SEPA
United States
Environmental Protection
Agency
Health Effects Research
Laboratory
Cincinnati OH 45268
EPA 600 1-79-023
July 1979
Research and Development
Asbestos and
Gastro-lntestinal
Cancer
Cell Culture Studies
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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-600/1-79-023
July 1979
ASBESTOS AND GASTRO-INTESTINAL CANCER:
CELL CULTURE STUDIES
by
B. Reiss, J.H. Weisburger and G.M. Williams
The Naylor Dana Institute for
Disease Prevention
American Health Foundation
1 Dana Road
Valhalla, New York 10595
Grant No. R-803998-01
Project Officer
James R. Millette
Health Effects Research Laboratory
Field Studies Division
Exposure Evaluation Branch
Cincinnati, Ohio 45268
HEALTH EFFECTS RESEARCH LABORATORY
OFFICE OF RESEARCH AND DEVELOPMENT
U.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.
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FOREWORD
The U.S. Environmental Protection Agency was created in response to
increasing public concern about the dangers of pollution of the health
and welfare of the American people and their environment. The complexities
of environmental problems require an integrated 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. A key segment of such a data bank is the knowledge of the effects
of a pollutant directly on a cell. Cell culture studies provide fundamental
data on the potential mutagenicity, fibrogenicity and carcinogenicity of
materials tested.
This report presents the results of a study to determine the relative
levels of cytotoxicity and mutagenicity for the different varieties of
asbestos fibers. An understanding of the effects of asbestos on the
cellular level is important in determining the potential health effects
of asbestos in drinking water.
Garner
Director
Health Effects Research Laboratory
iii
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ABSTRACT
Three forms of Union Internationale Centre le Cancer (UICC) as-
bestos: amosite, crocidolite and chrysotile, were assayed for their
cytotoxicity (inhibition of colony formation) and mutagenicity (in-
duction of mutants at the hypoxanthine-guanine phosphoribosyl trans-
ferase [HGPRT] locus) in cell culture. Using embryonic human intestine
derived (1-407) and adult rat liver-derived (ARL-6) epithelial cells,
the order of toxicity was chrysotile > amosite - crocidolite. All three
asbestos types were more toxic to 1-407 than to ARL-6 cells. Asbestos
was also tested for inhibition of colony formation in cultures of mouse
colon-derived epithelial-like cells (MCE-1); the toxic effects produced
in these cells were similar to those in 1-407 cells. Leaching the
asbestos for three days in sterile deionized water did not appreciably
affect the toxicity of the three asbestos forms. Leaching in hydro-
chloric acid, however, slightly increased the toxicity of amosite and
crocidolite and greatly decreased the toxicity of chrysotile. Curing
leaching in deionized water, substantial levels of Mg and Ca were
released from the asbestos fibers into the leaching fluid. Greater
titers of these ions were released during leaching in hydrochloric acid.
Mutagenesis assays, utilizing the toxic purine analog 6-thiogua-
nine for selection of HGPRT deficient mutants, revealed that high
concentrations of chrysotile, crocidolite or amosite were not mutagenic
in cultures of rat liver-derived epithelial cells.
Samples of contamination from six municipal water supplies were
also tested for cytotoxicity by measuring the inhibition of colony
formation in 1-407 cultures. The toxicity of these samples was much
less than that of equivalent amounts of standard asbestos materials.
This report was submitted in fulfillment of Grant No. R-803998010
by the Naylor Dana Institute for Disease Prevention, American Health
Foundation, under the sponsorship of the U.S. Environmental Protection
Agency. This report covers a period from January, 1976 to December,
1978 and was completed as of January 29, 1979.
IV
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CONTENTS
Forward i i i
Abstract i v
Figures and Tables vi
Abbreviations vii
Acknowl edgment vi i i
1. Introduction 1
2. Conclusions 3
3. Recommendations 5
4. Materials and Methods 7
5. Experimental Procedures 9
6. Results and Discussion 11
References 31
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FIGURES AND TABLES
FIGURES
Number Page
1 The cytotoxicity of chrysotile, amosite and
crocidolite in ARL-6 cells 12
2 The cytotoxicity of chrysotile, amosite and
crocidolite in 1-407 cells 13
3 The cytotoxicity of chrysotile, amosite and
crocidolite in MCE-1 cells 14
TABLES
Number Page
1 Effect on 1-407 colony formation of medium
harvested from twice-washed asbestos-treated
cultures. 16
2 Asbestos toxicity in cultures of ARL-6 cells
following leaching in sterile deionized water or
5N HC1 . 18
3 Asbestos toxicity in cultures of 1-407 cells
following leaching in sterile deionized water
or 5N HC1. 20
4 Release of Mg and Ca into sterile deionized
water or 5N HC1 by asbestos. 22
5 Asbestos-induced mutagenesis at the HGPRT locus in
ARL-6 cells. 26
6 Toxicity to the 1-407 cell line of samples of
contamination from municipal water supplies. 29
VI
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ABBREVIATIONS
ARL-6 Adult rat liver-derived epithelial cells
(transformed)
CFE Colony forming efficiency
EPA U.S. Environmental Protection Agency
HGPRT Hypoxanthine-guanine phosphoribosyl transferase
1-407 Embryonic human intestine-derived epithelial
eel 1 s
MCE-1 Mouse descending colon-derived epithelial-like
eel 1 s
UICC Union Internationale Centre le Cancer
VI 1
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ACKNOWLEDGMENT
The authors thank Dr. Robert Tardiff, National Academy of Sciences,
and Dr. Raymond Shapiro, National Institute of Environmental Health
Sciences, for their encouragement and advice on this program. We are
also grateful to Mrs. Sondra Solomon for her skillful technical as-
sistance, Mr. Andrew Soiffer for help with the atomic absorption spec-
trophotometric measurements and Dr. Charles Tong for advice on the
mutagenesis assay. Mrs. Bette Meyer and Miss Karen Brummett are thanked
for preparation of the manuscript, and Mr. C.Q. Wong is thanked for his
assistance with the figures.
viii
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SECTION 1
INTRODUCTION
The five main varieties of asbestos that are of economic
importance to various countries are chrysotile, crocido lite,
amosite, anthophyl1ite and tremolite. In the United States
where over 800,000 tons of asbestos are consumed annually,
chrysotile is the form of asbestos in greatest commercial use
(94%). Crocidolite (4.4%), amosite (1.1%) and anthophyl1ite
(0.1%) also have commerical significance. Natural asbestos
deposits occur worldwide, but in the United States, asbestos
is mined and milled primarily at five locations in California
(70%), Arizona and Vermont. However, most of the asbestos
consumed in the United States is imported (90%); chrysotile
is imported primarily from Canada (96%) and the Republic of
South Africa (3%). (12, 24)
Asbestos fibers can enter the environment from natural
geological deposits, particularly during mining and milling.
