Toxic. in Vitro Vol. 1, No. 2, pp. 59~65, 1987
Printed in Great Britain
0887-2333/87 $3.00+ 0.00
Pergamon Journals Ltd
M E C H A N I S M S OF CYTOTOXICITY OF ASBESTOS
FIBRES IN RAT T R A C H E A L EPITHELIAL
CELLS IN C U L T U R E
T. W. HESTERBERG*,D. G. RIRIE, J. C. BARRETTand P. NETTESHEIM
Environmental Carcinogenesis Group, Laboratory of Pulmonary Pathobiology, National Institute of
Environmental Health Sciences, Research Triangle Park, NC 27709, USA
(Received 14 August 1986)
Abstract--Little is known about the mechanism(s) of asbestos toxicity, especially in respiratory epithelium. Studies were carried out to elucidate some important aspects of the cytotoxic effects of asbestos,
using a tracheal epithelial cell line in culture. Chrysotile and crocidolite asbestos with similar aspect ratios
were used. Both induced in 2C5 cells a concentration-dependent inhibition of colony-forming ability, a
measure of proliferative capacity. In this respect, chrysotile (LCs0, 0.95 ~g/cm2) was about six times more
toxic than crocidolite (LCs0, 5.8/~g/cm2). Both types of asbestos caused only minor changes in membrane
permeability, measured by trypan blue exclusion and by [75Se]selenomethioninerelease, even at concentrations of asbestos that caused >90% inhibition in colony formation. Thus membrane damage was only
a minor component of the toxicity produced by the fibres. Chrysotile was phagocytized much more readily
than crocidolite and produced an approximately threefold greater increase in binucleated cells and
micronuclei than crocidolite, suggesting that phagocytosis was the rate-limiting step in fibre toxicity. Our
studies suggest a potentially important pathway of fibre toxicity involving binding, phagocytosis, nuclear
damage, disruption of mitosis, inhibition of proliferation and/or cell death. In this process, the fibre aspect
ratio is not the only determinant of fibre toxicity, since chrysotile and crocidolite fibres of similar length
and diameter exhibit very different degrees of toxicity. It appears that other fibre characteristics, such as
fibre surface charge, are of equal importance.
INTRODUCTION
Deposition of asbestos fibres in the airways elicits
two major disease processes: neoplastic disease
and inflammation-fibrosis (Churg & Golden, 1982;
Harington et al. 1975). In recent years many studies
have been conducted to elucidate the mechanisms
involved in these seemingly unrelated pathological
tissue reactions and to determine the fibre characteristics essential for the toxic activities of asbestos
(Beck & Bignon, 1985). These studies have led to two
major hypotheses. According to the one hypothesis,
the fibre length or diameter, rather than the chemical
composition of the fibres, is the key factor determining the degree of toxicity (Stanton et al. 1981).
The other hypothesis states that surface properties,
such as surface charge and surface area, are the most
important determinants of fibre toxicity (Bignon &
Jaurand, 1983). There is indeed evidence supporting
both hypotheses and it seems reasonable to speculate
that both factors are important (Beck & Bignon,
1985).
Previous studies from our laboratory have shown
that in Syrian hamster embryo (SHE) cells, concentrations of asbestos that inhibit cell proliferation also
induce chromosome changes (Oshimura et al. 1984)
and anaphase abnormalities (Hesterberg & Barrett,
*Present address: Department of Genetic Toxicology,
Chemical Industry Institute of Toxicology, P.O. Box
12137, Research Triangle Park, NC 27709, USA (tel.
(919) 541-2070).
Abbreviations: SHE=Syrian hamster embryo; cpm:=
counts per minute.
59
T L V I/2--A
1985). In addition, we have shown that fibre length
appears to be a critical factor in determining the
degree of fibre toxicity (Hesterberg & Barrett, 1984)
and phagocytosis of the fibres (Hesterberg et al.
1986). However, the type of target cell must also be
taken into consideration when the toxicity of various
agents is examined. Lechner et al. (1983) recently
demonstrated that marked differences exist between
pleural mesothelial cells, fibroblasts and bronchial
epithelial cells in their susceptibility to the toxic
effects of asbestos, the mesothelial cells being by far
the most sensitive targets. The basis of this difference
in susceptibility is not clear at present.
