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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
Rev. Int. Contam. Ambient. 11 (l), 9-12, 1995
INDUCTION OF MICRONUCLEI IN PERIPHERAL ERYTHROCYTES
OF CYPRINUS CARPIO FISH BY METHYL PARATHION
Júlio César NEPOMUCENO and Mário Antonio SPAN~
Universidade Federal de Uberlandia. Departamento de Biociencias, Bloco D, Carnpus Umuarama. CEP
38.400-
902 Uberlandia (MG)
,
Brasil
(Recibido mayo 1994, aceptado febrero 1995)
Keywords:
Cypinus cap,
micronucleus test, methyl parathion
ABSTRACT
Methyl parathion (MP) is a commonly used organophosphorus insecticide, commercially
available as Folidol 600. This chemical substance was tested to determine micronucleus
(MN) frequencies in peripheral erythrocytes of the fish
Cypnnus ceio
treated
in
vivo.
The concentrations tested were 1.25,2.50,5.00 and 7.50 ppm. Fish were killed after 24 or
48 h of treatment. There
was
a significant increase in the frequency of micronuclei in the
animals treated with 2.50 and 5.00 ppm after
24
h. At higher dosages this effect disap
peared probably due to the increased cell lethality. The frequency of MN induction was
lower at 48 than at 24 h after treatment. There
was
a dosedependent increase in MN fre-
quency which, however, was significant only at the highest concentration (7.50 ppm)
compared to control.
RESUMEN
El metil paratión (MP) es un insecticida organofosforado disponible comercialmente como
Folidol 600. Esta substancia fue probada para determinar la frecuencia de micronúcleos
(MN) en eritrocitos periféricos de peces
Cyprinus
ca+o
tratados
in vivo.
Las concen-
traciones utilizadas fueron 1.25,2.50, 5.00 y 7.50 ppm. Los peces fueron sacrificados 24 y
48 h después del tratamiento. Se notó un aumento significativo en la frecuencia de mi-
cronúcleos en los animales tratados con 2.50 y 5.00 ppm y sacrificados después de 24 h. A
concentraciones mayores este efecto desapareció probablemente debido al incremento
de la letalidad celular. La frecuencia de inducción de MN fue menor después de 48 h
que a 24 h del tratamiento. En la primera hubo una elevación en la frecuencia de
MN
de-
pendiente de la concentración que, sin embargo, fue significativa cuando se comparó
con el testigo solamente en la mayor (7.50 ppm).
INTRODUCTION
Methyl parathion (MP) is a broad-spectrum anticholines-
terase organophosphorus insecticide widely used against
crop pests. In Brazil it is also being employed at piscicul-
ture sites to select the zooplankton and to eliminate pre-
dators. Methyl parathion is commercially available as Foli-
do1 600.
Negative results have been reported in mutagenic assays
with MP on
Salmonella
and
Streptomyces
(Carere
et al.
1978), and positive results in
Salm¿rneIla
with and without
S9 mix (Waters
et al.
1980, Rashid and Mumma 1984,
Breau
et
al.
1985). The compound is probably mutagenic
in
Escha'chia coli,
inducing 5-methyltryptophan (5-MT)
resistance (Mohn 1973a, b). MP is mutagenic in somatic
and germ line cells of
Drosophila
and induces sex-linked
recessive lethals in immature maie germ cells (Tripathy
d
al.
1987).
The results from studies on mammais are also conflict-
ing. Negative results were obtained for chromosome abe-
rrations in bone marrow ceils and spermatogonia, and for
the frequency of pre and postimplantation fetal lethality
in a dominant lethal mutation assay in mice (Degraeve
and Moutschen 1984). MP caused no observable chromo-
some damage either in human cells in
uitro
or in mouse
cells in
vivo
(Huang 1973). However, an increased fre-
J.C. Nepomuceno and M.A. Spanó
quency of chromosome aberrations
was
described in mou-
se bone marrow cells
(Kuinnyi
1975) and of sister chroma-
tid exchanges in human lymphoid cells (Sobti
ei
al.
1982)
and in V79 Chinese harnster ovary cells (Chen
d
al.
1981).
The micronucleus test
(MNT)
was developed in marn-
malian polychromatic erythrocytes of the bone marrow
(Schmid 1975). Nevertheless, it has also been applied to
nucleated piscine erythrocytes in the genotoxicity evalua-
tion of test compounds
in vivo
(Hooftman and de Raat
1982, Hose
et
al.
