development
&EPA
Effects of Selected
Asbestos Fibers on
Cellular and
Molecular Parameters
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RESEARCH REPORTING SERIES
Research reports of the Office of Research and Development, U.S. Environmental
Protection Agency, have been grouped into nine series. These nine broad cate-
gories were established to facilitate further development and application of en-
vironmental technology. Elimination of traditional grouping was consciously
planned to foster technology transfer and a maximum interface in related fields.
The nine series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
6. Scientific and Technical Assessment Reports (STAR)
7. Interagency Energy-Environment Research and Development
8. "Special" Reports
9. Miscellaneous Reports
This report has been assigned to the ENVIRONMENTAL HEALTH EFFECTS RE-
SEARCH series. This series describes projects and studies relating to the toler-
ances of man for unhealthful substances or conditions. This work is generally
assessed from a medical viewpoint, including physiological or psychological
studies. In addition to toxicology and other medical specialities, study areas in-
clude biomedical instrumentation and health research techniques utilizing ani-
mals — but always with intended application to human health measures.
This document is available to the public through the National Technical Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/1-79-021
May 1979
EFFECTS OF SELECTED ASBESTOS
FIBERS ON CELLULAR AND MOLECULAR PARAMETERS
by
R. W. Hart, R. Fertel, H. A. I. Newman,
F. B. Daniel, J. R. Blakeslee
Chemical Biomedical Environmental Research Group (CBERG)
The Ohio State University
Columbus, Ohio 43210
Grant No. R-804201
Project Officer
James R. Millette
Exposure Evaluation Branch
Health Effects Research Laboratory
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
J.S. ENVIRONMENTAL PROTECTION AGENCY
CINCINNATI, OHIO 45268
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DISCLAIMER
This report has been reviewed by the Health Effects Research Laboratory,
U.S. Environmental Protection Agency, and approved for publication. Approval
does not signify that the contents necessarily reflect the views and policies
of the U. S. Environmental Protection Agency, nor does mention of trade names
or commercial products constitute endorsement or recommendation for use.
ii
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FOREWORD
The U. S. Environmental Protection Agency was created in response to
increasing public concern about the dangers of pollution to the health and
welfare of the American people and their environment. The complexities of
environmental problems originate in the deep interdependent relationships
between the various physical and biological segments of man's natural and
social world. Solutions to these environmental problems require an inte-
grated program of research and development using input from a number of
disciplines.
The Health Effects Research Laboratory was established to provide sound
health effects data in support of the regulatory activities of the EPA. Cul-
tural mammalian cell-line studies provide biological endpoints which can be
used to compare the potential health effects of various types of pollutants.
A multidisciplinary approach is necessary to evaluate the effects on biologi-
cal endpoints such as cytotoxicity, enhancement of virally directed cellular
transformation, alteration of cell membrane composition, changes in cyclic
nucleotide ratios, modification of chemical carcinogen metabolism, and asso-
ciation with genetic material.
The report that follows describes effects of varying time, type and
dosage of asbestos fibers on the above biological endpoints in cultured
mammalian cells. An understanding of the effects of asbestos on the indi-
vidual cell is important in determining t]ie potential health effects of
asbestos in drinking water.
R. J. Garner
Director
Health Effects Research Laboratory
iii
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PREFACE
Man and his biosphere must be protected from the adverse effects of
asbestos acting by itself or in conjunction with other carcinogens. Efforts
to protect the environment need appropriate biological endpoints. In regards
to asbestos, the CBERG program of The Ohio State University contributes to
this goal via a multidisciplinary approach involving studies of
cytotoxicity
enhancement of virally directed cellular transformation
the alteration of cell membrane composition
changes in cyclic nucleotide ratios
modification of chemical carcinogen metabolism and
association with DNA
The report that follows describes effects of varying both time and dos-
age of asbestos fibers on the above biological endpoints in cultured mammal-
ian cells. Unlike chemical carcinogens, asbestos did not induce any DNA
damage or decrease the rates of DNA replication.
Human cell failure to form colonies was 65 percent with wide ranges of
all asbestos fibers. Syrian hamster cells in a different test system exhib-
ited greater differential dose-dependent effects on cell death with different
asbestos fibers. At low concentrations of selected forms of asbestos fibers
there was differential enhancement of virally-directed cellular transforma-
tion. Differential changes in surface membrane sugar-containing lipid and
proteins are reminiscent of such changes in transformed cells. Asbestos also
induced elevations in the ratio cyclic AMP/cyclic GMP. This change in the
ratio may be characteristic of the actions of a promoter of cell transforma-
tion rather than the actions of an agent which interacts with DNA to initiate
transformation. This promoter concept is supported by the evidence that there
is an enhanced benzo(a)pyrene association with cellular DNA after cell treat-
ment with asbestos fibers.
Effects of Selected Asbestos
on Gellular and Molecu-
lar Parameters
Ronald W. Hart, Ph.D.
The CBERG Group
iv
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ABSTRACT
The purpose of this grant was to develop a cellular, biochemical and
molecular basis to compare the effects of five asbestos materials, short
chrysotile, intermediate chrysotile, crocidolite, tremolite and amosite.
The test systems employed were the normal human fibroblast cell strain
Detroit 550 and Syrian hamster embryonic cultures. The effects on 1) deoxy-
ribonucleic acid (DNA) damage, 2) DNA replication, 3) cytotoxicity, 4) vir-
ally directed cellular transformation, 5) cell membrane composition and 6)
cyclic nucleotide concentrations were studied.
Results of these studies were expressed as percentages of controls for
each of the variables measured. Neither induction of DNA per se nor replica-
tion of DNA was affected by treatment with phosphate buffered solution (PBS)
washed asbestos. Cytotoxicity (65 percent) was exhibited in human cells at
asbestos concentrations up to 10 ug/ml, but, in another system for measure-
ment of this factor (Syrian hamster cells), a greater dose and fiber (chryso-
tile intermediate > chrysotile mixed > chrysotile short _>_ crocidolite >
tremolite > amosite >. silica) dependence was observed. Virally induced
cellular transformation frequency increase was in the order amosite > chryso-
tile intermediate > crocidolite.
Cell membrane monosialoganglioside (GM..) is an index of a simpler cell
surface glycolipid pattern. For GM, the order was crocidolite > chrysotile
mixed > chrysotile intermediate > amosite. The reduction of molecular weight
of glycoproteins also is a sign of simplification of the cell surface. The
indicator of this process is the loss of a 85,000 molecular mass protein.
The greatest loss is after crocidolite treatment. The order for this loss
is crocidolite > chrysotile.mixed > chrysotile intermediate > amosite.
Cellular ratios of cyclic nucleotides increased toward controls in the order
crocidolite > chrysotile intermediate > amosite > tremolite > silica. These
results are consistent with asbestos acting as a promoter of carcinogenesis
metabolism of benzo(a)pyrene.
