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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. 6,69-74,1990.
CYTOGENETIC EFFECTS PRODUCED BY THE UREIC HERBICIDES DIURON AND
LINURON IN Vicia faba AND HUMAN LYMPHOCYTES CULTURES
Sandra G~MEZ
-
ARROYO, Josefina CORTES
:.
ESLAVA, Armando RAM~REZ
-
DOM~NGUEZ, Alfredo GUTIERREZ
-
ABAD and Rafael VILLALOBOS
-
PIETRINI
Laboratorio de Citogenética y Mutagénesis Ambientales, Centro de Ciencias de la Atmósfera, UNAM,
Coyoacán 04510
D.F.,
México
(Recibido julio 1990, aceptado octubre 1990)
Keywords: sister chromatid exchanges, ureic herbicides, diuron, linuron,
Vicicl
faba,
human lympho-
cytes
ABSTRACT
In order to evaiuate the cytogenetic effect of diuron and linuron, sister chromatid
exchanges (SCE) in cells of Vicia faba root tips and
in
humanlymphocytesin culture
were used as test systems. Both herbicides did not induce SCE in these biologicai
materids except at the lowest concentration of linuron
(1
0 ppm) which produced a
significant response only in Vicia.
RESUMEN
Se verificó el efecto citogenktico de los herbicidas ur6icos diurón y linurón, median-
te el análisis de intercambio de cromátidas hermanas (ICH) en las células de las
puntas de las raíces de Vicia faba y en el cultivo de linfocitos humanos, usadoscomo
sistemas de prueba. Ambos herbicidas no indujeron ICH en los materiales biológicos
empleados excepto el linurón a la concentración menor (10 ppm) que produjo
respuesta significativa solamente en Vicia.
INTRODUCTION
The ureic herbicides are derived from urea (H2NC(=O)NH2). They are solid crystals with
low solubility in water (Metcalf 197 1, Barberai 1976). It seems that the herbicide activity
is .a general property of the phenyl-N-N-dimethylurea structure.-The phenyl nuclei can be
substituted by chloro or nitro groups and its activity increased with ortho or para substitu-
tion (Metcaif 1971).
It is considered that the ureic herbicides disturb photosynthesis in which the primary
site of action is located in the photosystem 11 within, or near to, the phase envolving
oxygen (Asthon and Alden 1973).
Diuron (343,
4dichlorophenyl)-1-1-dimethylurea)
and linuron (343,4-dichlorophe-
ny1)-
1
-methoxi-
1
-methylurea) are Iireic herbicides.
Diuron is aiso known by its trade names karmex, marmer, DCMU, di-on, karmex-DL
and
sup'rflo. It is a compound of substituted ureas used as a herbicide in pre- and post-
emergent applications for such crops as cotton, apples, papaw, peas, alfalfa, bananas,
S. Gómez-Arroyo et
d.
pineapples, etc. In mice it caused irritability of the eyes and
skin
and the
LD5
o
was 3400
mg/kg (Thomson 1975, BarberA 1976). In protein deficient rats, diuron caused cholinergic
stimulation, depression of the central nervous system, local gastroenteritis, organ
inflarnmation and degenerative changes in the kidney, liver and pancreas (Boyd and
Kmpa 1970).
In relation to genetic effects, diuron did not induce mitotic gene conversion in yeast
(Siebert and Lemperle 1974), however it produced diuron resistant mutants in AnacystW
nidulans
RZ
(Golden and Haselkorn 1985), depressed thymidine incorporation in testi-
cular DNA of mice and in bacteria1 test demonstrated weak mutagenic activity (Seiler
1978). In some microbial systems (Salmonella and Escherichia) diuron did not induce
mutations (Moriya et al. 1983) and in the micronucleus test in mice it was inactive also
(Seiler 1978).
Linuron, with the trade names of lorox, afalon, prefalon, and sarklex, is a substituted
urea, used for pre- and post-emergent applications in crops of potatoes, cereals, sorghum,
cotton, celery, etc. At higher concentrations linuron is employed as soil sterilizer. In mice,
linuron causes irritation in the eyes, nose and skin and its
LD5,
is 1500 mg/kg (Thomson
1975, Barberá 1976).
