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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Conum. Ambient. 4. 57.61, 1988.
A THREE WAY DIFFERENTIAL STAINING PROTOCOL FOR
SCE ANALYSIS DURING THREE SUCCESSIVE CELL
DIVISIONS IN MURINE BONk MARROW CELLS
IN VIVO
PEDRO MORALES-RAMIREZ, TERESITA
VALLARINO-KELLY
AND
REGINA
RODRIGUEZ-REYES
Departamento de Radiobiología, Instituto Nacional de Investigaciones Nucleares. Insur-
gentes Sur No. 1079, 03720, MCxico, D.F. MCxico.
(Recibido abril 1988, aceptado noviembre 1988)
ABSTRACT
A method for the analysis of sister diromatid exchanges
(SCEs)
occurring in thrce subsequent
cell divisions in
uivo,
was developed in murine bone marrow cells. The method was based
on the three-way differential staining of sister chromatids. Mice were injected twice ¡.p.
with BrdU adsorbed to activated charcoal; the fint dose at O h (fint cell cycle) was varied
between 0.1 and 0.25 mg of BrdU/g of body weight and the second dose at 8 h (second
and third cell cycles), was either 1.0, 1.33 or 1.5 mg of BrdU/g of body weight.
Animals were injected with colchicine (¡.p.) two houn before sacrificing and chromosome
prepations were obtained by the usual method. After sister chromatid differentiation by
the method of fluomscence plus Giemsa the cells were scored for well and poor differential
staining and in some cases for SCE frequency in three subsequent cell divisions.
The results indicate that a higher percentage of well-differentiated three way stained cells
were obtained with the combination of BrdU doses of 0.2 and 1.5 mg/g of body weight.
The very low basal frequency of SCE observed in the first cell cycle (0.31-0.46) probably
increases the sensitivity to detected the mutagenic effect by SCE method. This method
can be used for both the analysis of
in uiuo
penistence of lesions involved in SCE in,duction
as well as the detection of sinergism between the mutagen action and the BrdU incorporatioa
to DNA.
RESUMEN
Se describe un metodo
in uiw
para el análisis de intercambio de cromátidas hermanas (ICH)
durante tres ciclos de división sucesivos. El mCtodo está basado en la tinción diferencial
de las cromatidas hermanas en tres tonos. Los ratones fueron inyectados dos veces intra-
peritonealmente con bromodesoxiuridina (BrdU) adsorbida en carbón activado; la prime-
ra dosis al tiempo cero (primer ciclo celular) se varió entre 0.1 y 0.25 mg de BrdU por
gramo de peso y una segunda dosis a las 8 horas (segundo y tercer ciclos celulares) con
1
.O,
1.33 6 1.5 mg de BrdU por gramo de peso corporal.
Los animales fueron inyectados con colchicina (¡.p.) dos horas antes de su sacrificio a las
35 h.
Las
laminillas obtenidas por la técnica usual fueron teñidas siguiendo el método de
fluorescencia más Giemsa. Se cuantificaron las cClulas con buena o pobre aiferenciación
y en algunos casos la frecuencia de ICH, en tres divisiones subsecuentes.
Los resultados indican que el mayor porcentaje de células con sus cromatidas hermanas
teñidas en tres tonos se obtuvo con una combinación de dosis de 6.2 y 1.5 mg de BrdU/g
de peso para la primera y segunda dosis, respectivamente. La frecuencia de ICHs tan baja
que se observó en el primer ciclo de división (0.31-0.46) probablemente aumente la sensi-
bilidad para detectar el efecto mutagénico mediante el análisis de
ICH.
Este método pue-
de ser usado para determinar
in
viuo
la persistencia de lesiones involucradas en la producción
de ICH y el efecto sinérgico entre la acción de los mutágenos y la BrdU incorporada
al
ADN.
INTRODUCTION
The induction of SCEs is considered a good index of the genetic damage caused by
the exposition to mutagens (Latt
et al.