This report was submitted in fulfillment of Grant No. R-804201 by The
CBERG Group under the sponsorship of the U.S. Environmental Protection Agency
covering the period April 15, 1976, to July 1, 1978, and completed as of
August 15, 1978.
v
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CONTENTS
Foreword
Preface iv
Abstract v
Figures xiii
Tables ix
Abbreviations and Symbols x
1. Introduction .' 1
Ob j ectives 1
Summary of results 1
2. Conclusions 6
Relevance of present studies to carcinogenic
effects of asbestos 6
Relative hazards of one form of asbestos versus
another 6
3. Narrative 7
Methods 7
Concentration effects 12
Time effects 18
4. Negative results 29
References 30
vii
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FIGURES
Number Page
1 The effect of amosite asbestos on ST FeSV transformation
of Detroit 550 cells 15
2 The effect of chrysotile asbestos on ST FeSV transforma-
tion of Detroit 550 cells 16
3 The effect of crocidolite asbestos on ST FeSV transforma-
tion of Detroit 550 cells 17
4 The effect of various concentrations of amosite on the
ratio of cyclic AMP to cyclic GMP in normal human
f ibroblasts 19
5 The effect of various concentrations of crocidolite on
the ratio of cyclic AMP to cyclic GMP in normal human
f ibroblasts 20
6 The effect of various types of asbestos on the ratio of
cyclic AMP to cyclic GMP in normal human f ibroblasts 21
7 The effect of benzo(a)pyrene on the ratio of cyclic AMP
to cyclic GMP in normal human f ibroblasts 22
8 The effect of nitrosodimethylamine on the ratio of cyclic
AMP to cyclic GMP in normal human f ibroblasts 23
9 Control and 2 hr electropheretic distributions of cell
surface 3n-glycoproteins 25
10 Control, 24 and 48 hr electropheretic distributions of
cell surface 3H-glycoproteins 26
11 Control and 75 hr electropheretic distributions of cell
surface ^H-glycoproteins 27
viii
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TABLES
Number Page
1 Comparative table of asbestos type vs. test at 1.0
or 10.0 pg/ml for a 24 hour exposure 4
2 Detroit 550 cell survival after treatment with
crocidolite asbestos 13
3 Detroit 550 cell survival after treatment with
amosite asbestos 14
4 Detroit 550 cell survival after treatment with
chrysotile asbestos 14
5 Relative distribution of surface labeled glycolipids
of Syrian hamster embryonic cells 24
ix
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ABBREVIATIONS AND SYMBOLS
ABBREVIATIONS
AMP
BP
BUdR
DABA
DEAE
DMSO
DNA
DN-HC
dsb
EDTA
EMEM
ESS
FBS
FFU
GD.
la
GL-4
GM1
GM2
GMP
GT1
3H-TdR
PBS
PAH
Rf
RNA
SDS
SHE
adenosine 3', 5, -cyclic monophosphate
benzo(a)pyrene
5 bromo deoxyuridine
3,5-diaminobenzoic acid
diethyl acetate ethyl
dimethylsulfoxide
deoxyribonucleic acid
DNA-hyd rocarbon
double strand break
ethylenedinitrilo-tetraacetic acid
Earle's minimal essential medium
endonuclease sensitive site
fetal bovine serum
focus forming units
disialoganglioside
globoside
monosialoganglioside 1
monosialoganglioside 2
guanosine 3', 5'-cyclic monophosphate
trisialoganglioside
tritiated thymidine
phosphate buffered saline
*
polycyclic aromatic hydrocarbon
ratio of compound migration to solvent front
ribonucleic acid
sodium dodecyl sulfate
Syrian hamster embryo
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ssb
ST FeSV
TCA
TLC
UDS
UV
single strand break
Snyder-Feline Sarcoma virus
trichloroacetic acid
thin-layer chromatography
unscheduled DNA synthesis
ultraviolet
SYMBOLS
T4
X
4X-174
co2
°2
mCi
pH
mM
DNA identifier, T, virus
DNA identifier, X virus
DNA identifier, *X-174 virus
carbon dioxide
oxygen
mi Hi Curie
-log hydrogen ion concentration
milliMolar
XI
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SECTION I
INTRODUCTION
OBJECTIVES
The primary objective of this grant was to develop a cellular, biochemi-
cal and molecular data base for the comparative effects of fibrous asbestos
materials (short chrysotile, intermediate chrysotile, crocidolite, treraolite
and amosite) which are being used in the animal feeding studies being con-
ducted by the National Institute of Environmental Health Sciences. (1)
Specifically, the purpose of this project was to evaluate the ability of
these same materials to:
1) induce deoxyribonucleic acid (DNA) damage
2) modify DNA replication
3) induce cytotoxicity in vitro
4) modify virally directed cellular transformation
5) alter cell membrane composition
6) modify cyclic nucleotide concentration
SUMMARY OF RESULTS
Induction of DNA Damage
None of the asbestos materials used at any concentration studied induced
either unscheduled DNA synthesis (as measured by autoradiography), endonu-
clease sensitive sites (utilizing S, endonuclease), single-strand breaks (as
measured by sedimentation in alkaline sucrose) or double-strand breaks (as
measured by sedimentation in neutral sucrose) when asbestos samples were
washed prior to use in phosphate buffered saline and autoclaved to reduce
any contamination by biological material. Nonwashed autoclaved samples pro-
duced a low level of unscheduled DNA synthesis in a dose independent fashion
from .01-10 ug/ml. Although this finding is reproducible, the mechanism
underlying it is unclear. Since this effect is induced only by short chryso-
tile and intermediate chrysotile and not by any of the other materials, and
also since none of the fibers induced repaired regions sensitive to 313 nm
light [5 bromo deoxyuridine (BUdR) - incorporated regions], strand breaks or
endonuclease sensitive sites, it is assumed that the unscheduled DNA synthe-
sis observed may have been the result of either alterations in the cellular
membrane resulting in a greater exchange of hot and cold thymidine (TdR)
or the leakage of.cells into scheduled DNA synthesis in spite of the hydroxy-
urea blockage used to prevent this event.
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Modification of DNA Replication
No retardation or stimulation of DNA replication (a recently developed
in vitro assay for chemical carcinogens) was observed for any of the asbestos
fibers studied between concentrations of 0.0001 and 10 yg/ml when fibers were
washed and autoclaved prior to use. Nonwashed, autoclaved fibers, while
cytotoxic to varying degrees depending upon fiber type (see following sec-
tion) , exhibit only a minor retardation of DNA replication for the short and
intermediate chrysotile. This response may relate to a modest contamination
of the asbestos which we have observed via elution washing and have identi-
fied as a possible hydrocarbon of unknown character.
Cytotoxicity
The cytotoxic potential of both washed and unwashed samples of all forms
of asbestos studied was extremely constant in human cells. Even at "doses"
as high as 10 yg/ml cytotoxicity following a 24-hour exposure using human
fibroblast cell cultures yielded only a 65 percent reduction in colony forma-
tion. Thus, human fibroblasts are relatively resistant to the cytotoxic
effects of asbestos and show no differential in cytotoxicity relative to the
asbestos type used. On the other hand Syrian hamster embryo (SHE) cell cul-
tures exhibit a cytotoxic curve dependent upon dose and asbestos type. The
SHE cell system is composed of several cell types and cytotoxicity in this
system can be measured only by measuring total cell number and correcting
for any difference in growth rates. In this system, however, it appears
that cytotoxicity is chrysotile intermediate > chrysotile mixed > chrysotile
short > crocidolite _>. tremolite > amosite > silica.