In the United States, there are five mining and milling
sites; of these, only one mine is underground while four are
surface mines which are responsible for maximal escape of
asbestos fibers into the atmosphere. Asbestos is also re-
leased during the manufacture of end-products, shipment,
usage and disposal. The mining of ores geologically associ-
ated with asbestos, such as talc and iron, is another major
source of environmental contamination with asbestos. (12,24)
Human exposure to asbestos can occur as a result of any
of these industry-related activites or from the use of as-
bestos-containing products. Inspired asbestos fibers are found
in the respiratory system, and in addition, these fibers can
migrate across internal membranes and have been detected in
thoracic and abdominal lymph nodes, liver, spleen, pancreas
and kidney (13, 22). Fibers also infiltrate the gastroin-
testinal tract via pulmonary clearance (11) and as the result
of ingesting asbestos-contaminated water and certain com-
mercially prepared beverages, foods and drugs (7, 8, 30, 32,
39). Data indicate that asbestos penetrates the gastroin-
testinal mucosa (48, 51-53, 32, 42) and produces biochemical
changes in the rat small intestine mucosa and lumen (18).
Asbestos is of grave concern as a health hazard because
of its cytotoxic, fibrogenic and carcinogenic properties
(24). Exposure to this mineral is associated with an increased
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incidence of bronchogenic carcinomata and pleural and peri-
toneal mesotheliomata in humans (3, 35). An increased in-
cidence of cancers of the gastro-intestinal tract in asbestos
workers has also been reported (36).
The objectives of this investigation were to develop and
utilize in vitro techniques with a variety of cell types for
the delineation of the biological effects of asbestos fibers,
particularly as they relate to cancer induction in the gastro-
intestinal tract. Part of this objective consisted in de-
veloping cytotoxicity and mutagenicity assays. The cyto-
toxicity of three forms of asbestos was examined in human
embryonic intestine-derived epithelial cells (1-407), adult
rat liver- derived epithelial cells (ARL-6) and mouse des-
cending colon mucosal epithelial-1ike cells (MCE-1). Muta-
genicity assays were performed on ARL-6 cultures, only. As a
corollary to these studies, samples of asbestos were leached
in water or hydrochloric acid, and the cytotoxicities of
leached samples of amosite, crocidolite and chrysotile were
compared with the toxicities of unleached material. Samples
of solid material isolated from municipal water supplies,
provided by the U.S. Environmental Protection Agency (EPA),
were also assayed for their cytotoxicity.
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SECTION 2
CONCLUSIONS
Three forms of Union Internationale Centre le Cancer
(UICC) asbestos: amosite, crocidolite and chrysotile were
analyzed for their cytotoxicity and mutagenicity using assay
systems developed in this laboratory. When assayed for their
effect on the colony forming efficiency of human embryonic
intestine-derived 1-407 and rat liver-derived ARL-6 epithelial
cells, all three forms of asbestos produced marked cytotoxi-
city. Chrysotile was approximately ten-fold more toxic than
the amphiboles. All three asbestos types were more toxic to
1-407 than ARL-6 cells. Chrysotile was found to have a simi-
lar toxic effect on MCE-1 and 1-407 cells; this suggests that
intestinal epithelial cells may be of particularly high sensi-
tivity to asbestos. The reproducibi1ity of the results from
the cytotoxicity assay demonstrates the usefulness of assaying
for the inhibition of colony formation as a reliable method
for the quantitation of cytotoxicity. This assay measured the
cytotoxicities of samples isolated from six different muni-
cipal water supplies and, therefore, appears useful for screen-
ing environmental samples.
UICC amosite, crocidolite and chrysotile were leached
for three days in sterile, deionized water. This procedure
did not appreciably affect the toxicity of the different
asbestos forms, although analysis of the leachates for Mg++
and Ca++ content by atomic absorption spectrophotometry
revealed that extraction of Mg++ and Ca++ had occurred.
Leaching in 5N HC1 greatly decreased the toxicity of chryso-
tile, while slightly increasing the toxicity of amosite and
crocidolite. Atomic absorption spectrophotometry demonstrated
a sizeable loss of Mg++ and Ca++ from all three asbestos
fibers, particularly chrysotile, due to 5N HC1-1eaching.
These results suggest a convergence of toxicity of chrysotile
and the amphiboles during prolonged emersion in a fluid i n-
vi tro; a similar process may occur in vivo. This phenomenon
could explain why the two classes of asbestos fibers, although
different in their short-term effects, are not clearly dif-
ferent in their long-term effects, i.e., delayed cytotoxicity
(25).
Assays for mutagenesis at the HGPRT locus failed to
demonstrate a consistant or significant increase in the
number of mutants resistant to 6-thioguanine after exposure
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to crocidolite, chrysotile or amosite. The lack of signi-
ficant mutagenicity that can be attributed to asbestos in
these experiments is consistent with the concept that asbestos
is not a genotoxic carcinogen characterized by interaction
with DNA but rather is an epigenetic carcinogen of the solid
state type (54). Solid state carcinogens appear to assert
their carcinogenic effects through physical interactions with
cells. An assay that measures physical interactions could
therefore be a reliable method for the quantitation of carcin-
ogenicity of a solid state carcinogen. If the physical
changes produced by asbestos are responsible for its cytotoxi-
city, then the cytotoxicity assay used in this study may be a
suitable assay for the quantitation of potential carcinogen-
icity of asbestoses as well as their cytotoxicity.
From the above results, it is concluded that chrysotile
and crocidolite are potentially the most hazardous forms of
asbestos. Furthermore, specific cell types, such as those of
intestinal origin, may be particularly sensitive to the toxic
effects of asbestos.
