Artículo en PDF
How to cite
Complete issue
More information about this article
Journal's homepage in redalyc.org
Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Contam. Amb. 1, 17-23, 1985
SISTER CHROMATID EXCHANGES INDUCED
BY THINNER IN
Vicia
faba
SANDRA
GOMEZ-ARROYO
AND
PRISCILA
CASTILLO-RUI Z
Laboratorio de Citogenética y Mutagénesis Am-
bientales, Centro de Ciencias de la Atmósfera,
Universidad Nacional Autónoma de México, Co-
yoacán 04510, México, D. F.
ABSTRACT
Root meristem cells of
Vicia faba
were used as the test system for the detection
of sister chromatid exchanges (SCEs) through the fluorescence Plus Giemsa tech-
nique. The results indicate that thinner composed of: 52.0% toluene, 25.5%
n-hexane, 12.5% ethanol, 6.0% ethly acetate, 2.0% isopropanol, 1.Wo benzene and
1.0% n-heptante applied at concentration of 0.003, 0.006, and 0.012% induced
a positive response in the frequency of SCEs.
RESUMEN
Se emplearon células meristemáticas de la raíz de
Vicia faba
para la detección de
intercambios de crometidas hermanas (ICH) mediante la técnica de fluorescencia
mas Giemsa. Los resultados indicaron que el tíner compuesto de: tolueno (52.00/0),
n-hexano (25.5%), etanol
(
12.3%), acetato de etilo (6.Wo), isopropanol (2.09Z0),
benceno
(
1.Wo) y n-heptano
(
I.O%), aplicado en concentraciones de 0.003, 0.006
y 0.012% indujo respuesta positiva en la frecuencia de IC H .
INTRODUCTION
The
use
of higher plants as test systerns for screening and monitoring environmental
mutagens is widely know. Different genetic and chromosomal alterations can be
analyzed in higher plants, and this fact together with other advatages, such as
their ease in handling and their relatively low cost, make them valuable biological
test rissay that could be iiicorporated into the tier testing system (Grant
et
al.
1981).
Vicia
faba
has long been used in numerous radiobiological and cytological studies.
Until now more than 80 different agents have been tested in this plant assay system
using chromosomal aberrations as indicators of genetic damage (Ma 1982).
~mon~
the chemical agents which have been studied in
Vicia
faba, are industrial
solvents which are notable potent toxic substances. Thinners are industrial product~
SANDRA GÓMEZ ARROYO AND PRISCILA CASTILLO RUIZ
that result from a balanced mixture of solvents. They can be used as ingredients of
paints
and
varnishes(Gutiérrez-Flores
1975).
Toxicological investigations with thinners have shown that acute exposure
to
such substances affects the central nervous system causing hypermobility and halu-
cinating conduct (Guzmán-Flores 1975). It has also been observed that thinners
produce anemia and hereditary defects in blood cells (Ferrara-Castre 1976).
In the root-tip meristematic cells of
Vicia faba,
thinner has proven to be an in-
ducer of chromosomal aberrqtions (Gómez-Arroyo 1980). Another cytogenetic tech-
nique for the study o€ low chemical doses is the sister chromatid exchange (SCE)
assay system. A hi& number o€ sister chromatid exchanges can be induced per chro-
mosome by agents known to produce chromosomal aberrations; therefore, Wolff
(1977) has propxed that the quantification of only 20 cells can give statistically
significant results.
Even though most of the investigations directed to analyze the frequency of SCEs
have been performed using animal cells (Latt 1974, Carrano and Wolff 1975), there
have been some studies carried out in higher plants such as
Vicia faha
(Kihlman
and Kronborg 1975, Scheid 1976),
Allium cepa
(Schvartzman and Cortés 1977),
Tradescantia $mludosa
(Grant and Goldstein 1983),
Sacale cereaie
(Friebe 1978)
and
Hordeum v,u¿gare
(Schubert
et al.
1980).
Before analyzing SCEs in plant cells, there are some obstacles which must be
overcome. One of these is the low incorporation capacity that mts have for 5-
bromodeoxyuridine (BrdUrd)
,
which results in poor differential staining. Aiiother
problem is the resistance offered by cell walls that have to be squashed, and finally
the high content o€
RNA
that causes an excessive staining o€ the cytoplasm (Kihl-
man and Andersson 1982).