Virally Directed Cellular Transformation
All forms of asbestos in the unwashed but autoclaved series increased
the frequency of virally directed transformation of human fibroblast cell
cultures with Snyder-Theiler Feline Sarcoma virus (ST FeSV) at dose levels
of .1 yg/ml or greater. The frequency of increase was similar for each form
of asbestos tested, with amosite > chrysotile intermediate > crocidolite.
Cell Membrane Composition
Membrane composition is modified by the various forms of asbestos tested
in a differential fashion. For example, after a 48-hour treatment with amo-
site at 10 yg/ml there was a significant increase in the labeling of mono-
sialoganglioside (GM.. ) and decreases in both disialoganglioside (GD, ) and
globoside GL-4. While chrysotile (intermediate) caused a similar change in
GM. , it caused a greater decrease in the gangliosides GD- and trisialogang-
lioside (GT..) than did amosite. The neutral glycolipid patterns of cells
treated witn amosite or chrysotile (intermediate) showed no difference from
one another. Crocidolite also induced similar changes in membrane structure as
did chrysotile; however, the time to manifestation at the same dose was
greater for the former than for the latter material. The asbestos fibers
examined also appeared to have differential effects on the surface labeling
of glycoproteins. The label patterns of untreated cells and amosite treated
cells were identical, whereas those cells treated with chrysotile intermed-
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late and chrysotile mixed were similar to one another but different from the
controls. The glycoprotein labeling pattern of crocidolite treated cells was
remarkedly different from that obtained with either amosite or chrysotile.
Modification of Cyclic Nucleotide Ratios
The normal human fibroblasts were incubated for 48 hours with various
types of asbestos and chemical carcinogens. At least three different con-
centrations were used with each type of asbestos. Cyclic adenosine 3',5,-
cyclic monophosphate (AMP) and cyclic guanosine 3',5'-cyclic monophosphate
(GMP) were determined by radioimmunoassay. The data are given only for that
particular concentration of carcinogen which gives maximum response in alter-
ing the ratio of cyclic AMP to cyclic GMP. It is quite clear that the effects
of asbestos are dependent upon the type of fiber used. Silica, which is a
weak carcinogen, also has an effect on the cells that is similar to the
effects of certain forms of asbestos. Asbestos increases the ratio of cyclic
AMP to cyclic GMP by 26 to 65 percent. This increase is largely due to an
increase in the concentration of cyclic AMP. (See Table 1)
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TABLE 1. COMPARATIVE TABLE OF ASBESTOS TVPE VS. TEST AT 1.0 (*) OR
10.0 (+) pg/ml FOR A 24 HOUR EXPOSURE EXPRESSED AS A % OF CONTROL
Test
Mixed Intermediate Short
Crocidolite Chrysotile Chrysotile Chrysotile
Tremolite
Amosite Silica
*DNA Damage
(washed)
a) ODS
b) ESS (Sx)
c) sab
dl dsb
e) BUdR
•*-.
* DNA Damage
(unwashed)
a^ .ugs
b) ESS(Si)
cl ssb
d) dsb
e) BUdR
Cytotoxicity
+ a) human
+ b) hamster °
+ Viral Trans-
formation
135
35
62
190
155
140
170
120
29
35
21
210
59
62
125
40
67
230
*cAMP/cCMP
154
143
111
111
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Crocidolice
Test
+Glycollpids
a) GM1 190
trt GM2 112
c^ GDla 21.9
dN Oil 22
e'Cerebroside 18. 8
fl GL-4 50.1
i-Glycoproteins
a^MW- 148000 99.6
b">MW=85000 28.0
c^MW-70000 33.5
•"Metabolism 98
of B&F
*DH-BF
association
al peak 3
b) peak 4
•*Non DN-BP
aasociacion
3) peak 1
bl peaU 2
Mixed Intermediate Short Tremolice Amos ice Silica
Chrysotile Chrysoeile Chrysotile
150 143 163
168 135 99
18.8 24.2 65.5
30.4 45,8 98.3
136 155 148
86.9 67.9 70.5
67.1 62.6 74.3
36.5 34.4 80.2
48.6 78.0 70.2
70 84 93 9?
-
750
250
700
°Ceil3 vere counted in trypan blue and thus cytotoxicity was determined
by vital dye exclusion.
* 1.0 ug/ail asbestos concentration.
+10.0 us/nl asbestos concentration.
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SECTION 2
CONCLUSIONS
RELEVANCE OF PRESENT STUDIES TO CARCINOGENIC EFFECTS OF ASBESTOS
1. Asbestos in and of itself does not act as do most carcinogens by
damaging cellular DNA.
2. Unwashed asbestos can and does contain trace levels of polyaromatic
hydrocarbons which can produce DNA damage.
3. Compounds such as benzo(a)pyrene do associate with asbestos.
4. To varying degrees, depending on the form of asbestos used, asbestos
does modify membrane structure in the same direction as does various chemical
carcinogens.
5. Asbestos of all forms enhances virally directed transformation but
the level of enhancement is relatively independent of the form of asbestos
used.
6. Asbestos acts as would a promoter relative to the cyclic nucleotides.
7. There is an uptake of asbestos in normal human fibroblasts in cul-
ture.
8. Iron-containing asbestos leaches iron intracellularly but not extra-
cellular ly.
Thus it is now apparent that specific forms of asbestos such as chryso-
tile can: (a) strongly associate with polyaromatic hydrocarbons; (b) be
taken up by fibroblasts as well as other cell types; (c) modify the metabo-
lism of polycyclic aromatic hydrocarbons (PAH) yielding a metabolite that
associates strongly with DNA; (d) alter membrane structure in the direction
of a transformed cell; and (e) modify the cyclic nucleotides in the same
manner as a classical promoter of carcinggen^esis would be expected to do.
RELATIVE HAZARDS OF ONE FORM OF ASBESTOS VERSUS ANOTHER
It appears that crocidolite causes the most changes at the lowest dosage.
Surprisingly, this asbestos fiber is the least cytotoxic. Perhaps the surviv-
ing cells are the most affected by the asbestos entering these cells. Amosite
conversely was the most cytotoxic fiber, but had little effect based on the
other biologic parameters.
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SECTION 3
NARRATIVE
METHODS
General
Cell Culture—
Cultures are maintained in conventional CO. incubators at 37 C and rou-
tinely tested for pleuro-pneumonia-like organisms. Cells were grown in
Earle's Minimal Essential Medium (EMEM) supplemented with 1 mM sodium pyruvate,
2 mM glutamine, 1 percent non-essential amino acids and 10 percent fetal bo-
vine serum. Confluent monolayers were dispersed with trypsin plus methyl
cellulose. The determination of the generation time and various .cell cycle
parameters was made by standard autoradiographic procedures (2 through 7).