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SECTION 3
RECOMMENDATIONS
The results of our studies suggest the following recom-
mendations:
First, further work should be undertaken concerning the
physical and chemical properties of the various asbestos
fibers and the biological effects of altering these proper-
ties. As can be seen from the leaching data in Section 6, the
chemical and physical properties of asbestos are readily
altered and it is relatively easy, in the laboratory, to
produce changes in these properties that have great biological
significance. Leaching can also occur i n vivo (19, 25) or
even geologically. In this regard, Langer (21) reports that
asbestos fibers found in older sediments of Lake Superior
show evidence of iron loss due to leaching and differ signi-
ficantly in composition from freshly mined fibers. Thus,
although asbestos is relatively heat-stable, asbestos fibers
are subject to change by means of leaching, in the laboratory
or in a biological or geological environment. Our studies and
those of others (16, 19, 25, 26, 28, 55) demonstrate that
this process may increase or decrease asbestos reactivity,
depending upon the type of asbestos leached and the leaching
conditions. Therefore, to insure the safe handling of as-
bestos and to further elucidate the toxic and carcinogenic
characteristics of asbestos, a thorough understanding of the
physical and chemical properties and the biological effects
of altering these properties is required. Such information
should be made readily available to laboratory investigators
and epidemiologists conducting asbestos-related studies, as
well as safety control officers in asbestos plants.
Secondly, detailed studies on the mechanism(s) of the
carcinogenic action of asbestos are important. Our studies
and the work of others (6,17) indicate that asbestos is not a
genotoxic carcinogen. Furthermore, removal of the surface
ligands from asbestos fibers by leaching in water or acid
appears to influence the cytotoxic (25, 26, 31) as well as
the tumorigenic (28) potential of asbestos fibers. Thus,
mechanisms of action have been proposed to explain asbestos
carcinogenicity which emphasize the reaction of surface
ligands, such as Mg++, of phagocytosed asbestos fibers with
lysosomal membrane sialoglycoproteins (9, 14, 15, 43, 44).
Such mechanisms, however, have not been confirmed nor do they
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include the ultimate production of tumors. It is, therefore,
necessary for specific reactive groups on asbestos fibers to
be identified and the effects of these groups on cells to be
elucidated. Related phenomena, such as the biological coating
of asbestos with protective proteins (10), also require explan-
ation. Once the basic mechanisms of asbestos carcinogenicity
are understood, it is possible that means for the reduction or
even elimination of asbestos carcinogenicity can be devised.
Thirdly, as an extension of the second recommendation,
further work should be undertaken concerning the biological
effects of asbestos in vivo and i n vitro, particularly as it
affects gastro-intestinal cells. Selikoff (36) reported an
excess of gastro-intestinal cancer in asbestos workers;
however, it is a more complex task to establish an increased
incidence that can be specifically attributed to asbestos
that is ingested during occupational exposure or from the
consumption of contaminated drinking water, medication, or
commercial food and beverages. Thus, i n vivo and i n vitro
cell and organ culture studies are needed to establish gastro-
intestinal cancer as a direct result of ingested asbestos.
Furthermore, recent studies (33, 40) indicate that pleural and
peritoneal tumors are produced by implanted fibers of a speci-
fic size, regardless of composition; it would be of interest
to verify these experiments in the gastro-intestinal tract, as
well as to determine whether ingested fibers can pass through
the walls of the digestive tract and produce tumors in more
distal organs. Also, the results in Section 6 of this study
indicating that two cell lines of intestinal origin are more
sensitive to asbestos than a liver-derived line deserve
confirmation and extension.
Finally, efficient methods for the bioassay of asbestos-
induced effects should be developed. In order to more ef-
fectively study the biological and epidemic!ogical effects of
asbestos, it is imperative that practical methods be develop-
ed for the identification and quantification not only of
amounts but also of the biological effects of asbestos. The
cytotoxicity assay used in this investigation provides a
rapid and reliable means for quantitating the inhibitory
effect of asbestos on colony formation of mammalian cells.
The results of this assay are a measure of the physical
alteration produced by asbestos in cells and may be further
used to evaluate asbestos as a solid state carcinogen.
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SECTION 4
MATERIALS AND METHODS
Asbestos Samples
The UICC standard reference samples provided by the EPA
were Canadian chrysotile B, crocidolite and amosite (45).
Before 2weighing, samples of asbestos were exposed to 130
ergs/mm/sec ultraviolet light for 2 hours in order to sup-
press viable contamination in a manner that would be least
likely to alter the physical or chemical properties of the
asbestos fibers. In order to eliminate inaccuracies in
weighing caused by the electrostatic character of asbestos,
aliquots of asbestos were weighed in a known weight of water
or culture medium immediately prior to use.
Cell Cultures
All cells were maintained at 37°C in Williams' Medium E
(Flow Laboratories, Rockville, Maryland) supplemented with 10
percent fetal bovine serum (Flow Laboratories) and contain-
ing 5.0 units/ml mycostatin (Gibco, Grand Island, New York)
and 100 yg/ml gentamycin (Schering, Kenilworth, New Jersey).
1-407 and ARL-6 cells were used for most of the studies
in this project. MCE-1 cells, isolated and maintained in
this laboratory, but, as yet, not fully characterized, were
also used for cytotoxicity assays.
For the isolation of the MCE-1 cells, the descending
colon mucosa of 6-12 week-old neomycin and mycostatin-pre-
treated mice was digested with 0.25 percent pronase and then
scraped gently to detach the cells. These cells were pelleted
atfi50xg for four minutes and washed twice. Yields of 1.5 x
10 viable cells (85 to 95 per cent trypan blue negative)
per cm colon were obtained. These cells were a mixture of
epithelial and fibroblast-1ike cells. Collagenase (50 units/-
ml) was used to enrich the primary cultures with epithelial
cells. When the primary cultures were at least 80 per cent
confluent, they were trypsinized and transferred. One of the
clones developed from these cultures was the MCE-1 cell line.
Mg++ and Ca++ Determinations
Atomic absorption spectrophotometry was utilized for the
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determination of Mg++ and Ca+ + concentrations in the post-
leaching supernatants (leachates). Both filtered and un-
filtered aliqots of leachate were analyzed.