1981). The phenomenon of sister chromatid
exchange (SCE), originally demonstrated by autoradiography (Taylor 1958), has been
amply studied since the development of techniques for the differential staining of sister
chromatids utilizing bromodeoxyuridine (BrdU) (Latt 1974, Perry and Wolff 1974).
The protocol commonly used for the differential staining of sister chromatids
involves the addition of BrdU during at least the first of two successive cell divisions.
The SCE is analyzed in the metaphases of the second cell division, assuming that the
frequency of the SCEs obtained is the sum of that occurred during the first and second
divisions.
Through use of the technique of three-way differential staining of sister chromatids
(TWD), it is possible to determine the frequency of SCE occurring in each of the three
successive cell cydes. Two protocols for TWD
in uitro
have been reported. In that of
Miller (1976), BrdU in decreasing quantities is added during the first two cell cycles
and the cells are analyzed in the third division; whereas, in the method of Schvartzman
and Goyanes (1980), BrdU is added in low concentrations during the first cell cycle
and in higher concentration during the last two divisions. The 1ater.procedure has been
used to analyze the persistence of lesions which induce SCE (Schvartzman
ct al.
1984),
to evaluate the effect of BrdU incorporation to DNA on SCE induction and to estimate
the effect of the BrdU incorporation to DNA on SCE production by mutangens (Morales-
Ramírez
et al.
1987). A modification of this
in vivo
procedure was used to stablish the
persistence of mutagen-induced lesions involved in SCE production (Morales-Ramírez
et al.
1988).
Herein, we report the adaptation of TWD technique (Schvartzman and Goyanes
1980) to murine bone marrow cells
in viuo
using BrdU adsorbed by activated charcoal.
MATERIALS AND METHODS
Animah.
Two to three months old male BalbIC mice weighing between 30 to 35 g, were
used. The animals were maintained in a temperature controled room and fed
ad
libitum
with Laboratory Purina Chow.
BrdU-actiuated charcoal.
BrdU was adsorbed by activated charcoal by mixing a BrdU
solution (20 mglml) with activated charcoal (200 mesh) for 5 min, similar to the method
previously described (Morales-Ramírez
ct al.
1984a).
For three-way diffeyntial staining of sister chromatids
(TWD),
mice were injected twice
(i.p.), with combinations of BrdU doses; the first dose, at O h, was either O. 1, 0.16,
0.2 or 0.25 mg of BrdU per gram of body weight, the second dose, at 8 h, was 1 .O,
1.33 or 1.5. The animals were injected with colchicine 15 fig per g of body weight (i.p.)
two hours before sacrifice at 35 h. The time of sacrifice was chosen to obtain a higher
percentage of cells in the third division, assuming an average generation time of 12.4
h (Morales-Ramírez
et
al.
1984b).
I
BrdU
low
dose
1
BrdU
high dose
ir
A
SCE
ir
!
:n'
:
::
:i
1:
:
*a
:;
1:
.-
-1
:
r:
:
zd
di vision
Ji
il
Fig. 1. Protocol for three-way differential staining of sister chromatids proposed by
Schvartzman and Goyanes (1980). DNA strands with high (dotted lines), low (dashed lines),
or no substitution (solid lines) with
BdU
are represented,
as
well
as
the sister chrornatid differential
staining obtained when SCEs occured in the first, second and third cell cycle
Stuining
and
SCE analysis. The bone marrow metaphases were obtained as was reported
above (Morales-Ramírez
ct
al. 1984a). The sister chromatids were differentially stained
by using a modification (Goto
et
al. 1975) of fluorescence plus Giemsa technique (Perry
and Wolff 1974). The SCE was analyzed in 30 cells per mouse and the exchanges that
had occurred in the first, second and third cell division were determinecl using the
protocol described by Schvartzman and Goyanes (1980) (Fig. 1).
The quality of three-way differential staining was determined from the analysis of two
hundred metaphases per mouse, in at least four anirnals, applying the following criteria:
poorly differentiated (PD) metaphases in which the sister chromatids were differentially
stained but not suficiently contrasted to permit the SCE analysis, and well differentiated
(WD) metaphases in which the contrast between sister chromatid ailowed SCE analysis.