DNA Damage
Unscheduled DNA Synthesis—
In_this technique cells are incubated in the presence of radiolabel (gen-
erally H-TdR) after exposure of the test cells to asbestos. The incorpora-
tion of radiolabel detected by autoradiography is a measure of excision re-
pair. Details of this procedure and its limitations have been explained
in detail elsewhere. Studies presented herein represent an average of 200
cells examined per point assayed. (2, 6, 7 through 11)
Strand-break Analysis—
Our method for determining DNA strand-breaks has been published in
detail and for specific procedural aspects see 13 through 17. Generally,
however, our procedure is a modification of the classical McGrath and
Williams technique (18). Cells are layered onto a solution of 1 _N NaCl,
0.01 M ethylenedinitrilo-tetraacetic acid (EDTA) on top of a 3.6 ml gradient
of 5-20 percent sucrose containing 2 M NaCl, 0.01 EDTA, and 0.33 M NaOH in a
4.0 ml polyallomer centrifuge tube. After lysis for 60 minutes at 23°C, the
denatured DNA is sedimented and fractions collected on paper strips. Our
analysis for weight-average molecular weights is done by a computer program
calibrated with single-stranded T,, X, and * X-174 DNA's.
BUdR Photolysis—
The method we use for BUdR photolysis was developed by Regan, Setlow and
Ley (19) and has be^n reported by us in several separate papers including ref-
erences 4, 5, 8, 17, and 20. Briefly, therefore, matched cell cultures are
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labeled with either H-TdR (experimental) or PO, (control), treated with
test agents and permitted to repair in the presence of either 10~ fl BUdR or
10 M TdR for either 4 hours (a measure of the rate of repair) or 20 hours
(a measure of extent of repair). Cells are then detached, adjusted for num-
ber of dpm/cell, mixed and exposed to high-intensity 313 nm light in the same
cuvette, thereby breaking the BUdR-containing regions but not the TdR-con-
taining regions of the DNA. The number of breaks detected by sedimentation
in alkali permits one to calculate the number of repaired regions, while the
fluence of 313 nm light (21) required to saturate all such sites permits a
determination of the size of such repaired regions.
Endosites—
Essentially our endonuclease sensitive site assay is that of Wilkins and
Hart (22), with the exception that S.. rather than ultraviolet (UV)-endo is
used, thus permitting recognition of single-stranded regions within the DNA.
DNA Replication
The number of cells performing DNA replication per unit time was deter-
mined by autoradiographic procedures described in previous pulications (3, 4,
5, 8, 17)._ In this procedure cells are exposed for a given length to the
agent and H-TdR added to the media for a prescribed length of time. After
varying lengths of time, coverslips to which the cells are attached are
removed and autoradiographs prepared. Those cells showing grains are assumed
to have gone through a round of DNA replication.
Cytotoxicity
Human Fibroblast Cell Cultures—
The procedure we use for determining cytotoxicity via colony formation
has been published in a paper by Blakeslee (25). In this procedure precon-
fluent monolayers are dispersed by trypsin and triturated to assure single
cell suspension. Two hundred and fifty cells were plated in 35 mm diameter
wells in EMEM supplemented with 10 percent fetal bovine serum, 1 mM sodium
pyruvate, 2 mM glutamine, IX nonessential amino acids (growth medium) and
incubated with the cells for 24 hours. Six to eight wells were used for
each test concentration. At the end of the incubation period, cells were
washed and refed with growth medium. Cell cultures were incubated for 12
days, fixed in formalin or methanol, stained with Giemsa and clones contain-
ing fifty or more cells enumerated. Absolute and relative plating efficien-
cies were determined and survival curves calculated from the data.
Syrian Hamster Embryo (SHE) Cell Cultures—
SHE cultures are composed of a number of cell types, each with its own
particular in vitro life span and cloning potential. It is used due to its
capacity to be transformed in vitro and its low degree of spontaneous trans-
formation (26, 27). Due to the fact that SHE cultures are composed of multi-
ple cell types, standard cloning cannot be accurately performed on this sys-
tem, and therefore cytotoxicity can only indirectly be measured by the change
in cell number compared to a control culture (26, 27).
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Modification of Virally Directed Transformation
Virus—
Briefly, 10 percent cell-free tumor homogenates from ST FeSV infected
cats were prepared and stored in L-15 medium + 15 percent fetal bovine serum
(FBS) at -70°C in 1 ml aliquots.
Infectivity Assay— ,
Cells were trypsinized and seeded onto either 16 mm (4 x 10 cells) or
35 mm (1 x 10 cells) diameter wells (Costar, Cambridge, MA) in 1.0 ml EMEM
medium for the former and four ml for the latter and incubated 18 hours prior
to treatment. Cells pre-treated with asbestos prior to virus infection were
incubated with designated concentrations of asbestos for two, six or 24 hours,
washed and treated with 0.2 ml (11 mm wells) or 1.0 ml (35 mm wells) of DEAE
dextran (40 jig/ml) in serum-free EMEM. After twenty minutes, the cells were
rinsed with EMEM + 5 percent FBS, infected at 0.05 ml per 16 mm wells or 0.2
ml per 35 mm wells with each of four twofold virus dilutions and allowed to
absorb for two hours. Four wells were used per dilution of virus. The
plates were rocked at 10 to 15 minute intervals to maintain an even distribu-
tion of inoculum and, after adsorption, the inoculum was removed and replaced
with two ml or four ml of growth medium. Cells post-treated with asbestos
after virus infection were incubated with designated concentrations of chemi-
cals two, six or 24 hours after virus adsorption. The medium was removed
from infected cells and the cells treated with medium containing only asbes-
tos for 24 hours, washed and refed with growth medium. Cells were refed with
fresh growth medium only on the sixth day after infection and subsequently
fixed with buffered formalin and stained with Giemsa three to four days later.
Foci appear as discrete areas consisting of round, hyperrefractile, enlarged
fibroblast cells. These foci were counted at 25 to 40 times with a dissect-
ing microscope.
Virus induced foci were counted in nontreated and asbestos treated wells.
The mean number of focus forming units and standard deviation was determined
for each treatment time and significance determined by Student's t-test.
Alterations in Cellular Membrane Composition
Surface Labeling—
For labeling in the presence of phosphate buffered saline (PBS) the media
was decanted, cells washed three times with PBS, and the incubation volume
adjusted to five ml with the same buffer (28,29). For labeling in the
presence of media alone, minimal essential medium (MEM) was employed for wash-
ing and final incubation volume adjustment. For labeling with media contain-
ing 10 percent FBS, the incubation volume was adjusted to five ml. Galactose
oxidase [Sigma Biochemical Type III (125 units/mg) or Grand Island Biological
Co. (100 units/mg)] (200-250 yg) was added to give a final concentration of
25 units per dish, and the plates were incubated in a Precision Scientific Co.
p Model 2 oven under C02-0~ atmosphere for three hours at 35°C. After incu-
bation, the dish was washed twice with either PBS or MEM medium and the
excess incubation mixture was aspirated. One (1) mCi of NaB H,, with S.A. 9
Ci/mmol (New England Nuclear; stored in 0.01 N NaOH solution at -40° C) was
added and allowed to stand with occasional shaking for 30 minutes at 37°C.