The Fluorescence Plus Giemsa technique (FPG) which has been adapted to
plants has overcome each of the above obstacles. Low incorporation o€ BrdUrd is
increased by the addition of 5-fluorodeoxyuridine (FrdUrd) (Haut and Taylor
1967), and cell wall resistance to sq&shing can be solved by using enzymes and
acids that soften tissue (=hlman
a@ Andersson 1982). In spite of
the fact
that these modifications result in more elaborate techniques, there are some aspects
of SCEs that can be studied in these sytems in great detail. This is partly due to
the large chromosomes possessed by some plants combined with their low chro-
mosome number
(Vicia faba
and
Trmdescantia 2n
=
12,
Secale cereale
2n
=
14,
Allium cepa
2n
=
16). Al1 these characteristics make higher plant systems escel-
lent biological resource material for analyses of environmental agents (Grant
et al.
1981). One of these assays using SCEs, can be used to determine chromosome
exchanges with great accuracy.
Since it has been shown that thinner induces chromosomal aberrations in
Vicia
faba,
this study was planned to use this same organism, and to determine the
effect of very low concentrations of thinner on meristematic root tip cells using
the sister chromatid exchange assay.
MATERIAL AND METHODS
Seeds of
Vic,ia faba
(var. minar) were set to germinate between two wet cotton
layers, at
21°C
in the dark. They were previously washed in tap water for two
hours, and soaked for 24 hours to accelerate germination. When radicles emerged
SCE INDUCED BY THINNER
(between the 4th and 5th day), the testa was removed to avoid fungus or bacteria1
infection. Those radicles with a length of 2-3 cm were selected and exposed for
one cell cycle time to the following mixture: 100 PM of 5-bromodeoxyridine
(BrdUrd), 0.1 yM of 5-fluorodeoxyuridine (FdUrd) and 5 pM of uridine (Urd).
The root tips were then treated for one hour with 0.003, 0.006 and 0.012%
thinner. The thinner components consisted of
:
toluene (52.0%), n-hexane (25.5%),
ethanol
(
12.5%/0), ethyl acetate (6.0%), isopropanol (2.0%), benzene
(
1.00/0) and
n-heptane
(
1
.O%).
The roots are grown during the second round of DNA replication in the presence
of 100 yM of thymidine (dThd) and 5
pM
of Urd.
The roots are exposed to 0.Ow0 colchicine the last 3 hours before fixation over-
night in cold methanol-glacial acetic acid (3: 1). During the treatmenb, the roots
were not exposed to light and the temperature was 23
+
1°C.
Root .meristem regions were squashed in pectinase on slides previously coated
with a 10: 1 mixture of gelatin and chrome alum (chromium potassium sulfate).
Cover slips were removed and the slides rinsed in 0.5
X
SSC and stained with
a "33258 Hoescht" solution, prepared by adding 1 mg of fluorochrome to 1
m1 of ethanol; then 0.1 m1 of this solution was dissolved in 100 m1 of SSC.
Differential contrast was improved by exposiig the slides to ultraviolet light
(Westinghouse type 5B-30, 250 v), 10 cm from the source for 30 minuta. Follow-
ing irradiation, the slides were incubated for 60 minutes at 55OC in 0.5
X
SSC.
Staining was carried out by using a solution containing 3% Giemsa in tap water
for 7-9 min. Finally, preparations were rinsed in distilled water, air dried, and
dehydrated with xylene. Canada balsam was used as a mounting medium.
For each concentration of the thinner tested, 250 subacrocentric chromosomes
(S) and 50 metacentric chromosomes (M) were scored. These chromosome num-
bers are equivalent to 25 metaphases. Slides were double blinded before scoring.
RESULTS
Each experiment was carried out in duplicate and a Student's "t" analyses was
used to test for statistical significance between the two experiments. No statistical
difference between the two experiments was found.
The results showed that the concentrations of thinner tested (0.003, 0.006 and
0.012%) induced an increment in the frequency of SCEs (Fig. 1).
The mean values obtained (Table 1) increased as concentration increased.
In every case the values proved to be statistically different to the control (p
<
0.001
)
.