RESULTS AND DISCUSSION
To determine the ratio between the first (first division) and second BrdU dose (second
and third divisions) which permits the most eficient three-way differential staining of
the chromatids, different combinations of doses were tested. The high doses used here
were at least the minimun (1 mg per g weight) required for two-way differential staining
in vivo
in bone marrow cells when using BrdU-activated charcoal method (Morales-
Ramírez
ct
al. 1984a). These doses were used to obtain a better contrast between the
unstained and the darkly stained chromatids, thereby increasing the range to better
distinguish the intermediate tone.
The metaphase figures of third division were classified as well differentiated (WD)
or poorly differentiated (PD) sister chromatids. In the latter category were included
metaphases in which the thiee tones were not suficiently contrkted to allow SCE
analysis. In the experiments, approximately 20 to 50% of the cells in third division
were found to have mitotic figures with only two tones. This was probably due to: the
particular generation time of &ese cells, the duration of BrdU disponibility, &e eficiency
of BrdU incorporation or a combination of these possibilities. Only the cells with well
or poorly differentially stained chromatids were considered for the estimate of the
eficiency of differential staining.
The relation of dose coeff~cient
(second/first) to the quality index (WDIPD) was not
proportional (Fig. 2). The maximum quality was obtained at a dose coeficient of
aproximately 7.5, and the optimal doses were 0.2 mg BrdU per
gram
weight for the
first and 1.5 mg per gram weight for the second.
The frequencies of SCE in the first, second and third cell cycles for the different
combinations of doses are shown in Table 1. The basal frequency of SCE was very low
in the first cycle, which possibly will permit a notable increase in the sensitivity for
detecting the mutagenic effect of genotoxic agents. A direct relation exist between the
doses of BrdU administered in the first and second cycles and their respective SCE
frequencies, this observation suggest that most if not al1 of the SCE were induced by
BrdU as was previously reported (Morales-Ramírez
ct
al. 1987).
It was found that by using this in in
vivo
procedure, a high percentage of cells having
their chromatids clearly differentiated in three tones can be obtained (Fig. 3). The
suggested protocol consists of a first dose of 0.2 mg BrdU (previously adsorbed by
activated charcoal) per gram of bociy weight at zero time, a second dose of 1.5 mg BrdU
IN
VIVO
SCE
ANALYSIS IN SUCCESSIVE CELL DIVISIONS
per gram of body weight eight hours later, and an injection of colchicine (15 pg per
gram weight) at 33 h, and the sacrifice of the animals two hours later.
DOSE COE FFlClENT
(2nd//rt)'
Fig.
2.
Graph of the three-way differentiation quality index (weU differentiatedlpoorly
differentiated)versus the dose coefficient
(2nd
dose/ls' dose). Each point represents the average
of values from four to six animals
TABLE
1.
EFFECT OF BrdU DOSE ON THE FREQUENCY OF SCE IN THE FIRST,
SECOND, AND THIRD CELL DIVISIONS
BrdU Dose
mglg weight
Number
of mice*
1
"dose
SCE/cell/mouse
(XI
SD)
Cell Division
4
0.20
1.5
0.31*0.20
2.3zt1.00
3.1I1.30
16
0.25
1.5
0.4010.15
2.310.78
3.010.66
8
0.25
1 .O
0.4610.12
1.7k0.30
2.610.60
'Thirty cells were scored per animal
This
in
vbo
pmedure may
be
used to stablish the persistente of DNA lesions eliciting
SCE (Morales-Rarnírez
ei
al. 1988), and to determine the effect that BrdU incorporation
to DNA has on the sensitivity to SCE induction by mutagens (Morales-Ramírez in
preparation).
ACKNOWLEDGMENTS
We whish to thank Jorge Mercader, Angel Reyes, Perfecto Aguilar, Felipe Beltrán arid
Enrique Fernández for their excellent technical assistance and Manuel Jiménez for
illustrations.
Fig. 3. Mouse bone marrow metaphases showing three-way differentiaiiy stained chromatids.
SCE occurring in successive cell divisions are indicated (see Fig. 1)
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$. ,
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