1984, Manna
d
al. 1985, 1987, Das and
Nanda 1986). This assay can detect agents which cause
chromosome breaks and agents which affect the mitotic
spindle. Thus, micronuclei (MN) are indicators of clasto-
genic or aneugenic effects.
Since micronucleated piscine erythrocytes have been
proved
to
be sensitive indicators of genetic damage, the
purpose of our study was to evaluate the cytogenetic (clas-
togenic or aneugenic) effects of methyl parathion in
Cy-
pznus carpo
fish using the MNT.
MATERLALS AND METHODS
Chemicai agent
Methyl parathion
(0,Odimethyl-0pnitrophenyl
phosphoro-
thioate)
(CAS
NQ 2980(M) is an organophosphoms insec-
ticide commercially available as Folidol 600 (Bayer). The
chemical structure of the compound is shown in
figure
1.
MP was dissolved in water collected from an IBAMA
pisciculture site and diluted to the desired concentrations.
C@'nus
carpa
fish were exposed to methyl parathion con-
taminated water for 24 and 48 h.
Zn
vivo
assay
on peripheral erytbrocytes of
Cyprinus car-
pio
for the detennhtion of micronucleus frequencies
Cyprinus carpio
fish weighing approximately 1.0 g were
purchased from an IBAMA pisciculture site, Uberlbdia
(MG) Brazil, and kept in laboratory aquaria containing
water collected from the same site, in order to maintain
zooplankton concentration and avoid chlorinated tap wa-
ter. The aquaria were aerated frequentiy and the animals
Fíg.
1. Strucairal
formula of the merhyl parathion
were allowed to acclimatize for one week prior to treat-
ment. A batch of 56 fish were equally released in four dif-
ferent aquaria (fourteen animals in each aquarium), con-
taining different concentrations (1.25, 2.50, 5.00 or 7.50
ppm) of MP dissolved in the aquarium water. Fish were
then killed, following 24 and 48 h of treatment. These
time periods were arbitrfly chosen. Fourteen fish kept
in the same kind of water, but without MP, and killed at
the same time intervals served as controls.
Seven fish were used for each concentration level and
for each sampling period, including the controls. Fish
were cut
in
the caudal region, and blood smears were im-
mediately prepared and fixed in absolute methanol for
15 min. On the following day, the material was stained
with Giemsa diluted 1:20 in sodium phosphate buffer, pH
6.8, for 10 minutes. A total of 2,000 cells, 500 per slide,
from each individual were examined microscopically un-
der 1000 X magnification. Coded and randomized slides
were scored using blind review by a single observer. The
number of micronuclei (MN) and micronucleated cells
(MNC) were expressed per 100 ceils.
The data were analyzed statistically by the conditional test
for the detection of rare events (Pereira 1991), with the
level of significance set at
a
=
0.05.
RESULTS AND DISCUSSION
The frequencies of MN and MNC observed in
Cyprinw
carpo
treated with MP for 24 h were significantly higher in
fish treated with 2.50 and 5.00 pprn (P
<
0.008 and 0.005,
respectively) than those in control fish. At a concentration
of 7.50 ppm, the increase in frequency was not significant
(P
>
0.07), probably due to the increased cell lethality
(Ta-
ble
1).
An inconsistent result was found in this experi-
ment. The frequency of MN and MNC observed in fish
treated with 1.25 pprn of MP for 24 h
was
significantly
le
wer than that in controls (P
<
0.005)
(Table 1).
High frequencies of MN and MNC were expected to
be found in the controls, based on previous observations
of high incidence of MN in fish
(Oreochromis mossambiczls)
collected from the same site of pisciculture (IBAMA-
Uberlhdia) due to environmental pollutants (Nepomu-
ceno and Spanó 1992).
On the other hand, the frequencies of MN and MNC
present in
Cypinw
capo
treated with MP for 48 h were
significantiy higher (P
<
0.005) only in fish treated with
7.50 pprn
(Table
11).
For 1.25, 2.50 and 5.00 pprn doses,
the data on MN and MNC did not differ significantly
from the respective controls (P
>
0.4, 0.08 and 0.06, res-
pectively)
(Table
U). The lower frequencies of micronu-
clei obtained after 48 h of treatment compared to those
obtained after 24 h maybe due to the toxicity of MP
leading to cell death.