DISCUSSION AND CONCLUSIONS
Vicia
faba has proved to be a sensitive and reliable system for the detection of
SCEs induced by various chemical agents. One aspect of induced
SCEs
that has
only been studied in plant cells is the existence of dot
SCEs.
Kihlman (1975)
re-
ported
this phenomenon in
Vicia faba
and later Schvartanan and Cortés (1977)
in
Allium cepa.
The presence of dot or minute SCEs in this work in clearly consistent with
the
Fig.
1.
Sister chromatid exchanges lrequencies induced by thinner.
CONCENTRATION
('/o)
SCE INDUCED BY THlNNER
TABLE 1. SISTER CHROMATID EXCHANGES INDUCED BY THINNER
IN
Vicia faba
Conccntration
SCE
crt'J
valuc
Control
0.003
0.006
0.012
above mentionzd findings. Dot SCEs were observed in control and treated cells,
and their size varied from a minute point to a narrow line that covered the width
of a chromatid.
The fact that minute SCEs have only been reported in plant cells could be
partly due to the large size of the chromosomes which occur in plants and which
make SCEs easy to identify.
The frequency of SCEs seems to be related to the number of rounds of DNA
s~nthesis
to which the cells are exposed to via the base analogue 5-BrdUrd (Schvartz-
man et
al.
1979). Therefore, Kihlman and Andersson (1982) have proposed that
chromosomes which have been exposed to BrdUrd for two rounds of replication
(TB-BB) will show a higher frecuency of SCEs than those exposed for only one
round of replication (TT-TB). In the latter case, the mean frequency of SCEs
would be 23 per metaphase. The results obtained in the present work showed a
higher "spontaneous" mean value of SCEs in chromosomes with a TT-TB
eond-
titution,
a5
these values went from 30 to 32 SCEs per metaphase. The discrepancies
in these studia could be the result of the authors using different varieties of
Vich
faba.
The frequency of
SCEs is also related to the DNA content. According
to Geard and Peacock (1969), the mean number of SCEs in the subwmentric
chromosomes (S) of
Vicia faba
is
1.64
+
0.04, while in the metacentnc
chrmo-
somes (M) of
Vicia faba
it is 4.05
rt
0.16 (taking into account that the content of
DNA is 44 picograms per cell, Baetcke
et
al.
1967). The spontaneous numbers of
SCEs found in this work is consistent with the reports of Geard and Peacock
(1969), in which a mean spontaneous frequency of five was found in the M chro-
mosornes, while a smaller frequency was observed for the S chromosomq.
Until now, a large number of substances have been reported to induce SCEs
in plant and animal systems. Most are classified as S-dependent agents since their
effect on chromosomes is only expressed as an aberration after DNA synthesis has
occurred. Some of the most well-known S-dependent agents are the alkyJating agents
(Bender et
d.
1973). However, two S-independent agents have also
been
reported
to be efficient inducers of SCEs: the antibiotic streptonigrin and long-wave U. V.
According to Kihlman and Andersson (1982) this fact suggests that their effect
is only partially S-independent.
The thinner used in this work has been reported to produce chromosomal aberrn-
tions in an S-independeni manner (Gómez-Arroyo et al. in press), and therefore,
thinner could also be included among those S-independent agents that are able
to induce SCEs. Various types of base damage rather than strand breaks seems
to be the result of DNA lesions induced by these agents so that the formation of
SCEs
occurs
later.
The high number of SCEs induced by different concentrations of thinner in
Vicia
faba means that this biological material is extremely sensitive and useful in
the detection of the demage caused by chemical agents on DNA. From this
data it can also be concluded that the thinner used must be considered a highly
toxic agent with mutagenic activity since genetic damage was induced at very low
concentrations.
ACKNOWLEDGEMENTS
We thank
Drs.
Rafael Villalobos-Pietrini and William F. drant for their critica1
cornments and suggestions to the manuscript, Teresa Romero for her valuable tech-
nical assistance and Erick Estrada Lugo, Subjefe Administrativo del Departamen-
to de Fitotecnia, Universidad Autónoma de Chapingo, for supplying the seeds
used for this study.
REFERENCES
Baetcke K. P., Sparrow A. H., Nauman C. H. and Schewemmer S. S. (1967). The relation-
ship of DNA content to nuclear and chromosomes volumes and to radioscnsitivity (LD,,).
Proc.