Mutagenesis Assay
The measurement of mutations at the HGPRT locus was
examined using a protocol described previously (46), as
follows:
1. Twenty-four hours after exposure to asbestos,
exposed and control cells were try,psi ni zed, ^counted, and
reseeded separately at 20 cells/cm in 25cm flasks for
colony formyig efficiency (CFE) determination and at 1.3 x
10 cells/cm in 75 cm flasks for maintenance during mutation
expression time. 2
2. Six days later, the cells seeded at 20 cells/cm
were fixed in formalin and stained for CFE determination.
3. After four and nine days, control and asbestos-
treated cells, seeded in step 1, were trypsinized and reseeded
separately at cell concentrations permitting optimal growth
(i.e. cell division occurring every 15 to 17 hours).
4. After fourteen days, control and treated cultures
were seeded for CFL determinations as in Step 1 and at 10
cells/ cm in 25 cm flasks for selection of HGPRT deficient
mutants.
5. Replacement of the culture medium with analog
(10yg/ml 6-thioguanine)-containing medium was initiated 24
hours after seeding. The analog-containing medium was re-
placed four times during the two-week selection interim.
6. One week after seeding, flasks for CFE determination
were fixed and stained.
7. Two weeks after treatment with 6-thioguanine was
initiated, selection flasks were fixed and stained for count-
ing of mutant colonies.
Municipal Water Contamination Samples
Samples of contamination obtained by filtration or
evaporation from municipal supplies were tested for their
cytotoxicity in 1-407 cultures. Before using, each sample
was exposed to ultraviolet light for two hours. Aliquots of
these samples were weighed in preweighed culture medium and
assayed for cytotoxicity as described in Section 5. Ultra-
violet 1 ijiht-irradiated amosite at an LD 50 concentration of
2.5 x 10" per cent served as a control for these experi-
ments.
8
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SECTION 5
EXPERIMENTAL PROCEDURES
Cytotoxicity Assays
The cytotoxicity of asbestos samples was quantified by
measuring the inhibition of 1-407 and ARL-6 colony formation
following exposure to asbestos.2 For this assay, 10 cells/
cm I-4072 cells or 20 cells/cm ARL-6 cells were inoculated
into 25cm culture flasks. Twenty-four hours after the cells
were seeded, the medium was replaced with medium containing
asbestos. Following three days exposure to asbestos, the
cells were washed twice and reincubated in asbestos-free
medium. Between one and two weeks after the initiation of
treatment, the cells were fixed in ten per cent formalin and
stained with Giemsa for the determination of colony forma-
tion.
Leaching of U.I.C.C. Asbestos
Approximately 50 mg samples of UICC asbestos (chrysotile,
amosite or crocidolite) were weighed in a pre-weighed aliquot
of sterile deionized water or 5N HC1 . Each weighed sample
was further diluted to 0.5 per cent (w/v), stirred thoroughly
to disperse the fibers throughout the fluid phase, and leach-
ed for one 96-hour or three consecutive 24-hour intervals at
25°C.
Following leaching, the asbestos samples were pelleted
by centrifugation at 10,000 x g for 15 minutes, and the
supernatants (except approximately 0.5 ml directly above the
pellet) were removed. The asbestos pellets were resuspended
in the remaining fluid and adjusted to the proper concentra-
tions for quantification of asbestos toxicity. When three
consecutive Teachings were performed, each leaching was follow-
ed by centrifugation and resuspension of the asbestos pellet
in sterile water or 5N HC1 after the first two Teachings and
in culture medium after the final leaching. As a control,
unleached asbestos samples were weighed in a pre-weighed
aliquot of culture medium and adjusted to concentrations
comparable to the leached asbestos.
Following this protocol, no significant loss of asbestos
weight occurred during leaching. This was established in
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control experiments in which weighed samples of asbestos were
leached for one 96-hour or three consecutive 24-hour inter-
vals, dried at 50-75°C and reweighed in a known weight of
water.
Mutagenesis Experiments
ARL-6 cells were exposed to asbestos and then assayed
for mutants at the HGPRT loojs. Cells were seeded in 75cm
flasks at 16 and 32 cells/cm . Twenty-fou/ hours later, the
medium in the flasks seeded at 32 cells/cm was replaced with
fresh medium containing asbestos and the medium in the flasks
seeded at 16 cells/cm was replaced with fresh asbestos-free
medium. Following six days of exposure to asbestos, control
and asbestos-treated cultures were washed two times and as-
sayed for mutagenesis (see Section 4).
10
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SECTION 6
RESULTS AND DISCUSSION
C.ytotoxicity of UICC Asbestos
When added to macrophages i n vitro,asbestos fibers
damage plasma and lysosomal membranes and cause cell death
(1, 2, 27). This form of short-term cytotoxicity, as well as
hemolysis (10, 15, 16, 34) occurs more readily in the pre-
sence of chrysotile than the amphiboles, amosite and croci-
dolite; however, long-term (delayed) cytotoxicity is equally
induced by the serpentine and amphibole asbestos forms (20,
41, 49-51). In order to study the cytotoxic effects of chry-
sotile and the amphiboles and to further our understanding of
the differences between the short-term and long-term effects
of these fibers, cytotoxicity assays were performed on fresh
samples of asbestos using ARL-6, 1-407 and MCE-1 cells. The
results were compared with those obtained from assays of
asbestos leached in water or hydrochloric acid.
The colony forming efficiencies (CFE's) of untreated
ARL-6 and 1-407 cells were 50 per cent and 40 per cent ,
respectively; exposure of these cells to chrysotile, amosite,
or crocidolite, after attachment, resulted in an inhibition
of colony formation that was dependent upon dose and duration
of treatment (see Figures 1 and 2). The order of toxicity
was chrysotile > amosite jv crocidolite. At least five-fold
higher concentrations were required to produce a comparable
inhibition of colony formation in the ARL-6 line as in the I-
407 line. Although periods of exposure to the asbestos
fibers were extended to include seven days, cumulative data
indicated that three days' exposure produced reliable and
consistent toxic inhibition. For example, after three days'
treatment with 2.5 x 10" % chrysotile, the CFE was reduced to
56% ± 16 of that in control cultures in three repeated experi-
ments.
The CFE of untreated mouse colon-derived MCE-1 cells was
60 to 75 per cent. When exposed to asbestos and then
assayed for colony forming inhibition, the MCE-1 cells ex-
hibited a level of susceptabi1ity to all fiber types similar
to that of 1-407 cells (see Figure 3). This may indicate a
general sensitivity to asbestos for cells of intestinal ori-
gi n.