Therefore, we decided to extend our studies on the
mechanisms of fibre toxicity to another cell type,
namely epithelial cells. Since the airway epithelium is
the major target for asbestos fibres, the rat tracheal
epithelial cell culture system used in our laboratory
(Nettesheim & Barrett, 1984) seemed particularly
appropriate. The specific purposes of the studies
presented here were (1) to examine the sensitivity of
epithelial cells to chrysotile and crocidolite fibres of
similar physical dimensions; (2) to determine the
extent of phagocytosis of these fibres by the epithelial
cells; (3) to determine whether the fibre toxicity
results primarily from cell membrane damage or from
damage to the mitotic apparatus; (4) to determine
whether fibre characteristics other than length and
diameter are important determinants of fibre toxicity.
Some of these studies have been briefly presented in
a preliminary report (Ririe et al. 1985). The major
conclusions we have reached on the basis of these
studies are that fibre characteristics other than fibre
size play a major role in determining how readily the
T. W. HESTERBERGet al.
60
fibre is phagocytized and how toxic it is and that the
major mechanism of chrysotile and crocidolite toxicity is not related to membrane damage but to
interference with cell division.
MATERIALS AND M E T H O D S
Cells and culture conditions
These studies were conducted using a cell line (2C5)
derived from rat tracheal epithelial cells isolated from
an 8-wk-old male Fisher 344 rat (Steele et al. 1978).
Cultures were initiated from frozen stocks of cells at
passage 20 and all experiments were conducted between passages 21 and 25. The culture medium
consisted of Waymouth's Medium (MD705, Grand
Island Biological Co., Grand Island, NY), 5% foetal
bovine serum (GIBCO), amino acids (L-alanine,
17.8#g/ml; L-arginine, 347/~g/ml; L-asparagine,
30/t g/ml; Na pyruvate, 220/~ g/ml; L-serine,
21/zg/ml; GIBCO), insulin (10/zg/ml; Sigma Chemical Co., St Louis, MO), hydrocortisone (10-6M,
Sigma), penicillin (100 units/ml, GIBCO) and streptomycin (100#g/ml, GIBCO). Cultures were incubated at 37°C in a humid atmosphere of 5% CO 2 and
95% air. Cells were gently trypsinized with 0.1%
trypsin (GIBCO) and 0.02% ethylendiaminetetraacetic acid in Ca2+-Mg2+-free phosphate-buffered
saline (PBS), pH 7.4, for 5 min at 37°C.
Asbestos preparation
International Union Against Cancer (UICC)
chrysotile and crocidolite asbestos were obtained
from V. Timbrell (Medical Research Council, UK).
Asbestos was weighed and mixed directly with complete medium to reach a stock concentration of
1 mg/ml. Asbestos was readily suspended by pipetting
back and forth using a 10-ml pipette.
To determine the physical dimensions of the fibres,
suspensions of asbestos fibres were diluted to
100ktg/ml in 95% ethanol, and 10#1 of the suspension was spread on a carbon disc that had previously been coated with 100#1 of 95% ethanol to
promote spreading. The discs were dried in a desiccator and then coated with 100 A of gold. The lengths
and diameters of 100 fibres/sample were measured
using scanning electron microscopy at magnifications
of x 6000 and × 10,000, respectively.
Determination of colony-forming efficiency
To determine the effects of asbestos on colonyforming efficiency, cells were seeded in complete
medium at 300 cells/60-mm culture dish (Falcon
Plastics, Oxnard, CA) in 3 ml complete medium.
Appropriate dilutions of asbestos were made 24 hr
later and 2 ml of the suspension was added to the
cultures. Concentrations of mineral fibres were expressed in ktg/cm 2, since the asbestos precipitated to
the bottom of the culture dish. After treatment,
cultures were incubated for 7 days at 37°C in a humid
atmosphere of 5% CO2 and 95% air. The cultures
were then fixed in absolute methanol (Fisher
Scientific Co., Fairlawn, NJ) and stained with 10%
Giemsa (Fisher) and the number of colonies per dish
was counted. The colony-forming efficiency was determined by dividing the number of colonies per dish
by 300 (the number of cells seeded per dish).
Assays for membrane damage
Two different methods were used to measure the
induction of membrane damage by mineral fibres.
The first method involved measuring the exclusion
of trypan blue (Sigma) from cells. Cultures were
treated with mineral fibres 24 hr after 105 cells had
been seeded in 5 ml complete medium in 60-mm
culture dishes (Falcon Plastics). After a further 24 hr,
the medium was removed, 0.5 ml 0.4% trypan blue
stain was added directly to the cultures and, after
1-2 min, the cultures were rinsed three times with
PBS. Four l-mm 2 fields were randomly selected on
each dish and cells were evaluated for trypan blue dye
exclusion. These determinations were not performed
using a haemocytometer because it was felt that
trypsinization and centrifugation of asbestos-containing cells could cause further membrane damage.