Natl. Acad. Sci. (U. S. A.) 58, 533-539.
Bender M. A., Griggs H. C. and Walker P. L. (1973). Mechanisms of chromosomal aberra-
tions production. 1. Aberration induction by ultraviolet light. Mutation Res. 20, 387-402.
Carrano A. V. and Wolff S. (1975). Distribution of sister chromatid exchanges in the
euchromatin and heterochromatin of the Indian muntjac. Choromosoma 53, 361-369.
Ferrara-Castro L. (1976). Estudio de un grupo de menores que inhalan tíner y la obser-
vación de rasgos de personalidad. Cuadernos Científicos CEMEF 5, 3-40.
Friebe B. (1978). Untersuchungen zum
Schwesterchromatidenaustausch
bei Secale cereale.
Microsc. Acta 81, 159-165.
Geard C. R. and Peacock W. J. (1969). Sister chromatid exchanges in Vicia faba. Muta-
tion Res. 7, 215-223.
t
Gómez-Arroyo S. (1980). Efectos cro~bsómicos del tíner y algunos de sus principales com-
ponentes en Vicia faba. Tesis Doctoral. Facultad de Ciencias, UNAM, MCxico.
Grant W. F. and Goldstein L. D. (1983). Sister chromatid exchanges in Tradescantia. A
fit
report. Genetics Soc. Canada Bull. 14, 51.
Grant W. F., Zinov'eva-Stahevitch A. E. and Zura K. D. (1981). Plant genetic test systems
for the detection of chemical mutagens. In: Short-term tests for chemical carcinogens
(H. F. Stich and R. H. C. San, Eds.) Spnnger-Verlag, New York, pp. 700-216.
Guti6rrez-Flores R. (1975). Solventes industriales. Cuadernos Científicos CEMEF 2, 35-48.
Guzmán-Flores C. (1975). Neurobiología del tíner: alteraciones conductuales prodiicidas a
largo plazo. C,uadernos Científicos CEMEF 2, 49-58.
Haut W. F. and Taylor J. H. (1967). Studies of bromouracil deoxyriboside substitution in
DNA of bean mts (Vicia faba). J. Mol. Biol. 26, 389-401.
Kihlman B. A. (1975). Sister chromatid exchanges in Vicia faba. TI. Effects of thiotepa,
caffeine and 8-ethoxycaffeine on the frequency of SCEs. Chromosoma 51, 11-18.
Kihlman B. A. and Andersson H. C. (1982). Sister chromatid exchanges in plants. In:
Skter chromatid exchanges. (S. Wolff, Ed.). Wiley, New York, pp. 243-265.
SCE
INDUCED
BY
THINNER
23
Kihlman B. A. and Kronborg D.
(1975).
Sister chromatid exchanges in
Vicia faba.
1.
Demostration by modified fluorescent plus Giemsa (FPG) technique. Chromosoma
51,
1-10.
Latt S. A.
(1974).
Sister chromatid exchanges, indices of human chromosome damage and
repair: detection by fluorescente and induction by mitomycin C. Proc. Natl. Acad. Sci.
(U. S. A.) 71,
3162-3166.
Ma T. H.
(1982).
Vicia
cytogenetic tests for environmental mutagens. A report of the U. S.
Environmental Protection Agency Gene-Tox Program. Mutation Res. 99,
257-271.
Scheid W.
(1976).
Mechanism of differential staining of BrdU-substituted
Vicia faba
chro-
mosomes.
Exp.
Cell
Res.
101,
55-58.
Schubert
l.,
Kunzel G., Bretschneider H., Rieger R. and Niwloff H.
(1980).
Sister chro-
matid exchanges in barley. Theor. Appl. Genet.
56,
1-4.
Schavartzman J. B. and Cortés F.
(1977).
Sister chromatid exchanges in
Allium cepa.
Chro-
mosoma
62,
119-131.
Schvartzman J. B., Cortés F., Guti6rrez
C,.
and López-Sáez J. F.
(1979).
Dn the nature of
sister-chromatid exchanges in
5-bromodeoxyuridine-substituted
chromosomes. Genetics 92,
1251-1264.
Wolff S.
(1977).
Sister chromatid exchange. Ann. Rev. Genet.
11,
183-201.
logo_pie_uaemex.mx