-------
Finally, the cells were detached from the plates with a scraper having a
wide rubber blade. The reaction mixtures obtained from the various plates
were washed five times with PBS pH 7.4, and centrifuged at 1500 rpm for 10
minutes each time. The pelleted cells were suspended in 200 yl PBS, and
aliquots were taken for the glycolipids and glycoproteins determinations.
For glycoproteins the cells (10 ) were digested in PBS (100 ul) containing
one percent sodium dodecyl sulfate (SDS) and five percent 0-mercaptoethanol
and heated in a water bath at 85°C for 10 minutes.
SDS-Polyacrylamide Gel Electrophoresis—
Electrophoresis was performed according to the method of Weber and
Osborn (30). B-galactosidase, urease, albumin, peroxidase and lysosyme
with molecular masses of 130,000, 83,000, 68,000, 44,050 and 13,930 respec-
tively were used. The gels were sliced and each slice radioassayed.
Extraction of Syrian Hamster Cells Glycolipids (31)—,
An aliquot (100 pi) of surface labeled cells (10 ) was homogenized with
1 ml of methanol for five minutes at room temperature, and chloroform (2 ml)
was added so that the final ratio was 30 volumes of chloroform: methanol (2:1,
v/v) to 1 volume of cells. The homogenate was left overnight at room tem-
perature for efficient extraction, and then centrifuged at 1500 rpm for ten
minutes and the precipitate washed twice with chloroformmethanol (1:1, v/v).
Isolation of Glycolipids (32)—
Following the extraction of lipid from the cells, polar glycolipids
(gangliosides) were separated from neutral glycolipids by the procedure of
Folch (33). After Folch partition the total upper layer was reduced in vol-
ume to one to two ml and dialyzed at 4°C against distilled water for 24 hours.
The dialized upper phase was evaporated to dryness under N~, and the residue
extracted with a small volume of chloroform:methanol (2:1, v/v). This frac-
tion represents a major part of the gangliosides and contains some neutral
glycolipids in minor quantities. The analysis of gangliosides was performed
by thin-layer chromatography (TLC) (Kontes/quantum, precoated TLC plates)
and developed in a solvent system (Tetrahydrofuran:0.5 percent aqueous KC1,
7:1, v/v). Standard gangliosides were run separately in the same system at
the same time. The standard gangliosides were detected with resorcinol reag-
ent. Zones having the same Rf as the standards were scraped with a razor
blade and counted for radioactivity. The lower phase, containing the neutral
glycolipids, sulfatides, neutral lipid and phospholipid, was evaporated to
dryness and the glycolipid fraction separated according to Laine et al (34).
The isolated glycolipid fraction was separated into individual neutral gly-
colipids by TLC as described previously (35, 36).
Determination of Cyclic Nucleotide Levels
Incubation with Asbestos and Preparation of Cell-free Extracts—
Asbestos was suspended in the medium at the desired concentrations.
Cells were incubated in the medium containing asbestos for various time
periods (37, 38, 39). At the end of the incubation period, the medium was
poured off, and the cells were washed twice with five ml phosphate-saline
buffer (pH 7.4). Cells were harvested in two ml of five percent trichloro-
acetic acid (TCA) with the help of a rubber scraper. The cells in TCA were
10
-------
sonicated for 10 seconds and centrifuged at 4°C at 3000 rpm for 20 minutes.
The precipitate fraction was dissolved in 1 N NaOH and saved for measurement
of protein, which was determined by the method of Lowry (40). The TCA from
the supernatant fraction was removed by extracting three times with three
volumes of water-saturated diethyl ether. The ether layer was aspirated and
any traces of ethyl ether remaining after the last extraction removed by
heating the aliquots for 10 minutes at 50°C. The pH of the extracts was
adjusted to 6.2 by adding 0.1 volume of 1 M acetate buffer, pH 6.5.
Radioimmunoassay of Cyclic Nucleotides—
Succinylated cyclic nucleotides were coupled to keyhole limpet homocya-
nin as described by Steiner (41). This antigen was injected in rabbits to
produce the antiserum to cyclic nucleotides. The antiserum obtained has very
high specificity for cyclic nucleotides. Succinylated cyclic nucleotide-
tryosyl methyl ester derivatives are iodinated with I-labeled cyclic nu-
cleotide derivative (5000 cpm) followed by 50 ul of 1:2000 dilution of
antiserum (cyclic GMP) or a 1:4000 dilution of antiserum (cyclic AMP). The
other details of this procedure are identical to those described by Steiner
(42). This method permits the measurement of cyclic nucleotides in the femto-
mole range (37, 38, 39, 43, 44).
For each measurement, five plates of cells were harvested individually.
The concentrations of cyclic nucleotides were measured in duplicate for each
plate. The standard errors of the mean were calculated from 10 values.
Measurement of PAH Metabolism and Binding to Cellular DNA
Isolation of DNA—
DNA was isolated by either phenol extraction (45) or hydroxylapatite
chromatography (26, 27, 46, 47).
Degradation of DNA—
DNA was degraded to deoxynucleosides by either enzymic or chemical means
as appropriate: (i) the enzymic methods we employed were modified from those
used by Dipple, et al (48). In this method purified DNA is converted to mono-
nucleotides by incubation with bovine deoxyribonuclease I and snake venom
phosphodiesterase (12 hours, pH 7.5, 37 C). Hydrolysis to nucleosides is
accomplished by either wheat germ (pH 6) or bacterial (pH 8.5) phosphatases
after pH adjustment. In our hands, this system gave quantitative hydrolysis
of carcinogen bound DNA as judged by cellulose TLC; (ii) when necessary, DNA
was hydrolyzed by incubation in 0.1 IT HC1 at 37°C (24 hours). Conversion to
bases was accomplished by boiling nucleosides in 0.1 N HC1 for 20 minutes (26,
27).
Analysis—
(i) Protein was quantitated by the Lowry method using either a BSA or
lypholized microsomal standard curve; (ii) DNA was quantitated, as appro-
priate, by either UV spectotroscopy, diphenylamine reaction, fluorescene or
total DNA phosphate content; (iii) the extent of RNA contamination of DNA
samples was determined by the orcinal reaction (27).
11
-------
Chromatography of Deoxynucleosides and Deoxynucleoside-hydrocarbon Products—
Separation of the normal deoxynucleosides from the deoxynucleoside prod-
ucts was accomplished by chromatography on Sephadex LH-20 employing a water-
methanol gradient. Eluting fractions are monitored by ultraviolet spectro-
scopy continuously and, where appropriate, individual fractions are moni-
tored for radioactivity or fluorescence. Several scintillation counting
systems are presently employed in our laboratories.and are used as appro-
priate. Double label fractions are converted to C0_ and H.O by a Packard
306 sample oxidizer and analyzed radiometrically in separate vials or are
analyzed directly employing a computer program (26,27).
