Carcinogenesb vol.6 no.3 pp.473-475, 1985
Short Communication
Induction by asbestos fibers of anaphase abnormalities: mechanism
for aneuploidy induction and possibly carcinogenesis
Thomas W. Hesterberg1 and J. Carl Barrett
Environmental Carcinogenesis Group, Laboratory of Pulmonary Function
and Toxicology, National Institute of Environmental Health Sciences,
Research Triangle Park, NC 27709, USA
Syrian hamster embryo cells were treated with crocidolite
asbestos at a dose (1 /ig/cm2) which was shown in previous
studies to induce cell transformation and aneuploidy in these
cells. Treatment of cells with asbestos induced a > 20-fold
increase in the incidence of cells in anaphase with abnormalities, including lagging chromosomes, bridges, and sticky
chromosomes. Asbestos fibers were observed in mitotic cells
and appeared, in some cases, to be interacting directly with
the chromosomes. From these studies, we propose that the
physical interaction of the asbestos fibers with the
chromosomes or structural proteins of the spindle apparatus
causes missegregation of chromosomes during mitosis
resulting in aneuploidy. These findings provide a mechanism,
at the chromosomal level, by which asbestos and other
mineral fibers might induce cell transformation and cancer.
Asbestos has been associated with an increased incidence of lung
cancer and mesothelioma in man (1—3). However, the
mechanism(s) of cancer induction by asbestos is unknown. It has
been proposed that asbestos acts by epigenetic mechanisms
because it is inactive in gene mutation assays (4), and there is
evidence that asbestos can act as a promoter or cocarcinogen
(5,6). However, results from animal studies have shown that
asbestos treatment alone causes broncogenic carcinomas and
mesotheliomas (7,8). Thus, asbestos may act by direct as well
as indirect mechanisms.
We have recently shown that asbestos and other mineral dusts
induce a dose-dependent increase in morphological transformation of Syrian hamster embryo cells in culture (9). Milling of
glass fibers, which decreases their length without affecting
diameter, reduces the transforming potency of the fibers, implying
that- fiber length rather than chemical composition is important
in the induction of this preneoplastic change. These findings agree
with in vivo studies in which fiber dimension was important in
the induction of mesotheliomas in rats (7).
We have also shown that transforming doses of asbestos fail
to induce mutations at two specific genetic loci but induce a dosedependent increase in chromosome aberrations, especially
numerical chromosome changes (10). Other authors have also
reported chromosome changes following asbestos treatment of
mammalian cells in culture (11,12). We have also shown that
milling of glass fibers not only reduces their transforming potency
but also reduces their ability to induce chromosome changes (10).
Thus, we have proposed that the induction of aneuploidy by
© IRL Press Ltd., Oxford, England.
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'Present address: Department of Genetic Toxicology, Chemical Industry
Institute of Toxicology, Research Triangle Park, NC 27709, USA
mineral fibers is mechanistically important in the induction of
tumorigenicity. In the present study, we attempted to gain a better understanding of the mechanism by which asbestos induces
aneuploidy.
Syrian hamster embryo cell cultures were established from
13-day gestation fetuses (Lakeview Hamster Colony, Newfield,
NJ), cryopreserved in liquid nitrogen, and secondary cultures
were initiated from frozen stocks as described in detail elsewhere
(9). All experiments were performed with tertiary cultures. The
culture medium was IBR-modified Dulbecco's Eagle's reinforced
medium (Grand Island Biological Co., Grand Island, NY) supplemented as described previously (9). International Union
Against Cancer crocidolite asbestos was obtained from V. Timbrell (Medical Research Council, Llandough Hospital, United
Kingdom). Asbestos was weighed and suspended in complete
medium by pipeting back and forth using a 10 rru- pipet.