MICRONUCLEI IN FISH ERYTHROCYTES BY METHYL PARATHION
TABLE
1.
FREQUENCY OF MICRONUCLEATED CELLS (MNC) AND MICRONUCLEI (MN) IN PERIPHERAL
ERYIHROCYTES OF
CYPRINUS
CARPIO
FISH EXPOSED TO METHYL PARATHION CONTAMINATED WATER
FOR
24
HOURS
Dose
No. of
Total
Total
Total
%(%)
I
SD
(pprn)
indiv.
ceiis
MN
MNC
MN
MNC
0.00
7
14000
72
7 1
0.51
I
0.35
0.50
+
0.35
1.25
7
14000
41*
41*
0.29
I
0.20
0.29
+
0.20
2.50
7
14000
105*
102*
0.75
I
0.32
0.72
I
0.30
5.00
7
14000
278*
275*
1.98
I
2.58
1.96
I
2.55
7.50
7
14000
9 1
9 1
0.65
*
0.57
0.65
?
0.57
*
Significantly different from the control according to the conditional test for the detection of rare events (Pereira 1991) with the leve1 of significancr set
at
a
=
0.05.
Most of the earlier studies with organophosphates
used bacteria1 test systems and the reports on mammalian
test systems were mostly negative possibly due to the toxi-
city and the rapid degradiation of the test chemicals
in
in
riirio
and
in vitro
experiments (Wild 1975).
The induction of 5-MT resistance mutations in E.
coli
shows that MP is probably mutagenic. However, under
the experimental conditions descnbed, the compound is
not significantly mutagenic at doses which do not inacti-
vate the cells, whereas at higher doses an increase in rela-
tive mutation frequency occurs with a parallel decrease in
survival (Mohn 1973a).
Incubation of human lymphoid cells (M-007) with
20 p/ml caused a significant increase in SCEs. However,
cytotoxic effects of MP were dose related and led to cell
death (Sobti et al. 1982).
Wild (1975) described the general aspects of the che-
mistry, toxicity and metabolism of organophosphates.
According to Breau
et
al. (1985), the most mutagenic phos-
phorothioates are those containing strong electron-with-
drawing substitutes (as in MP)
.
Kurinnyi (1975) observed chromosome lesions in bo-
ne marrow of mice treated with 10 mg/kg MP, consider-
ing this compound to be slightly clastogenic.
There is a lack of information about the formation
and frequency of micronuclei in the haeinatopoietic
tissues of
Cypnnus carpio
fish. There is no information
about the time taken for the completion of the mitotic
cycle in blood cells, nor about the time taken for the blood
cells to reach the peripheral circulation. Because of this,
we cannot assure that micronuclei found had been formed
during haematopoiesis at different times (24 or 48 h) of
TABLE
11.
FREQUENCY OF MICRONUCLEATED CELLS (MNC) AND MICRONUCLEI (MN) IN PERIPHERAL
ERYTHROCYTES OF
CYPRINUS
CARPIO
FISH EXPOSED TO METHYL PARATHION CONTAMINATED WATER
FOR
48
HOURS
Dose
No. of
Total
Total
Total
x
(%)
I
SD
(PP~)
indiv.
ceiis
MN
MNC
MN
MNC
7.50
7
14000
168*
168*
1.20
I
1.85
1.20
I
1.85
*
Significantly different from the control according to the conditional test for the detection of rare wenu (Pereira 1991) with the leve1 of significance set at
a
=
0.05.
J.C. Nepomuceno and MA. Spanó
treaunent with MP. However, in spite of these considera-
tions, based on our data and on previous reports, we may
conclude that MP has genotoxic effects.
Although organophosphates are not persistent in the
environment and do not pose a serious residue problem
(Wild
1975),
the continued and increased use of these
pesticides may lead to unintentional deleterious effects
on the environment and on animal and human health.
ACKNOWLEDGMENTS
We are grateful to IBAMA (Uberlhdia, MGBrasil) which
provided the animais for this study. This work was suppor-
ted by Universidade Federal de Uberlhdia and
CNPq.
The authors are aiso indebted to two anonyrnous referees
for criticai reading of the original manuscript.
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