Determination of DNA-Hydrocarbon Adducts—
These studies were performed at two concentrations of benzo(a)pyrene and
four concentrations of each form of asbestos. The duration of benzo(a)pyrene
exposure was held constant and the duration of asbestos exposure varied
between zero and 48 hours at 12 to 24 hour intervals. The methodologies
employed in these studies are as follows: confluent monolayers of Syrian
hamster embryo cells and/or human fibroblast cells cultured as described
previously were treated with selected asbestos fibers at the desired con-
centration (0.1 to 1 ng/ml). At various times subsequent to the addition of
the asbestos, the media were inoculated with H-benzo(a)pyrene (1-1.5 mCi/
mmole) at final concentration of one to two mM. Twenty-four hours after the
hydrocarbon addition the medium was decanted, the monolayers rinsed three
times with PBS and the cells harvested in four ml of lysing solution (eight
M urea, containing 1 percent SDS, 0.01 M EDTA, and 0.24 M sodium phosphate,
pH 6.8). The DNA was then isolated by hydroxylapatite chromatography. The
isolated DNA was dialyzed and its concentration determined by DABA fluores-
cent techniques. The total amount of bound benzo(a)pyrene was determined by
liquid scintillation counting. The purified DNA was then hydrolyzed enzyma-
tically and the nature of the benzo(a)pyrene-deoxynucleoside adducts deter-
mined with Sephadex LH-20 chromatography (26,27).
CONCENTRATION EFFECTS
DNA Damage and DNA Replication
Only one concentration of asbestos (10 pg/ml) was employed to elicit
maximal response. Since little or no increase in DNA damage was observed
at this high concentration, no additional studies of concentration effects
were undertaken (49,50).
Cytotoxicity
For human Detroit 550 skin fibroblasts the dose response of plating
efficiency for amosite asbestos ranged from 58 to 90 percent of control.
A bimodal response was seen with greater survival in the middle range of
amosite concentrations and lower survival in the upper and lower ranges
(Table 2). Crocidolite had no effect at any concentration on cell plating
efficiency (Table 3). Chrysotile asbestos was in general more cytotoxic
at higher (0.1 - 10 wg/ml) doses of asbestos (Table 4)(51).
12
-------
T/VBI.K 2. DETROIT 550 CELL SURVTVAL AFI'KR TREATMENT WITH CROCrDOT.ITE ASBESTOS
No. clones
per 250
cells (a)
Absolute
Plating
efficiency (?) 0>)
Relative
plating
efficiency (%)
-------
TAIU.K 3. liBTROIT 550 CELT. SURVIVAL AFTER TREATNKNT WITH AMOSITE AUUBHTUS
No. clones
per 250 cells
Absolute
plating
efficiency (*)
Relative
plating
efficiency (%)
Concentration
ug/ml
101
23.8+4.3
9.5
70"
10°
24.8+J.8
9.9
72.9
ID'1
28.2+4.9
11.3
82.9
ID'2
30.7+3.5
12.3
90.3
10-3
30.513-3
12.2
89.7
lo-1-
26.3+4.2
10.5
77.lt
ID'S
20.0+5.7
8.0
58.8
Control
34.0+1.0
13.6
(P)-0.05-0.001 determined by student t test.
TABLE 1|. DETROIT 550 CELL SURVIVAL AFTER TREATMENT WITH CHRYSOTILE ASBESTOS
Concentration
ug/ml
No. clones
per 250 cells
Absolute
plating
efficiency U)
Relative
plating
efficiency (%)
10'
19.3+3.5
7.7
58.0*
.0°
23 +3.1
9.3
69.4
10"'
22.5+4.6
9.0
68.0
ID'2
26.7+4.4
10.7
80.2
10-3
33.2+4.9
13.3
100
io-4
22.1+4.0
8.8
66.4
10-5
29.0+3.0
11.6
87.1
Control
33.3+5.0
13.3
* -(P) 0.050-0.001 determined by student t test
14
-------
IjJ
O 4 I-
-------
If)!.
UJ
S
LU
O
4.0
3.0
2.0
I 0
0
20.0
^ 40.0
^ 60.0
UJ
£800
UJ
°- 100.0
Q
d
2
O
E
CD
CONCI Ni
-0.0001//g/m>!
*- SIGNIFICANCE - 0.001 - 0.025
.*
I
I I I
I
-24
-6 -20+2 +6
UNTREATED CONTROL
+ 24
TREATMENT TIME IN RELATION TO VIRUS INFECTION (HOURS)
Figure 2. The effect of chrysotile asbestos on ST FeSV tranformation of Detroit 550 cells.
-------
CONCENTRATION
LU
I
2
LL)
I
Q
g
b
CD
UJ
oc
LJ
Q.
6
5
4
3
2
I
0
20
40
60
80
100
0.
^-SIGNIFICANCE- 0.001-0.025
UNTREATED CONTROLS
I
I I I
-24 -6 -20+2 +6
TREATMENT TIME IN RELATION TO VIRUS INFECTION (HOURS)
+ 24
Figure 3. The effect of crocidolite asbestos on ST FeSV transformation of Detroit 550 cells,
-------
exhibit a dose response t6 tremolite. This finding is the opposite of that
found with cells incubated with the known chemical carcinogens, benzo(a)-
pyrene and nitrosodimethyl amine (Figures 7 and 8)(39, 43, 44).
Modification of Chemical Carcinogen Metabolism and Association with DNA
This study was initiated at the beginning of the last grant period, so it
is too early to include such data.
TIME EFFECTS
DNA Damage and DNA Replication
Since neither extent of DNA. damage nor amount of DNA replication was
altered after treatment of cells with PBS-leached asbestos, no time studies
were attempted in this area.
Cytotoxicity
Not determined at this time.
Enhancement of Virally Induced Cellular Transformation
Amosite asbestos fibers inhibited ST FeSV directed transformation if the
asbestos was introduced six hours prior to or two hours after viral infec-
tion. At all other times transformation frequency did not differ from con-
trol values (See Figure 1). Chrysotile fibers enhanced virally of 0.001
yg/ml) at all times of asbestos fiber exposure relative to viral infection
(See Figure 2). Crocidolite, in general, showed enhancement of virally
directed transformation at all times of asbestos incubation relative to viral
infection times (See Figure 3). At 10 yg/ml the enhancement was most notable
when the ST FeSV infection occurred either simultaneously with or within two
hours of the asbestos induction (See Figure 3)(51).
Alteration of Cell Membrane Composition
The alteration of the cell membrane glycolipids (Table 5) and glycopro-
teins (Figures 9 to 11) by chrysotile asbestos was tested at two, 24, 48 and
72 hours of incubation (28,29). At two hours there was little change in the
glycolipids or glycoproteins but at 24, 48, and 72 hours the ganglioside com-
position in the cell membrane progressively lost sialic acid moieties. This
loss is reflected in a shift from GD. and GT.. to GM- and GM«, simpler gang-
liosides. Surface glycoproteins after two hours of asbestos incubation were
unchanged. At 24 and 48 hours there was a loss of total glycoprotein as
well as a reduction in the higher molecular mass proteins. At 72 hours
there was a rebound in the total surface glycomolecular mass constituent
(Figure 11).