11
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\ \ /A
\ \ / N
* A\
\
» /i
\ / \
\ t •
* / »
V-/ \
i
CMociuoiirr \
• • 10 :l% \
• • fiOnio 3'>;, *A
A A 10 2%
, , , , , , .
I 2 3 4 9 • 7
DUHATION ()l I Xl'OSUni , d.ivs
121456
nUHAHON (11 I XI'OSUHI il.iy
OUIIAI ION ()l I XI'OSUMt . il.iys
Figure 2. The cytotoxlclty of chrysotHe, amoslte and croddollte 1n I-4U7 cells
-------
100-1
90-
80
70
eo-
50
UJ
40
30
20
1O
^ v-"s 1
X S ^. '
^ — /
•\ A
\\ /\
It/ \ CHRYSOTILE
I w \ m • 10 4%
I \ • • 10 3%
' \ A A 10 2%
1 •
1 \
\ \ '
\ \ '
'< /\ L — -''
v \ * —
\
\
\
\ ^
^ ^ ** MM A*
[v
l\
i'VA -''^
v-
1 \
M '
I, AMOSITE
|« •- - -• 10 3%
• I • • 10 2%
M A- - -A 10 1%
\\
1
t
\ A
l/\ ^N
'/ A ^' x»
/V-"^x
f
/
/
y
~*x
\ y»x
\ / Sti
1 \/
« ft V /
II /\ V
II / \
11 / \
n* •
/ \
II / \
II / \
IV « -—
!/' \
k ^S -A
CROCIDOLITE
• • 10 3%
« • 10 2%
A A 10 '1%
1 2 3 4 S 6
DURATION OF EXPOSURE, days
1234567
DURATION OF EXPOSURE, clays
2345
DURATION OF E XI'OSUR E .
-------
To examine the mechanism of inhibition of colony form-
ation by asbestos, studies were performed to insure that the
toxicity results were not affected by an alteration in the
surface of the culture flasks produced by interaction with
the asbestos fibers. In these, experiments, flasks without
cells were exposed to 2.5 x 10" % chrysotile in Medium E and
incubated for three days at 37°C. Following incubation, the
flasks were alternately scraped with a rubber policeman and
washed twice and then washed agaJn to remove all fibers
before seeding with ten cells per cm . No significant differ-
ence was found in the CFE of cells grown in asbestos-pretreat-
ed flasks compared to the CFE of those grown in untreated
flasks, indicating that inhibition of colony formation by
asbestos was due entirely to a toxic effect on the cells and
not an alteration of the culture surface.
In order to determine whether inhibition of colony
formation was due to asbestos fibers remaining in the flasks
following the two changes of medium routinely used to wash
out asbestos after treatment, the toxicity of a third change
of medium was examined. Colony formation of 1-407 cells
maintained for three or seven days in the third wash from
chrysotile, crocidolite or amosite-treated 1-407 cells was
not supressed, indicating that the third wash did not contain
levels of asbestos high enough to alter the results in this
assay system (see Table 1).
Our results correspond with the data of other investi-
gators indicating chrysotile to be more inhibitory than the
amphiboles to epithelial cell and macrophage growth (2, 29,
49) and epithelial cell colony formation (29). Chamberlain
and Brown (5), however, using the cloning efficiency of
Chinese hamster lung cells as a criterion, found the order of
asbestos cytotoxicity to be amosite > crocidolite > chryso-
tile. In contrast, they found that chrysotile was more in-
hibitory than crocidolite to human alveolar lung cell growth;
growth inhibition in the presence of amosite was not deter-
mined by this group. A possible explanation for the reversed
order of toxicity obtained by Chamberlain and Brown in their
cloning assay might be the simultaneous addition of cells and
asbestos fibers to the culture dishes (in some cases there was
a 2 1/2 hour delay between seeding and addition of the fi-
bers). We have found that the presence of fibers on the
surface of a flask is inhibitory to cell attachment; further-
more, the order of adherence of asbestos fibers to the culture
surface is amosite > crocidolite > chrysotile. Thus it would
appear that the values obtained by Chamberlain and Brown for
cloning efficiency are actually values for attachment ef-
ficiency in the presence of asbestos fibers and do not re-
present true asbestos cytotoxicity. The cytotoxicity of
amphiboles (crocidolite, amosite and anthophyl1ite) contain-
15
-------
TABLE 1
EFFECT ON 1-407 COLONY FORMATION OF MEDIUM HARVESTED
FROM TWICE-WASHED ASBESTOS-TREATED CULTURES3
Days of Exposure to
Harvested Medium^
Experiment Harvested Medium 3 7
1 Control
Chrysotile
34
39
42
42
Control 95 115
Crocidolite 98 118
Control 243 255
Amosite 255 233
2
a 1-407 cells were seeded at 5-10 cells/cm ; 24 hrs later, the nutrient
medium was replaced with medium harvested.from unexposed (control) I-4Q7
cultures or cultures exposed to 2.5 x 10" % (w/v) chrysotile, 5 x 10" %
(w/v) crocidolite or 2.5 x 10 (w/v) amosite for 3 days. After 3 or 7
days, the harvested medium was removed from the flasks and the cells were
washed twice before adding fresh medium. Colonies were allowed to develop
for one week before quantification.
b Values represent the average number of colonies per flask in each
group.
ing decreasing percentages of long fibers were also compared
by these workers, using the same methodology (4). Decreased
colony number due to inhibition of cell attachment and
cytotoxicity was largely dependent upon the presence of
fibers of at least 6.5 ym in length.
Neugut et al. (29) recently assayed two epithelial cell
lines for growth inhibition in the presence of asbestos.
They found, particularly in cultures of Chinese hamster
16
-------
ovary, an "escape" from chrysotile toxicity after five to six
days of exposure. Re-exposure to chrysotile, however, did not
reveal the presence of a subpopulation of cells resistant to
asbestos. Our cytotoxicity results do not indicate an escape
from toxicity even 7 days after the initiation of exposure.
Furthermore, these workers found that cells exposed to chryso-
tile for one to two days, but not longer, and then trypsinized
and reseeded at cloning density in chrysoti1e-free medium
exhibited a decreased cloning efficiency. We have observed a
decreased cloning efficiency even after 6 days of exposure
when cells were trypsinized and reseeded in the absence of
chrysoti1e.