In order to obtain a large number of mitotic cells, cells were
synchronized by techniques described previously (13). Briefly,
8 x 103 cells in 2 ml of complete medium containing 10% serum
were plated in each chamber of tissue culture chamber/slides
(2-chamber, Lab-Tek Products, Miles Laboratories, Inc., Naperville, IL). After overnight incubation at 37 CC, complete medium
was replaced with medium containing 1 % serum and cultures
were incubated for — 36 h. At this time the medium was replaced with 2 ml of complete medium with 10% serum containing
2 /ig/ml crocidolite asbestos and 0.32 mM hydroxyurea. Hydroxyurea arrests the cells at the G,/S border (13) and allows uptake
of the asbestos fibers. Since the surface area of the bottom of
each chamber was 4 cm2, the final asbestos dose was 1 /tg/cm2.
After 12 h, the cultures were released from hydroxyurea block
by washing once with complete medium and then prewarmed
medium was added.
Between 6 and 9 h after release from hydroxyurea block, when
the maximum number of mitotic cells were present (13), cells
were fixed and stained with safranin-O by a modification of
methods described by Parry et al. (14). Brilliant blue R, which
was used in that study (14), was not employed in the present
study because staining of cytoplasmic proteins obscured asbestos
fibers and small displaced chromosomes. Cells were gently rinsed
by dipping the slides in phosphate-buffered saline and fixed directly with fresh 3:1 methanol:acetic acid containing 4 mM MgCl2
and 1.5 mM CaCl2. After three fixations for 14 min each, the
slides were air-dried overnight and placed in 5 % perchloric acid
solution at 4°C for 24 h. (Note: Caution should be employed
in the handling of perchloric acid because its metal salts, which
can form in metal drain pipes, are explosive.) The slides were
rinsed in several changes of distilled water for 10 min and airdried again. The cells were then stained with a solution of 0.5%
safranin-O (Sigma Chemical Co., St. Louis, MO) in 10% acetic
acid for 24 h, washed in distilled water, and air-dried. Cells in
anaphase were randomly selected and examined for missegregated
T.W. Hesterberg and J.C. Barrett
4
M
B
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c
A
\
Fig. 1. Normal and abnormal anaphases in asbestos-treated Syrian hamster embryo cells. A. Normal anaphase, arrows denote asbestos fibers. B. Anaphase
with lagging chromosome as denoted by arrow. C + D . Anaphases with lagging chromosomes and chromosome bridge. E. Anaphase with sticky chromosomes
and chromosome bridge. F. Anaphase with lagging chromosome.
chromosomes, i.e., chromosomes that were not associated with
the two polar condensations of chromosomes in each anaphase.
The types of anaphase abnormalities that were scored as
missegregated chromosomes were bridges, sticky chromosomes,
and lagging chromosomes. A detailed description of these types
of anomalies is found elsewhere (15).
474
A normal SHE cell in anaphase is shown in Figure 1A. Only
one anaphase containing a single lagging chromosome was
observed in 120 anaphases of cells from control cultures (Table
I). No other anaphase abnormalities were observed in untreated
cells. In contrast, missegregated chromosomes were observed
in 22 out of 120 anaphases in asbestos-treated cultures. Many
Induction by asbestos fibers of anaphase abnormalities
Table I. Induction of anaphase abnormalities by crocidolite asbestos
Treatment
Control
Crocidolite
Total abnormal
anaphascs"
1 (0.8)
22 (18)
Number of anaphases with a given
number of displaced chromosomes
1
2
3
1
8
0
7
0
1
0
6
"Anaphases were selected at random and scored for missegregated
chromosomes, i.e.. chromosomes thai were not associated with the two
polar condensations of chromosomes in each anaphase. For each group
120 anaphases were examined. The percentage of anaphases containing
displaced chromosomes is in parentheses
h
Anaphases having 1. 2. or 3 missegregated chromosomes were relatively
easy to discern. In anaphases having 4 or more displaced chromosomes,
individual chromosomes were difficult to distinguish. These anaphases were
assigned to the £ 4 category. These data represent the combined results of
two separate experiments, the results of which were similar.
Receiwd on 28 September 1984; accepted on 21 November 1984
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475
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of the asbestos-treated anaphases contained more than one displaced chromosome (Table I, Figure 1). Multipolar mitoses were also
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There are a number of mechanisms by which asbestos could
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