18
-------
VO
ALTERATION IN THE RATIO OF CYCLIC AMP TO CYCLIC GMP
IN NORMAL HUMAN FIBROBLASTS EXPOSED TO AMOSITE
200
12 18 24 30
INCUBATION TIME (HOURS)
36
48
Figure U. The effect of various concentrations of amosite on the ratio of cyclic AMP to
cyclic GMP in normal human fibroblasts. The ratio of cyclic AMP to cyclic GMP in control
cells is expressed as 100$ and the ratio in the asbestos-treated cells is expressed as a
percent (%) of control value. , control; o o, 0.05 yg/ml, A A, 0.5 yg/ml;
D O, 5.0 yg/ml.
-------
ALTERATION IN THE RATIO OF CYCLIC AMP TO CYCLIC GMP
IN NORMAL HUMAN FIBROBLASTS EXPOSED TO CROCIDOLITE
a.
2
o
o
0«
U.5?
O
o
<
200
180-
160-
140-
120-
100-
80-
60-
40
0.05 /ig/mt
I
t
5.0 jig/ml
18 24 30
INCUBATION TIME (HOURS)
42
Figure 5. The effect of various concentrations of crocidolite on the ratio of cyclic AMP
to cyclic GMP in normal human fibroblasts. The ratio of cyclic AMP to cyclic GMP in the
control cells incubated without asbestos is taken as 100$. The ratio of cyclic AMP to
cyclic GMP in the asbestos-treated cells is expressed as a percent (%) of control value.
, control; o o, 0.05 yg/ml; A A, 0.5 yg/ml; a a, 5-0 pg/ml.
-------
to
Q. 170
O
2 150-
£ 130 H
o
110-
O 90-
o
0 70-
O
o: 5O-
30
CHRYSOTILE
(SHORT)
CHRYSOTILE TREMOLITE
(INTMD)
0.01 O.I 1.0
ASBESTOS CONCENTRATION (>ag/ml)
10
Figure 6. The effect of various types of asbestos on the ratio of cyclic AMP to cyclic
GMP in normal human fibrotlasts. o o, chrysotile short type; • •, chrysotile inter-
mediate; A A, tremolite.
-------
a.
2
O
o
ALTERATION IN THE RATIO OF CYCLIC AMP TO CYCLIC GMP IN
NORMAL HUMAN FIBROBLASTS EXPOSED TO BENZO(A)PYRENE
to
120-
100-
<^8°1
_J O
60-
40-
U. J* 20
O
<
oc
CONTROL
12 18 24 30 36
INCUBATION TIME (HOURS)
42
48
Figure 7. The effect of "benzo(a)pyrene on the ratio of cyclic AMP to cyclic GMP in
normal human fibroblasts. Benzo(a)pyrene (BP) was dissolved in dimethyIsulfoxide (DMSO).
The ratio of cyclic AMP to cyclic GMP is expressed as 100$ of the control value. ,
control DMSO only;
1 yM BP; A A, 10 jiM BP; O 0, 100 yM BP.
-------
O
O
to
U)
ALTERATION IN THE RATIO OF CYCLIC AMP TO CYCLIC GMP IN NORMAL
HUMAN FIBROBLASTS EXPOSED TO NITROSODIMETHYLAMINE
>- LL.
100-
80-
60-
40-
20-
0
<
IT
CONTROL
200 mM
-T—
18
—i—
30
12 18 24
INCUBATION TIME (HOURS)
-i—
36
—i—
42
—r
48
Figure 8. The effect of nitrosodimethylamine on the ratio of cyclic AMP to cyclic GMP
in normal human fibrotlasts. The ratio of cyclic AMP to cyclic GMP in the control cells
is expressed as 100$. The ratio of cyclic AMP to cyclic GMP in nitrosodimethylamine-
treated cells is expressed as a percent (%) of control value. , control; o o,
2 mM; A A, 20 mM; a D, 200 mM nitrosodimethylamine.
-------
TABLE 5. RELATIVE DISTHIBUT10M OF SURFACE LABKLKD OLYCOUPIDS
OP SYRIAN HAMSTER EMBRYONIC CELLS
KJ
Compounds
Polar glycollpids
Monoaialoganglioaide (d )
Monoaialoganglioside (0.-_)
Dinlaloganglioside (ODI )
Tristaloganglioslde (Om,)
Neutral glycoliplds
"Olucocerebroside"
Cerebrosl de
Oloboslde QL-1)
Untreated
cells
2>l.9
30.3
20.7
2l).0
32.8
31.1
35-9
± 2.31
± 3.7l)
t 1.97
* 3.39
i 1.69
± 1.56
± 0.1)2
Percentage labeling*
Chrysotlle asbestos treated cells
2 hr 2l| hr 1)8 lir
25.2
29.8
20.2
21). 7
30.5
36.0
33.1)
±
t
±
±
i
i
i
2.05
1.76
2.96
3.25
1.20
1.20
2.1)0
26.0
1)8.1
6.9
18.8
21.2
35.1)
1)3.3
1 2.1)7
± 2.89
± 1.89
± 1.1(8
± 1.76
i l).2l)
± 2.1)7
37.5
51.2
3-9
7-3
26.1)
1)2.1)
31.2
i 1.62
t 0.91
1 1.12
± 1.86
± 0.56
± 1.69
± 2.26
72 hr
1)8.1
1)3.1
lt.li
1).2
29.6
56.5
13.8
i 2.05
i 1.20
i 1.68
i 0.89
± 1.06
± 1.62
± 0.56
* Based on TLC comparison with known glyuolipld standards
Mean i standard deviation, n • 2
-------
10 -i
M
2 4-
X
2-
Control
Cells 8 Asbestos (2 hr)
20 40
SLICE NUMBER
60
Figure 9- Control and 2 hr electropheretic distributions of cell surface H-glycoproteins
-------
10 -i
N>
Control
Cells & Asbestos (24 hr)
— Cells a Asbestos (48 hr)
*•••••••
20 40
SLICE NUMBER
60
Figure 10. Control, 2k & ^8 hr electropheretic distribution of cell surface "Tl-glycoproteins.
-------
to
•si
10 n
8-
Q. ft J
•n O
° 4
2-
Control
Cells a Asbestos (72 hr)
20 40 60
SLICE NUMBER
Figure 11. Control and 12 hr electropheretic distributions of cell surface 3H-glycoproteins,
-------
Changes in Cyclic Nucleotide Ratios
Brief (six hours) exposures of Detroit 550 human fibroblasts to either
amosite or crocidolite asbestos decreased the cyclic AMP/cyclic GMP below the
control value (See Figures 4 and 5). At 24 hours the ratio for both amosite
and crocidolite fibers bracketed the control ratio. At 48 hours the ratio
was almost universally greater than that of the control (39, 43, 44).
Modification of Chemical Carcinogen Metabolism and Association with DNA
No time studies of alteration of this factor are presented here.
28
-------
SECTION 4
NEGATIVE OR NEGLIGIBLE RESULTS OF STUDIES WITH ASBESTOS
All asbestos fiber types at the highest concentration employed had little
or no effect on induction of DNA damage based on unscheduled DNA synthesis if
the samples were washed. Unwashed fibers caused small amounts ofJDNA damage.