Membrane damage was also assessed by measuring
755e release after asbestos treatment. Cells (104) were
seeded in 1 ml complete medium in 16-mm multiwell
culture dishes, and 24 hr later the medium was replaced with medium containing 75Se-labelled selenomethionine (5/~Ci/ml, 0.6-4Ci/mmol, Amersham
Corp., Arlington Heights, IL; Leibold & Bridge,
1979). After 3 hr, the labelling medium was removed
and fresh medium was added to the cultures. Cultures
were allowed to incubate for 18 hr, after which time
the medium was changed and the cells were treated
with various concentrations of mineral fibres. After a
further 24 hr, the medium was removed and the
cultures were rinsed three times with PBS. The incubation medium and the rinse solution were combined
and counted in a gamma counter to determine the
755e released (in counts/min; cpm). To determine the
intracellular cpm, the cells remaining on the dishes
were solubilized by adding 1.0 N-NaOH to the cultures and the solution was counted. The total cpm
comprised the released cpm plus the intracellular
cpm. To calculate the fraction of radioisotope remaining in the cells, the intracellular cpm was divided
by the total cpm.
Inhibition of cell proliferation 24 and 48hr after
asbestos treatment
To make a direct comparison between the ability
of asbestos to induce membrane damage and its
ability to inhibit cell proliferation, both endpoints
should be measured at the same time after treatment.
Therefore a method was developed to measure cell
proliferation 24 hr after treatment. As in the colony
formation experiments, cells were seeded into culture
dishes at a density of 300 cells/60-mm culture dish.
After 24 hr, the cells were treated with 1/tg chrysotile
asbestos/cm 2 or with complete medium, and after
another 24 hr, the cultures were fixed in methanol,
stained with 10% Giemsa and analysed for cell
microcolonies. The number of cells/microcolony was
determined for each of three control and three
asbestos-treated cultures.
Measurement of phagocytosis and perinuclear accumulation of asbestos
Methods used in measuring the phagocytosis and
perinuclear accumulation of asbestos fibres have been
described in detail elsewhere (Hesterberg et al. 1986).
Asbestos cytotoxicity in epithelial cells
Briefly, 2C5 cells were seeded at a density of
105 cells/60-mm culture dish containing a 25-mm
Thermonox disc (Lux Scientific Corp., Newbury
Park, CA) and 24 hr later the cultures were treated
with either chrysotile or crocidolite asbestos at
1 #g/cm 1. After a further 24 hr, the colonies on the
Thermonox discs were fixed and stained for scanning
electron microscopy as previously described
(Hesterberg et al. 1986). Scanning electron microscopy was used to observe asbestos fibres on the cell
surface, while electron backscatter imaging was used
to observe asbestos fibres that had been phagocytized. In each treatment group, 25 cells were randomly selected and used to measure the following
parameters: (1) the total number of fibres associated
with a cell (cell-associated fibres); (2) the number of
fibres on the cell surface; (3) the number of fibres
partially phagocytized; (4) the number of fibres
totally phagocytized; (5) the number of fibres located
in the perinuclear region of the cell (any portion of
fibre ~<1 p m from the nucleus); (6) the number of
non-perinuclear fibres (no portion of fibre ~<1 # m
from the nucleus); (7) the length (at x6000
magnification) and diameter (at x 10,000 magnification) of each asbestos fibre.
61
40
30
~_ 20
o
d
10
0
I
5
10 15 20 25 )30
Length (p.m)
30
Determination of the incidence of micronuclei and
binuclei
Cells were seeded at 2.5 x 10s/75-cm 2 culture flask
(Falcon Plastics) and 24 hr later the culture medium
was removed and 10 ml of either complete medium or
medium containing suspended asbestos was added to
the flasks. The cultures were then incubated for 48 hr.
The cells were then trypsinized, collected by centrifugation, treated with 0.075 M-KC1 for 5 min and fixed
in methanol-acetic acid (3 : 1, v/v). The suspension of
cells in fixative was dropped onto a glass slide wetted
with 100% ethanol and then air dried. This procedure
allowed nuclei to remain intact in the cell cytoplasm.
The slides were stained with Giemsa and 1000 cells
were scored for each experiment.