Cytotoxicity of Leached U.I.C.C. Asbestos
Samples of asbestos were leached in sterile deionized
water. The cytotoxic effects of water-leached chrysotile,
amosite and crocidolite were not significantly different from
unleached samples in ARL-6 cells (see Table 2) or 1-407 cells
(see Table 3). In a further attempt to diminish the cytotoxi-
city of asbestos, samples were leached in 5N HC1 for three
consecutive 24-hour or one 96-hour interval. Unleached and
water-leached chrysotile were significantly more toxic than
chrysotile leached with 5N HC1 ; however, 5N HCl-leached amph-
iboles were slightly more toxic than water-leached amphiboles.
Despite the fact that water-leaching did not produce a
distinct change in the cytotoxicity of the three types of
asbestos, atomic absorption spectrophotometry indicated that
Mg++ and Ca++ were released from the asbestos fibers into the
supernatant (see Table 4). Thus, leaching in water did alter
the_+ chemi stry of the asbestos fibers. Similar amounts of
Ca were released from the amphiboles aipd chrysotile; how-
ever, a significantly greater titer of Mg was released from
chrysotile than from either crocidolite or amosite.
Leaching in 5N HC1 for a 24-hour interval removed more
Mg++ from all three asbestos types, particularly chrysotile.
Ca++ removal was also increased in 5N HC1 , especially from
crocidolite. A longer interval (96 hours) of leaching in 5N
HC1 further increased the removal of Mg++ but not Ca++ from
al 1 asbestos types.
These results suggest a direct relationship between
excessive Mg++ and Ca++ depletion of chrysotile and loss of
cytotoxicity and an inverse relationship between Mg++ and
Ca++ depletion of the amphiboles and loss of cytotoxicity.
Thus, with prolonged dispersion of asbestos fibers in a fluid
environment, the cytotoxicity of chrysotile decreases while
the cytotoxicity of the amphiboles increases. Similarly,
Light and Wei (25) have found that leaching progressively
17
-------
TABLE 2
ASBESTOS TOXICITY IN CULTURES OF ARL-6 CELLS FOLLOWING LEACHING IN STERILE
DEIONIZED WATER OR 5N HCL
Asbestos Experiment
Amosite 1
2
3
_, Crocidolite 1
00
2
3
Chrysotile 1
2
3
Concentration
(% w/v)
2.5 x 10"2
2.5 x 10"2
2.5 x 10"2
2.5 x 10"2
2.5 x 10"2
2.5 x 10"2
2.5 x 10"3
2.5 x 10"3
3.3 x 10"3
Un leached
79
74
25
62
12
93
95
51
Toxicity3
H20-leached
55
70
25
42
63
19
96
96
51
5N HC1 -leached
54
98b
42b,c
45
96b
36b'C
29
75b
gb,c
a Inhibition of CFE of ARL-6 cells following three days exposure to asbestos; each value is expressed
as the percentage inhibition of CFE for control cultures not exposed to asbestos. In experiment 1,
samples were leached in water for 3 consecutive 24-hour intervals or in 5N HC1 for one 96-hour
interval. In experiment 2, both water-leaching and 5N HCl-leaching were performed for three con-
secutive 24-hour intervals. In experiment 3, both water-leaching and 5N HC1- leaching were per-
formed for one 96-hour interval.
Cont'd.
-------
Table 2 (cont'd.)
_ b Before addition to the culture medium, these asbestos samples were washed in 200 volumes sterile
10 deionized water and recentrifuged for 15 minutes at 10,000 X g
c 5N HC1 was neutralized with IN NaOH before addition of asbestos to the culture medium.
-------
ro
o
TABLE 3
ASBESTOS TOXICITY IN CULTURES OF 1-407 CELLS FOLLOWING LEACHING IN STERILE
DEIONIZED WATER OR 5N HCL
Asbestos
Amoslte
Crocldolite
Chrysotile
Experiment
1
1
2
3
4
1
1
2
3
4
1
1
2
Concentration
(% w/v)
io-3
2.5 x IO"3
2.5 x TO"3
5 x IO"3
3.3 x 10~3
10~3
2.5 x 10~3
2.5 x 10~3
5.0 x IO"3
3.3 x 10~3
io-4
2.5 x IO"4
2.5 x 10"4
Unleached
35
54
58
97
80
45
23
97
62
24
31
25
Toxicity3
H20-leached 5N HCl-leached
35
56
47 23
96 99C
74 76b>c
39
27 16b
88 92
60 66b)C
40
39
35 5b
Cont'd.
-------
TABLE 3 (Cont'd.)
ro
Toxicity3
Asbestos Experiment
3
4
Concentration
(% w/v)
-4
5 X 10 H
5 x 10"4
Unleached
91
87
H00-leached
c.
94
71
5N HC1 -leached
f
48C
31b'C
a Inhibition of CFE 1-407 cells following three days exposure to asbestos; each value is expressed
as the percentage inhibition of CFE for control cultures not exposed to asbestos. All samples
were leached for 3 consecutive 24-hour intervals except in experiment 2 where one 96-hour interval
of 5N HC1-leaching was used and experiment 4 where one 96-hour interval of water and 5N HC1-leaching
were used.
b Before addition to the culture medium, these asbestos samples were washed in 200 volumes sterile
deionized water and recentrifuged for 15 minutes at 10,000 x g.
c 5N HC1 was neutralized with IN NaOH before addition of asbestos to the culture medium.
-------
TABLE 4
RELEASE OF Mg++ AND Ca++ INTO STERILE DEIONIZED WATER OR 5N HYDROCHLORIC ACID BY ASBESTOS'
IVi
ro
Asbestos
AmosHe
Crocidolite
Experiment
1
2
3
4
1
2
3
4
Leaching Mg h
Fluid Unfiltered Filtered0
sterile - 34.9
deionized water
sterile 22.4 92.3
deionized water
5N hydro-
chloric acid 394.0
5N hydro- 473.8 546.0
chloric acid
sterile 10.0 27.4
deionized water
sterile 10.0 59.9
deionized water
5N hydro- 221.9
chloric acid
5N hydro- 281.8 344.0
chloric acid
Ca+t „
Unfiltered Filtered0
61.7 107.0
94.7 90.5
119.3
90.5 205.8
102.9 107.0
144.0 86.4
1,172.8
806.6 2,448.6
Cont'd.