Crocidolite caused no cytotoxicity at fiber concentrations of 10 - 10
Vg/ml.
29
-------
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determinations by dodecylsulfate-polyacrylamide gel elecrophoresis. J.
Biol. Chem. 226:497-509.
31. Saat, Y. A., H. A. I. Newman and R. W. Hart. 1977. Surface glycolipids
distribution on Syrian hamster embryonic cells, Abstract. American
Physiological Society.
32. Saat, Y. A., H. A. I. Newman, R. E. Gibson and R. W. Hart. 1977. Short-
term effects of asbestos on surface glycolipids and glycoproteins of
Syrian hamster embryonic cells, Abstract. American Physiological So-
ciety.
33. Folch, J., M. Lees and G. H. Sloone-Stanley. 1957. A simple method for
the isolation and purification of total lipides from animal tissue. J.
Biol. Chem. 226:497-509.
34. Laine, R. A., K. Stellner and S. I. Hakomori. 1974. Isolation and
characterization of membrane glycosphingolipids. In: Methods of Membrane
Biology, E. Korn, ed. Plenum Press, New York. pp. 205-244.
32
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35. Saat, Y. A., H. A. I. Newman, D. K. Allison and R. W. Hart. 1978.
Effects of chrysotile asbestos fibers on Syrian hamster embryonic
cell membrane glycolipids, Abstract. American Society of Biological
Chem. and the American Assoc. of Immunology. Atlanta, Georgia.
36. Saat, Y. A., H. A. I. Newman, D. K. Allison and R. W. Hart. 1978.
Putative inhibitory effect of chrysotile asbestos on glycoproteins and
complex glycolipids biosynthesis in cell culture. Clinical Chemistry.
24:1013.
37- Tejwani, G. A., R. Fertel, R. W. Hart and R. E. Gibson. 1977. The
effects of ultraviolet radiation on cyclic nucleotide concentrations
in human diploid fibroblasts and cells derived from xeroderma pigmen-
tosum patients, Abstract. In: Proceedings of the 5th Annual Meeting
of the American Society for Photobiology. p. 109.
38. Tejwani, G. A., R. Fertel and R. W. Hart. 1977- The effect of ultravio-
let irradiation on cyclic nucleotide concentrations in human diploid
fibroblasts. Federation Proceedings. 36:688.
39. Tejwani, G. A., R. Fertel, R. W. Hart and D. K. Allison. 1979. Effects
of asbestos and chemical carcinogens on the cyclic nucleotide system
of human fibroblasts. J. Environ. Path. Tox. In press.
40. Lowry, 0. H., H. J. Rosebrough, A. L. Farr and J. Randall. 1951. Pro-
tein measurement with the folin phenol reagent. J. Biol. Chem. 192:
265-275.
41. Steiner, A. L., C. W. Parker and D. M. Kipnis. 1972. Radioimmunoassay
for cyclic nucleotides. I. Preparation of antibodies and iodinated
cyclic nucleotides. J. Biol. Chem. 247:1106-1113.
42. Steiner, A. L., A. S. Pagliara, L. R. Chase, L. Rani and D. M. Kipnis.
1972. Radioimmunoassay for cyclic nucleotides. II. Adenosine 3',5'-
monophosphate and guanosine 3',5'-monophosphate in mammalian tissues
and body fluids. J. Giol. Chem. 247:1114-1120.
43. Tejwani, G. A., R. Fertel, R. W. Hart and D. K. Allison. 1979. The
differential effect of asbestos and chemical carcinogens on cyclic
nucleotide concentrations of normal human fibroblasts. Cancer Research.
Submitted.
44. Tejwani, G. A., R. Fertel, R. W. Hart, D. Eneanya and D. Allison. 1978.
Asbestos induced changes in the concentration of cyclic nucleotides in
normal human fibroblasts, Abstract. American Society of Biological
Chemists and the American Association of Immunology Joint Meeting.
Atlanta, Georgia.
45. Kirby, K. S., and E. A. Cook. 1967. Isolation of deoxyribonucleic acid
from mammalian tissues. Biochem. J. 104:254-257.
33
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46. Meinke, W., D. A. Goldstein and M. R. Hall. 1974. Rapid isolation of
mouse DNA from cells in tissue culture. Anal. Biochem. 58:82-88.
47. Markov, G. G., and 1. G. Ivanov. 1974. Hydroxyapatite column chroma-
tography in procedures for isolation of purified DNA. Anal. Biochem.
59:555-563.
48. Dipple, A., D. A. Brookes, D. S. MacKintosh and M. P. Rayman. 1971
Reaction of 7-bromomethylbenz(a)anthracene with nucleic acids, poly—
nucleotides and nucleosides. Biochemistry. 10:4323-4330.
49. Hart, R. W. 1979. Biochemical, molecular and subcellular effects.
To be published in: Proceedings of the Workshop on the biological
Effects of Mineral Fibers and Particulates. Washington, D.C.
50. Hart, R. W., H. A. I. Newman, R. Fertel, J. R. Blakeslee and 0. Kendig.
1979. Differential effects of a series of asbestos fibers on membrane
structure, cyclic-nucleotide metabolism and fiber breakdown in vitro.
To be published in: Proceedings of the International Workshop on the
JEn Vitro Effects of Mineral Dusts. Penarth, Wales.
51. Blakeslee, J. R., 0. Kendig and R. W. Hart. 1979. EDAX and SEM analy-
sis of asbestos-treated human cells. To be published in: J. Environ.
Path. Tox.
34
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/1-79-021
2.
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
EFFECTS OF SELECTED ASBESTOS FIBERS ON CELLULAR AND
MOLECULAR PARAMETERS
5. REPORT DATE
May 1979 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
R. W. Hart, R. Fertel, H.A.I. Newman, F.B. Daniel,
J.R. Blakeslee
8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Chemical Biomedical Environmental Research Group
The Ohio State University
Columbus, Ohio 43210
10. PROGRAM ELEMENT NO.
614B(d)
11. CONTRACT/GRANT NO.
Grant No. R-804201
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory-Cincinnati, Ohio
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
13. TYPE OF REPORT AND PERIOD COVERED
14.
/ 15/7 6-7/1/7 8
AGENCY Cdl5£
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Embryonic cultures were used to compare the effects of five asbestos mater-
ials on a cellular, biochemical and molecular basis. Chrysotile was found
to be the most cytotoxic followed by crocidolite, tremolite, amosite, and
silica. Results of tests involving cellular ratios of cyclic nucleotides
were consistent with asbestos acting as a promoter of carcinogenesis.
Washed asbestos fibers had little or no effect on induction of DNA damage
based on unscheduled DNA synthesis tests.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Asbestos
Serpentine
Amphiboles
Cellular materials
Deoxyribonucleic acids
Health effects
06F
18. DISTRIBUTION STATEMENT
Release to public
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
47
20. SECURITY CLASS (This page)
Unclassified
22. PRICE
EPA Form 2220-1 (Rev. 4-77)
35
£• I). S. GOVERNMENT roiHTING OFFICE: 1979-657-060/1666 Region No. 5-11
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