= 20
.*,
~6
6 1(3
]
J
0 .10 .15 .20 25 .30 .35 .40
Diometer (/~m)
.45 ~.50
RESULTS
Physical dimensions of chrysotile and crocidolite
asbestos
To determine the potential contribution of the
physical dimensions of the asbestos fibres to their
toxic effects, the lengths and diameters of 100 fibres
of each of the two asbestos types were measured.
Frequency histograms of the length and diameter
distributions are shown in Fig. 1. The mean
l e n g t h _ SEM of UICC chrysotile asbestos was
7.8+0.5/~m
and
the mean diameter was
0.20+ 0.02#m. The corresponding dimensions of
UICC crocidolite asbestos were 7 . 2 _ 1.0/~m and
0.22+0.01/~m. Thus the physical dimensions of
these two asbestos types were very similar.
~C-(c )
4(]
~ 3C
L,.
"6
2o
10
Comparison of asbestos effects on colony formation
and membrane integrity
One of the purposes of these studies was to define
the toxicity of two asbestos types of roughly equal
fibre length/diameter ratios using two different cytotoxic endpoints, inhibition of colony formation and
I
0 i
.I
5 I0 15 20 25 >30
Length (~.m)
Fig. 1. For legend, see overleaf.
62
T.W. HESTERBERGet al.
30
50
A
2O
>o
L
u~
10
6
z IO
n.-
0 .10 .15 20 25 30 .35 .40 .45 >.50
Diometer (/zm)
(a)
lo ;.o 2'.o 3'.o ,'.o d.o 8'.o/o d.o
Fig. 1. Frequency distributions of lengths (a, c) and diameters (<0.I0- > 0.50; b, d) of chrysotile asbestos (a, b)
and crocidolite asbestos (c, d), in 100 fibres of each asbestos
type examined using scanning electron microscopy at a
magnification of × 6000 for length and x 10,000 for diameter. The data on chrysotile asbestos are reproduced, with
the publisher's permission, from Hesterberg & Barrett
(1984) and are included here for comparison with crocidolite
fibre dimensions.
induction of membrane damage. Inhibition of colony
formation of 2C5 cells was determined by plating
cells at low density (300 cells/60-mm dish) 24hr
before treatment with asbestos. The relative survival
of cells after asbestos treatment was calculated by
dividing the colony-forming efficiency of treated cells
by that of untreated control cells. Both chrysotile and
crocidolite asbestos induced dose-related decreases in
the colony-forming ability of 2C5 cells (Fig. 2a).
However, chrysotile asbestos was more toxic than
crocidolite asbestos. The LCs0 values for chrysotile
and crocidolite asbestos were 0.95 and 5.8 #g/cm 2,
respectively. Correction of the data for number of
fibres/cell, taking into account the densities of the two
types of asbestos--2.55 × 10 -6 #g//~m 3 for chrysotile
(Roggli & Brody, 1984) and 3.07 x 10-6#g/jim 3 for
crocidolite (Roggli e t al. 1986)---does not significantly
change the outcome of these experiments.
In Fig. 2b, the results obtained with three different
assays for measuring chrysotile asbestos toxicity,
namely dye exclusion, isotope release and colonyforming ability, are compared. Trypan blue exclusion
was slightly more sensitive than 75Se release in detecting changes in cell membrane permeability induced by asbestos. Both assays showed far less
cytotoxicity resulting from fibre exposure than was
demonstrated by the colony-forming assay. Following incubation of 2C5 cells with chrysotile asbestos at
4 #g/cm 2, > 70% of the cells remained viable according to the dye exclusion test (the isotope release assay
yielded values 80% of controls) compared to only 3%
survival measured by the colony-forming assay. Since
the colony-forming ability depends on the capacity of
cells to undergo several rounds of cell division, we
conclude that fibres dramatically inhibit cell division
at concentrations that have little measurable effect on
cell membrane permeability.
Dose
( / ~ g / e m 2)
E
o
IO
E
(3-
(b)
o ,Io
6'.o
Dose (/~g/crn 2)
Fig. 2. (a) Effects of different doses of chrysotile asbestos
(O) and crocidolite asbestos (©) on the relative survival of
2C5 cells in culture. Relative survival was calculated by
dividing the total number of surviving colonies in five
asbestos-treated cultures by the total number of colonies in
five control cultures. Points are means + SD for at least four
separate experiments. (b) Comparison of the effects of
chrysotile asbestos on three cytotoxic end points in 2C5 cells
in culture: (gl) relative 75Se release, calculated by dividing
the percentage of 75Se remaining intracellular (intracellular
cpm/total cpm) in control cultures by that in asbestostreated cultures; (I-q) relative trypan blue exclusion, calculated by dividing the percentage of dye-excluding cells in
asbestos-treated cultures by that in control cultures; (0)
relative survival, calculated by dividing the colony-forming
efficiency of asbestos-treated cells by that of control cells.