-------
TABLE 4 (Cont'd.)
ro
CO
Asbestos Experiment
Chrysotile 1
2
3
4
None 1
2
3
4
Leaching
Fluid Ui
sterile
deionized water
sterile
deionized water
5N hydro- 20
chloric acid
5N hydro- 34
chloric acid
sterile
deionized water
sterile
deionized water
5N hydro-
chloric acid
5N hydro-
chloric acid
Mg*+ b
nfiltered Filtered
548.6 498.8
498.8 1,034.9
,448.9
,663.3 33,915.2
0.0 2.5
0.0 0.0
0.0
0.0
Ca'
Unfiltered
37.0
37.0
49.4
65.8
0.0
0.0
0.0
0.0
r+
Filtered
205.8
234.6
-
181.1
16.5
0.0
-
-
Cont'd.
-------
Table 4 (Cont'd.)
Values for the concentrations of Mg and Ca were obtained in parts per million (ppm) by atomic
absorption spectrophotometric analysis of leaching-fluid samples following 24 hours (experiments
1,2 and 3) or 96 hours (experiment 4) of leaching a|+25°C, || 0.5 per cent (w/v). Each sample
was monitored using reference standards+o.f known Mg or Ca ppm a.pjj then converted to
y moles by division with 0.0401 (for Mg values) or 0.0243 (for Ca values).
Leachate filtered through a Nalgene 0.2y (experiments 1 and 3) or 0.45p (experiments 2 and 4)
filter unit before atomic absorption spectrophotometric analysis.
-------
decreases the surface charge and hemolytic activity of chryso-
tile but increases these properties for the amphiboles. Such
convergence of activities would explain the equal effects of
the serpentine and amphibole forms of asbestos in delayed
cytotoxicity, following a prolonged interval of in vivo leach-
ing. These results correspond with those of Pelfrene (31) who
also noticed a preferential Mg++ leakage from chrysotile and a
preferental Ca++ leakage from the amphiboles, particularly
crocidolite, during saline leaching.
Mutagenicity of UICC Asbestos
Chrysotile and crocidolite have been reported to induce
chromosome damage in cultured Syrian hamster embryo cells
(23), Chinese hamster ovary cells (38), murine 3T3 cells (37)
and Chinese hamster lung cells (17). Although no mutagenic
activity was found to be associated with amosite, crocidolite
or chrysotile in mutation tests using E. coli or S. typhi-
murium (6), crocidolite was reported to have weak mutagenic
activity at the hypoxanthine-guanine phosphoribosyl trans-
ferase (H6PRT) locus in cultures of Chinese hamster lung cells
(17). Therefore, in order to develop further information on
the possible genetic effects of asbestos, the three forms of
asbestos were examined for mutagenicity using the HGPRT muta-
genesis assay on ARL-6 cells, developed in this laboratory.
-4
Ex_ppsure to chrysotile at concentrations of 7.5 X 10
and 10 per cent , resulted in a statisically significant
greater mutant recovery than was obtained in nonexposed cul-
tures _yi one out of _f^ve experiments (Table 5,a). Crocidolite
at 10 and 2 X 10" per cent yielded a greater mutant re-
covery in three out of seven experiments (Table 5,b). Amosite
exposure did not produce an increased mutant recovery in two
experiments (Table 5,c). Although the slight increase in
mutant recovery after exposure to high concentrations of
asbestos was in some cases statisically significant, the
results of these experiments cannot be taken as evidence of
mutagenicity because the mutant incidences after asbestos
exposure in all but one experiment did not exceed the range of
incidences (i.e., up to 17.3 mutants per 10 colony forming
cells) observed in control experiments. Furthermore, the 98%
confidence level for increased incidences in this line has
been established to be 11.8 +_ 3.7 mutants per 10 colony
forming units (47), and none of the incidences in exposed
cultures significantly exceeded this value. Thus, the
results indicating increased mutant recovery following ex-
posure to asbestos must be considered to be due to fluctuations
in spontaneous mutant recovery between experiments. Therefore,
we conclude that in this series of experiments, asbestos was not
mutagenic.
25
-------
ro
en
Table 5
Asbestos-Induced Mutagenesis at the HGPRT Locus in ARL-6 Cells'
Concentr
Asbestos (% w/
a.
b.
c.
chrysotile 7
7
crocidol ite
2
2
2
3
amosi te
2
.5
.5
.0
.0
.0
.0
.0
X 10
10":
10"J
X IQ
10 3
TO'2
x iq
10 ••
x iq
10 <•
X 10
X 10
10"2
X 10
ation
v)
-4
-4d
ft
A
-2d
-2d
Mutants/10 colony forming units
As
4
26
6
7
2
10
5
bestos-Exposed
.2
.0
.7
.5
.0
.0
.9
±
±
±
0
0
±
±
0
0
±
0
0
0
±
4.6 _
20. 7C
11.3
4.7
4.9
12.7
14.6
Control
0
0
0
0
0
0
0
0
0
17.3 ± 12.1
17.3 ± 12.1
17.3 ± 12.1
0
0
P Val
N.S.
<0.02
N.S.
N.S.
N.S.
ueb
5
<0.005
N.S.
N.S.
N.S.
N.S.
<0.01
<0.01
N.S.
N.S.
This table represents the results of fourteen experiments. For these experiments,
cells were exposed to asbestos for six days as described in Section 5 and assayed
for mutagenesls as described in Section 4. Preliminary experiments performed
before our selection technique was refined showed high spontaneous mutant
incidences; these results are not presented.
b Significance was determined by means of the Student's t-test
Cont'd.
-------
Table 5 (Cont'd.)
c This value exceeded 11.8 ± 3.7, the 98% confidence limit for
increased incidences with p< 0.1.
d The cells used in this experiment were non-transformed ARL-6 cells
-------
These results are in agreement with bacterial mutation
tests demonstrating no mutagenicity for amosite, crocidolite
or chrysotile (6). They do not differ greatly from the weak
mutagenicity results obtained for crocidolite at the HGPRT
locus in Chinese hamster lung cells (17); in this study, a
slight increase in mutant incidence was observed in only one
experiment. Since the range of variation in spontaneous
mutant recovery was not presented, it cannot be determined
whether this result, unlike the present results, was outside
the 98% confidence limit for spontaneous mutant incidence.
