United States
Environmental Protection
Agency
Office of Health and
Environmental Assessment
Washington DC 20460
Research and Development
EPA/600/S8-86/006 Apr. 1986
Project Summary
Aneuploidy Test Development:
Kinetochore Staining in
Mammalian Systems
Raymond R. Tice and Vicki L Dellarco
The purpose of this project was to
determine the feasibility of using
human-derived antibodies against the
chromosomal kinetochore region cou-
pled with immunof luorescence staining
as a method for evaluating the induction
of aneuploidy in mammalian cells in
vitro and in vivo. This technique was
applied to (1) Chinese hamster cells
(V79 cells), human fibroblasts, and
peripheral lymphocytes interphase cells
in vitro; (2) mouse bone marrow cells in
vivo; and (3) mature human and rat
sperm. Although kinetochore staining
can be accomplished on a routine basis
on mammalian interphase in vitro and in
vivo cells, the technique does not appear
to offer the staining intensity and/or
persistence to allow for an accurate
enumeration of the total genomic com-
plement of a Chinese hamster, mouse
or human cell. It was found that kineto-
chore structures are not visible in nature
before or after techniques to cause
sperm head enlargement. Kinetochore
structures can, most likely, be detected
in micronuclei.
This Project Summary was developed
by EPA's Office of Health and Environ-
mental Assessment. Washington, DC.
to announce key findings of the research
project that is fully documented in a
separate report of the same title (see
Project Report ordering information at
back).
The ability of chemicals to induce
chromosomal malsegregation during cy-
tokinesis in mammalian cells constitutes
a serious human health hazard (Dellarco
et al., 1985). Numerical chromosomal
anomalies (i.e., aneuploidy, the loss or
gain of one or more whole chromosomes
from the chromosomal complement of a
somatic or germ cell) are the major cause
of early gestational spontaneous abor-
tions in humans (Hook, 1986). In addition,
there is increasing evidence that aneu-
ploidy in somatic cells may play a role in
the etiology of carcinogenesis (Barrett et
al., 1986). While short-term tests for the
detection of aneuploidy-inducing agents
exist, few approaches using mammalian
cells in culture, or more importantly, in
the intact mammal, are available. The
majority of mammalian assays involve
direct cytogenetic analysis of the chro-
mosomal complement of metaphase cells.
The validity of this approach is frequently
confounded by technical artifacts associ-
ated with the loss and, to a lesser extent,
the gain of isolated chromosomes during
slide preparation. Thus, there is a need
for additional mammalian cell oriented
tests, especially those that are reliable,
sensitive, and cost-effective.
The recent availability of human-
derived antibodies against the chromo-
somal kinetochore region, as described
by Brenner et al. (1981), coupled with the
use of immunofluorescence staining tech-
niques, suggests another approach for
evaluating the induction of aneuploidy
cells in vitro and in vivo. The purpose of
this project was to determine the feasi-
bility of using the kinetochore antibody
staining technique as developed by B. R.
Brinkley (Baylor Medical College, Hous-
ton, TX) (personal communication) to
enumerate the number of chromosomes
in (1) mammalian interphase somatic
cells in vitro, (2) mammalian interphase
cells in vivo, (3) mature sperm, and (4)
micronuclei in vitro and in vivo.
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It was found that kinetochore staining
can be accomplished on a routine basis
on mammalian interphase cells obtained
from in vitro and in vivo material. How-
ever, the current FITC-dependent immu-
nofluorescence technique does not
appear to offer the staining intensity
and/or persistence to allow for an
accurate enumeration of the total genomic
complement of a Chinese hamster,
mouse, or human cell. This is primarily
due to the three-dimensional structure of
the interphase nucleus on the coverslips.
Suggested future directions for further
research in this area include: (i) the use of
Indian Muntjac cells (n = 5,6); (ii) technical
manipulations to diminish cell thickness;
and (iii) other staining techniques (e.g.,
immunoperoxidase, biotynilation).
The present experimentation showed
that mammalian interphase somatic cells
from in vivo tissues can be examined for
kinetochores. However, if the cells are
not grown on coverslips, additional tech-
nical manipulations are required such as
the use of poly-L-lysine coated coverslips
to increase cell adhesion (Rajendra et al.,
1980). The exact conditions under which
the cells are attached (type of fixative,
etc.) are cell-type-specific and require
additional experimental analyzing to op-
timize conditions.
Kinetochore structures are not visible
in mature sperm that are either untreated
or increased in size by various manipula-
tions to break down sulfhydryl bonds.
Whether the absence of staining results
from an antigenic difference in the kin-
etochore in mature sperm or a technical
artifact remains to be elucidated.
Kinetochore structures can, most likely,
be detected in micronuclei, suggesting an
extremely important role for this assay
system in identifying aneuploidy-inducing
agents that do not destroy the ki netochore
(antigenically speaking). Methods have
been developed for making smears of
mouse peripheral blood for use in the
kinetochore staining technique. However,
the absence of large numbers of micro-
nucleated cells will probably limit the
usefulness of this approach. Collection of
micronuclei by cell sorting or differential
centrifugation after destruction of eryth-
rocytes, may provide a method for col-
lecting appropriate quantities of micro-
nuclei for use in kinetochore evaluation.
It is suggested that these observations
on kinetochore counting in interphase
cells indicate the necessity of other
technical developments and the initial
observations on kinetochore staining in
micronuclei suggest a possible research
tool for the future.
References
Barrett, J. C., Oshimura, M., Tanaka, N.,
and Tsutsui, T. (1986). Role of aneu-
ploidy in early stages of neoplastic
progression of Syrian hamster embryo
cells in culture. In: Dellarco, V. L,
Voytek, P. E., and Hollaender, A. (eds.).
Proceedings of symposium on aneu-
ploidy: Mechanisms and etiology.
Plenum, New York (in press).
Brenner, S., Pepper, D., Berns, M. W., t
Tan, E., and Brinkley, B. R. (1981). "
Kinetochore structure, duplication, and
distribution in mammalian cells: Anal-
ysis by human autoantibodies from
scleroderma patients. J. Cell. Biol.
91:95-102.
Dellarco, V. L, Mavournin, K. H., and Tice,
R. R. (1985). Aneuploidy and health risk
assessment: Current status and future
directions. Environ. Mutagen. 7:405-
424.
Hook, E. B. (1986). The impact of aneu-
ploidy upon public health: Mortality and
morbidity associated with human chro-
mosome abnormalities. In: Dellarco, V.
L, Voytek, P. E., and Hollaender, A.
(eds.). Proceedings of symposium on
aneuploidy: Mechanisms and etiology.
Plenum, New York (in press).
Rajendra B. R., Sciorra, L. A., and Lee, M.
(1980). A new and simple technique for
chromosomal preparations from peri-
pheral-blood lymphocytes, amniotic
cell-cultures, skin fibroblasts, bone-
marrow and single cell clones when
the yields from harvests are low. Hum.
Genet. 55:363-366.
Raymond R. Tice is with Brookhaven National Laboratory, Upton, NY 11973; and
the EPA author Vicki L. Dellarco (also the EPA Project Officer, see below) is
with the Office of Health and Environmental Assessment, Washington, DC
20460.
The complete report, entitled "Aneuploidy Test Development: Kinetochore
Staining in Mammalian Systems," (Order No. PB 86-163 334/AS; Cost: $9.95,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
Office of Health and Environmental Assessment
U.S. Environmental Protection Agency
Washington. DC 20460
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
«'• -*• j/iw !
Official Business
Penalty for Private Use $300
EPA/600/S8-86/006
OC003?9 PS
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