Points are means + SD for four separate experiments.These
results are reproduced, with the publisher's permission,
from Ririe et al. (1985) and are included here to allow
comparison with the other data presented.
Asbestos cytotoxicity in epithelial cells
Inhibition of cell proliferation 24hr after asbestos
treatment
The results presented so far suggest that asbestos
fibres cause inhibition of the proliferation of 2C5 cells
in culture at concentrations much lower than those
required to induce significant measurable changes in
membrane permeability. However, it could be argued
that the greater effect of the fibres on colony formation, compared to membrane permeability, is a
result of the longer duration of exposure in the colony
assay (7 days compared to 24 hr in the dye exclusion
and isotope release assays). Therefore, we measured
inhibition of cell proliferation after a 24-hr exposure
of cells to asbestos.
Cells were seeded at low density and treated 24 hr
later with complete medium or with medium containing chrysotile asbestos. Periodic examination of
the cultures revealed that formation of microcolonies
of cells resulted from cell division and not from
migration of cells together. The number of cells/
microcolony was determined 24hr after treatment
(Fig. 3). Single-cell 'microcolonies' comprised 13% of
the control microcolonies and 52% of the microcolonies in the asbestos-treated cultures at that time.
At the same time, 37% of the control microcolonies
contained eight cells/microcolony, while only 10% of
the asbestos-treated microcolonies were of this size.
Thus 1 #g asbestos/cm 2 clearly inhibits cell proliferation during the first 24 hr of exposure.
The relationship between inhibition of cell proliferation at 24 hr and inhibition of colony formation
at 7 days was determined by allowing parallel cultures
to incubate for 24 hr or 7 days, at which times they
were scored for microcolonies or colonies, respectively. In the 7-day colony-forming assay, colonies of
i> 50 cells were routinely scored, while in the 24-hr
microcolony assay, microcolonies containing four or
more cells were scored. The relative survival in the
7-day colony assay was 24% and compared well with
the relative survival of 29% obtained in the 24-hr
microcolony assay. Thus, the duration of exposure to
asbestos does not affect relative survival significantly.
It is therefore legitimate to compare membrane
63
damage and inhibition of colony formation with the
assays used in these studies.
Phagocytosis of asbestos by cells in culture
The foregoing experiments suggested that fibre-cell
membrane interaction was probably not sufficient to
explain the severe toxicity of asbestos fibres. Previous
studies with SHE cells suggested that severe cell
damage occurred once the fibres were phagocytized
and had become intracellular (Hesterberg et al. 1986).
Therefore, the phagocytosis of asbestos fibres by cells
in culture was examined 24 hr after treatment with
either chrysotile or crocidolite asbestos. As can be
seen in Table 1, cells treated with chrysotile asbestos
contained 2.7-times more fibres at 24 hr than cells
treated with crocidolite asbestos. There appeared to
be little difference in the percentage of perinuclear
fibres for the two asbestos types, but chrysotiletreated cells contained nearly twice as many perinuclear fibres as did crocidolite-treated cells. The
average lengths and diameters of the cell-associated
chrysotile and crocidolite asbestos fibres differed only
slightly from one another, if at all (Table 1). Thus, a
major difference between chrysotile and crocidolite
fibres is how readily they are phagocytized by the
epithelial cells.
Induction of binuclei and micronuclei by asbestos
We showed previously that the frequency of tetraploidy correlated with the frequency of binuclei and
that the frequency of near-diploid aneuploidy correlated with frequency of micronuclei in asbestostreated cells in culture (Oshimura et al. 1984). Since
binuclei and micronuclei are much more convenient
to measure, we used these endpoints to monitor
chromosome changes induced by asbestos in 2C5
cells.
The frequency of binuclei showed dose-dependent
increases over controls 24-hr after treatment with
either chrysotile or crocidolite asbestos (Table 2). A
concentration of 1/~g chrysotile asbestos/cm 2 induced a threefold greater increase in the incidence of
binuclei than an equal concentration of crocidolite
60
SO
o
4o
30
20
10
1
2
3
4
F~
n~
FI~
S
6
7
~
~n
9
10
r~
11
12
r~
13
J--'L~ r ~
14
15
n
16
No. of celts / c L u s t e r
Fig. 3. Inhibition of cell proliferation 24 hr after treatment with chrysotile asbestos. The cells were plated
at 300/dish and treated 24 hr later with chrysotile asbestos (I/~g/cm2). After another 24 hr the total
number of cells/microcolony was counted in three cultures: ( n ) control microcolonies; (1~) asbestostreated microcolonies.