Although it has been reported that asbestos fibers pro-
duce major chromosome breaks and aberrations in Syrian hamster
cells (23), Chinese hamster ovary cells (38) and murine 3T3
cells (37), these breaks are dependent upon the presence of a
specific size range of fibers, either asbestos or glass, and
are not necessarily related to the production of point muta-
tions.
Cytotoxicity of Municipal Water Contamination
Six samples, provided by the EPA, were tested for cyto-
toxicity in 1-407 cultures. Before testing, all samples were
irradiated with ultraviolet light to elimate contamination
with micro-organisms. Amosite, at an LD 50 concentration
of 2.5 x 10" per cent, served as a positive control. The
results (see Table 6) demonstrated that, by assaying the
inhibition of 1-407 cell colony formation, the cytotoxic
levels of the six samples could be easily evaluated. The
order of toxicity of these samples, as determined by the
cytotoxicity assay, was: sample no. 8>3>2>ljv5>4.
Project Officer's Note
Samples of particulates, some extracted directly from drinking
waters were sent to the grantee referenced only by a code number. The
cytotoxicity testing was then done "blind" using the 1-407 cultures.
Sample No. 8 was a sample of the amosite fibers currently being used to
study the effects of ingested asbestos on rats and hamsters by the
National Institute of Environmental Health Sciences (NIEHS). The
feeding study is partially supported by the Environmental Protection
Agency. Not surprisingly the cytotoxicity of the NIEHS amosite was very
similar to that of the UICC amosite used as a positive control. Sample
Nos. 3,2, and 1 were particulates collected by filtration from drinking
waters from San Francisco, Seattle, and Duluth (prior to the instal-
lation of the filtration plant at Duluth) respectively. Chrysotile
fibers have been identified in the particulate samples from San Francisco
and Seattle; amphibole fibers were identified among the particulates
from the Duluth sample. Sample No. 5 was a sample of attapulgite clay,
a non-asbestos mineral, which consisted of fibers in the same range as
chrysotile asbestos. Attapulgite fibers have been identified in some
28
-------
water supplies in Georgia and Florida. Sample No. 4 was a sample of the
less than 2 micrometer size fraction of taconite tailings which had been
prepared by a sedimentation separation procedure. Amphibole fibers were
identified among the particulates in sample No. 5.
All the particulate samples were less cytotoxic than the commercial
asbestos variety samples. Details concerning the characteristics of the
particulate in the various samples and further discussion of the dif-
ferences in cytotoxicity will be forthcoming in a subsequent paper.
29
-------
TABLE 6
TOXICITY TO THE 1-407 CELL LINE OF SAMPLES OF CONTAMINATION FROM
MUNICIPAL WATER SUPPLIES
Percent Inhibition of Colony Formation
Concentration of Sample Number
Sample (% wTvT 123458
f4
2.5 x 10
10"4 8 18
-4
5.0 x 10"4 21 - - - 95
7.5 x 10"4 ____--
10~3 - 10 - 1 16 54 ± 13b
2.5 x 10"3 21 - - 0 - 72 ± 12
5.0 x 10"3 0 1-86
7.5 x 10"3 - 16 - - -
10"2 - 9 - 9
2.5 x 10"2 - 6 3
5.0 x 10"2 6 - - - 5
7.5 x 10"2 - 49 6 -
10"1 1 37b 96 2 3 -
2.5 x 10"1 30 73 100 33 35
5.0 x 10"1 88b 91 - 41 43 -
a These values represent the results of a total of seven experiments;
many of these values are averages from duplicate experiments. Where
more than two experiments were averaged, the standard deviation is
included. Each value is the per cent inhibition of CFE of 1-407
cells following exposure to water contamination samples. The average
CFE for all experiments of 1-407 cells not exposed to water contam-
ination samples or amosite was 112.5% ± 41.2. An amosite standard at
(Cont'd)
30
-------
TABLE 6 (Cont'd)
_o
2.5 x 10 % (w/v) was tested simultaneously with each experiment;
the average per cent inhibition of CFE for all experiments of _»<*
amosite-exposed cells was 59.2 ± 15.1. Chrysotile at 2.5 x 10"
(w/v) exhibited approximately a 50% inhibition of CFE in I-
407 cell cultures.
A result widely divergent from this value was omitted because it
was inconsistent with the trend of increasing toxicity with
increasing concentration of the sample.
31
-------
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing}
1. REPORT NO.
EPA-600/1-79-023
3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE
Asbestos and Gastro-Intestinal Cancer:
Cell Culture Studies
5. REPORT DATE
July 1979 issuing date
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
B. Reiss, J.H. Weisburger and G.M. Williams
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
The Naylor Dana Institute for Disease Prevention
American Health Foundation
1 Dana Road
Valhalla, New York 1Q595
10. PROGRAM ELEMENT NO.
614B(d)
11. CONTRACT/GRANT NO.
Grant No. R-S03998-01
12. SPONSORING AGENCY NAME AND ADDRESS
Health Effects Research Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Cincinnati, Ohio 45268
CIN
13. TYPE OF REPORT AND PERIOD COVERED
Final - 1/76 - 12/78
14. SPONSORING AGENCY CODE
EPA/600/10
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Three forms of asbestos: amosite, crocidolite, and chrysotile, were
assayed for their cytotoxicity and mutagenicity in cell culture. Using
embryonic human intestine derived and adult rat liver derived epitelial
cells, the order of toxicity was chrysotile > amosite = crocidolite. Leaching
in acid slightly increased the toxicity of amosite and crocidolite and
greatly decreased the toxicity of chrysotile. High concentrations of all three
asbestos forms were not mutagenic in cultures of rat liver-derived epithelial
cells.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
c. COSATI Field/Group
Asbestos
Serpentine
Amphiboles
Cellular Materials
Health Effects
06F
18. DISTRIBUTION STATEMENT
Release to Public
19. SECURITY CLASS (This Report)
Unclassified
21. NO. OF PAGES
46
20, SECURITY CLASS (This page)
Unclassif ledT
22. PRICE
EPA Form 2220—1 (Rev. 4—77)
PREVIOUS EDITION is OBSOLETE
37
657-060/5361
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