64
T.W.
HESTERBERG et al.
Table 1. Cellular distribution and dimensions of chrysotile and crocidolite asbestos
24 hr after treating 2C5 cells with a concentration of 1/ag/cm2
Mean _+ SEM (and %) for cells
treated with asbestos*
Cell-associated fibres
(location/dimensions)
Chrysotile
Total no./cell
No. of fibres/cell in
different stages of phagocytosist
Surface (not phagocytized)
Partially phagocytized
Totally phagocytized
No. of perinuclear and
non-perinuclear fibres/cell S
Perinuclear
Non-perinuclear
Dimensions§
Length (/am)
Diameter (/am)
Crocidolite
43.8 _+ 0.8 (100)
16.4 _+ 0.4 (100)
9.4 ,+ 0.5 (21)
22.0 ,+ 0.7 (50)
12.5 ± 0.4 (29)
4.4 _+ 0.3 (27)
7.6 _+ 0.4 (47)
4.3 _+ 0.3 (26)
18.8 ,+ 0.6 (43)
25.1 ,+ 0.8 (57)
9.8 _+ 0.3 (60)
6.6 _+ 0.5 (40)
8.4 __%0.4
0.42 ,+ 0.01
10.5 _+ 0.7
0.56 _+ 0.02
*For each asbestos type, 25 randomly selected cells were examined using scanning
electron microscopy. Electron backscatter imaging was used to detect intracellular fibres. This experiment was repeated with similar findings. The mean
number of fibres/cell_+ SEM are presented, with percentage of total cellassociated fibres in brackets.
tSurface = no part of the fibre below the cell surface; partially phagocytized = part
of the fibre inside the cell and part on the surface; totally phagocytized = the
entire fibre inside the cell.
:~Perinuclear = some part of the fibre ~<1/am from the nucleus; nonperinuclear = no part of the fibre ~<1/am from the nucleus.
§Fibre dimensions were determined using electron backscatter imaging at a
magnification of x 6000 for length and x 10,000 for diameter.
asbestos. Thus, at a concentration of 1 /~g/cm2, the
relative potencies of the two asbestos types in the
induction of binuclei reflected their relative potencies
in the inhibition of colony formation (2.8-fold difference) and also how readily they were phagocytized by
the epithelial cells (2.9-fold difference). Chrysotile but
not crocidolite asbestos induced significant increases
in micronuclei at concentrations of 1 and 2 #g/cm 2
(Table 2).
DISCUSSION
One of our objectives was to determine whether rat
tracheal epithelial cells show a sensitivity to asbestos
fibre toxicity similar to that of SHE cells, which
were studied extensively in previous investigations
(Hesterberg & Barrett, 1984 & 1985; Hesterberg et al.
1986; Oshimura et al. 1984). We used two types of
assay to measure asbestos toxicity--a colony-forming
assay and membrane permeability assays (either a
dye exclusion test or an isotope release assay). Using
the colony-forming assay, we found that cells of the
Table 2. Induction of binuclei and micronuclei in 2C5 cells by
chrysotile and crocidolite asbestos 48 hr after treatment
Frequency of binuclei/micronuclei (%) in
cells treated with fibre concns (/ag/cm2) of:
Type of
asbestos
0.0
0.5
Chrysotile
Crocidolite
1.5,+0.5
1.5,+0.0
Binuclei
5.4+1.4"
2.6_+0.7*
Chrysotile
Crocidolit¢
1.2 -+ 0.7
1.2,+ 0.7
Mleronnclei
2.3 -+ 1.4
1.1 -+0.5
1.0
2.0
9.3-+2.3* 10.1 + 0.1"
3.1 ± 1.3" 3.8-+ 1.7"
6.2 -+ 2.7*
1.4_+ 1.1
6.2 _+ 1.9"
2.0-+0.4
In each treatment group 1000 cells were examined. Values are
m e a n s , + S D and an asterisk indicates values significantly
different from the control: P < 0.05 (Mann-Whitney test).
tracheal epithelial cell line 2C5 are highly sensitive
to asbestos toxicity, particularly to the toxicity
of chrysotile asbestos (LCs0, 0.95/~g/cm2), similar to
that (0.9#g/cm 2) found in SHE cells. Crocidolite
asbestos, however, was less toxic to 2C5 cells (LCs0,
5.8pg/cm 2) than to SHE cells (LCs0, 1.7pg/cm2),
perhaps reflecting a differential specificity for phagocytosis of the two cell types. Others have also shown
that chrysotile asbestos is more toxic than crocidolite
asbestos (Chamberlain & Brown, 1978; Davies et al.
1974; DiPaolo et al. 1983; Haugen et al. 1982;
Jaurand et al. 1983; Neugut et al. 1978; Reiss et al.
1980), but the reason for this difference is not known.
The membrane permeability assays showed little
evidence of toxicity, suggesting that membrane damage is only a minor component of the overall asbestos
toxicity in this cell system. The reduction in colony
formation is a sign of mitotic inhibition. Fibres that
have been internalized by cells apparently interfere
with the mitotic process. This was shown to occur in
SHE cells following phagocytosis of fibres (Hesterberg & Barrett, 1985). The end result is formation of
binuclei and micronuclei and, in many cells, mitotic
arrest (and cell death).
Phagocytosis appears to be an essential prerequisite for fibre toxicity, at least in these epithelial cells,
which are surprisingly competent to internalize a
large number of fibres. It follows that fibres that are
not readily phagocytized are less toxic than fibres that
are. This was indeed supported by the finding that
crocidolite, which is much less readily phagocytized
than chrysotile is also much less toxic to 2C5 cells.
As was shown previously (Hesterberg et al. 1986),
phagocytosis and the toxicity of fibres are dependent
on fibre length; short fibres were not readily phagocytized and were less toxic than long fibres. In the
present study we tested another fibre variable; we
compared chrysotile and crocidolite fibres of roughly
Asbestos cytotoxicity in epithelial cells
equal length and diameter. Chrysotile fibres were
readily phagocytized, caused a great amount of
nuclear damage (as evidenced by formation of binucleated cells and of micronuclei) and were highly
toxic, reducing the colony-forming efficiency of 2C5
cells 2.8 times more than occurred with crocidolite.
This is a clear demonstration that, besides fibre size,
other characteristics are also crucial determinants of
fibre toxicity. The important differences between
chrysotile and crocidolite that are responsible for this
difference in toxicity are not known. The two fibres
differ in chemical composition, fibre structure and
surface charge (Harington et al. 1975). It is conceivable that the surface charge is of major importance.
Chrysotile fibres are positively charged and crocidolite fibres are negatively charged. It is possible
that the positively charged fibres bind more readily to
the negatively charged cell membrane (Takata et al.
1981 & 1982; Weiss & Zeigel, 1970) and are consequently more readily phagocytized.
These and the previous studies (Hesterberg
& Barrett, 1984 & 1985; Hesterberg et al. 1986;
Oshimura et al. 1984) seem to establish clearly several
important points regarding fibre toxicity: (1) the
major mechanism of toxicity is mediated by phagocytosis, leading to nuclear damage and mitotic arrest
(this seems to be true at least in those conditions in
which phagocytosis can occur); (2) whether or not
phagocytosis and subsequent nuclear damage occur
depends not only on fibre size (length v. diameter),
but also, to a very large extent, on other fibre
characteristics--it is not clear which characteristics
are critical determinants, but conceivably fibre surface charge may be o f considerable importance.
REFERENCES
Beck E. G. & Bignon J. (Editors) (1985). In Vitro Effects o f
Mineral Dusts. NATO ASI Series G, Vol. 3. SpringerVerlag, New York.
Bignon J. & Jaurand M. C. (1983). Biological in vitro and
in vivo responses of chrysotile versus amphiboles. Envir.
Hlth Perspect. 51, 73-80.
Chamberlain M. & Brown R. C. (1978). The cytotoxic
effects of asbestos and other mineral dust in tissue culture
cell lines. Br. J. exp. Path. 59, 183-189.
Churg A. & Golden J. (1982). Current problems in the
pathology of asbestos-related disease. Path. Ann. 17 (2),
33-66.
Davies P, Allison A. C., Ackerman J., Butterfield A. &
William S. (1974). Asbestos induces selective release
of lysosomal enzymes from mononuclear phagocytes.
Nature, Lond. 251, 423-425.
DiPaolo J. A., DeMarinis A. J. & Doniger J. (1983).
Asbestos and benzo(a)pyrene synergism in the transformation of Syrian hamster embryo cells. Pharmacology
27, 65-73.
Harington J. S., Allison A. C. & Badami D. V. (1975).
Mineral fibers: chemical, physicochemical, and biological
properties. Adv. Pharmac. Chemother. 12, 291-402.
Haugen A., Schafer P. W., Lechner J. F., Stoner G. D.,
Trump B. F. & Harris C. C. (1982). Cellular ingestion,
toxic effects, and lesions observed in human bronchial
epithelial tissue and cells cultured with asbestos and glass
fibers. Int. J. Cancer 30, 265-272.
65
Hesterberg T. W. & Barrett J. C. (1984). Dependence of
asbestos- and mineral dust-induced transformation of
mammalian cells in culture on fiber dimension. Cancer
Res. 44, 2170-2180.
Hesterberg T. W. & Barrett J. C. (1985). Induction by
asbestos fibers of anaphase abnormalities: mechanism
for aneuploidy induction and possibly carcinogenesis.
Carcinogenesis 6, 473-475.
Hesterberg T, W., Butterick C. J., Oshimura M., Brody
A. R. & Barrett J. C. (1986). Role of phagocytosis in
Syrian hamster cell transformation and cytogenetic effects
induced by asbestos and short and long glass fibers.
Cancer Res. 46, 5795-5802.
Jaurand M. C., Bastie-Sigeac I., Bignon J. & Stoebner P.
(1983). Effect of chrysotile and crocidolite on the morphology and growth of rat pleural mesothelial cells. Envir.
Res. 30, 255-269.
Lechner J. F., Tokiwa T., Curren R. D., Yeager H. & Harris
C. C. (1983). Effects of asbestos on cultured human lung
epithelial and mesothelial cells. Proc. Am. Ass. Cancer
Res. 24, 58.
Leibold W. & Bridge S. (1979). 75Se-release:a short and long
term assay system for cellular cytotoxicity. Z. Immun.
Forsch. 155, 287-311.
Nettesheim P. & Barrett J. C. (1984). Tracheal epithelial cell
transformation: a model system for studies on neoplastic
progression. CRC Crit. Rev. Toxicol. 12, 215-239.
Neugut A. I., Eisenberg D., Silverstein M., Pulkrabeck P. &
Weinstein I. B. (1978). Effects of asbestos on epithelial cell
lines. Envir Res. 17, 256-265.
Oshimura M., Hesterberg T. W., Tsutsui T. & Barrett J. C.
(1984). Correlation of asbestos-induced cytogenetic
effects with cell transformation of Syrian hamster embryo
cells in culture. Cancer Res. 44, 5017-5022.
Reiss B., Solomon S., Weisburger J. H. & Williams G. M.
(1980). Comparative toxicities of different forms of asbestos in a cell culture assay. Envir. Res. 22, 109-129.
Ririe D. G., Hesterberg T. W., Barrett J. C. & Nettesheim
P. (1985). Toxicity of asbestos and glass fibers for rat
tracheal epithelial cells in culture. In In Vitro Effects o f
Mineral Dusts. Edited by E. G. Beck & J. Bignon. NATO
ASI Series G, Vol. 3. pp. 177 184. Springer-Verlag, New
York.
Roggli V. L. & Brody A. R. (1984). Changes in numbers and
dimensions of chrysotile asbestos fibers in lungs of rats
following short-term exposure. Expl Lung Res. 7,
133-147.
Roggli V. L., George M. H. & Brody A. R. (1986).
Clearance and dimensional changes of crocidolite asbestos fibers isolated from lungs of rats following short-term
exposure. Envir. Res. In press.
Stanton M. F., Layard M., Tegeris A., Miller E., May M.,
Morgan E. & Smith A. (1981). Relation of particle
dimension to carcinogenicity in amphibole asbestoses and
other fibrous minerals J. natn. Cancer Inst. 67, 965-975.
Steele V. E., Marchok A. C. & Nettesheim P. (1978).
Establishment of epithelial cell lines following exposure of
cultured tracheal epithelium to 12-O-tetradecanoylphorbol-13-acetate. Cancer Res. 38, 2563-2565.
Takata K., Nishiyama F. & Hirano H. (1981). Double
labeling study of anionic sites and concanavalin A
binding sites in monkey macrophages. J. Histochem.
Cytochem. 29, 858-863.
Takata K., Nishiyama F. & Hirano H. (1982). The endocytosis and the intracellular fate ofcationized ferritin in monkey macrophages. Acta histochem, cytochem. 15, 129-138.
Weiss L. & Zeigel R. (1970). Cell surface negativity and the
binding of positively charged particles. J. Cell Physiol. 77,
179-186.