Linuron is a compound that cytologicaliy prevented ceil wall formation(Grant 1973),
in maize induced chlorophyll mutations (Morgun 1982), in mice inhibited thymidine in-
corporation into testicular DNA (Seiler 1978), and chomosome clumping (Grant 1973).
However in bacteria1 systems linuron did not induce mutations (Andersen et al. 1972,
Marshall et al. 1976, Seiler 1978, Moriya et al. 1983) nor induced micronuclei in mice
(Seiier 1978).
Due to the fact that these herbicides are widely used in MBxico and because of the
few studies and the contradictory results obtained on their effect on the genetic material,
in this study the cytogenetic damage of these two herbicides is analysed by means of the
sister chromatid exchange (SCE) assay which is very sensitive in evaluating the genetic
activity of chemicals (Perry and Evans 1975). For thk purpose root tip meristems of
Vicia faba and human lymphocytes in culture have been used, as the SCE assay has been
shown to be most useful for this type of study (G6mez-Arroyo et al. 1987, 1988).
ViSia faba
MATERIAL AND METHODS
Seeds were germinated between two cotton layers wetted with tap water.The primary
roots that reached 2-3 cm in length were introduced into a solution containing 100
ph4
5-bromo-2'deoxyuridine (BrdUrd), 0.1
M.4
5-fluorodeoxyuridine (FdUrd) and 5
phí
of
uridine (Urd) for 20 h. Then they were treated for 2 hin solutions containing the following
concentrations of diuron (Dupont) and linuron (Helios): 10,20,40,50,75, 100,150 and
300 ppm. Fresh solutions of BrdUrd, FdUrd and Urd were apglied for a second replicate
cycle (20 h). The treatments were carried out in the dark at 19 C. Controls were exposed
to distilied water and underwent the same procedure.
After this, the meristems were cut and treated with colchicine 0.05% for 3 h and
stained
wing
the Feulgen differential technique described by Tempelaar et
al.
(1982)
CYTOGENETIC EFFECTS PRODUCED BY UREIC HERBICiDES
modified as follows: cuttims were fmedowith glacial acetic acid for 1 h, then put in
ethanol-acetic acid (3: 1) for 2 days at -20 C and later
in
ethanol70%
for 15 mirl and after
hydrolysis in 5N HCl for 80 rnin at 28'~. They were then washed 3 times with distilled
water and stained with the Sbhiff reagent (Feulgen staining) for 12 rnin in the dark. Then
cuttings were treated with pectinase 2% disolved'in 0.01M citrate buffer (pH 4.7) for 15
rnin at 28O~,
followed by acetic acid 45% for 10 rnin and then fmaily transferred to cold
ethanol70%
for 30 min.
The squash was done in 45
%
acetic acid and the slides were made permanent by the
dry-ice technique (Conger and Fairchild 1953), dehydrated by means of two changes
in
absolute butano1 and then mounted in Canada balsam.
Human lymphocy tes
Cultures of peripheral blood of healthy individuals were made using the same donor
for each experiment and its replicate.
Eight drops of blood were put on a culture glass having 3 m1 of McCoy'gSA (Micro-
lab) plus 0.2 m1 of phytohemagglutinin (Gibco). This was incubated at 37 C for 24 h
then BrdUrd was added to the culture medium at a final concentration of 5 &m1 simul-
taneously with the corresponding concentrations of diuron and linuron (10, 20, 40, 50,
75, 100, 150 and 300 ppm). 70 h after the begining of the culture, 0.04 pg/ml of colchi-
cine (Merck) was added and two hours later the cells were harvested by centrifugation
and the peilet resuspended in hypotonic solution of 0.75M KCl for 20
min.
Cells were
again centrifuged and fmally fixed in methanol-acetic acid (3:l). The slides were made
by placing a drop of cell suspension onto the slide and ailowing this to dry in air and were
immersed for 20 rnin in a solution of 0.05 pdml of Hoechst-33258 in deionized water in
the dark, then rinsed and mounted in phosphate buffer of 6.8 pH with a coverslip, then
irradiated with UV light for one hour, after removal of the cover-glass, slides were stained
in Giemsa distilled water (1 50) for 30 min.
The sconng of
SCE
was made on 25 metaphase cells for each experiment and its
replicate in both
Vicia faba
and human lymphocytes. Statistical analysis was applied by
means of the Student
t
test. Slideswere manipulated in code so that it was not known to
which group they belonged.
RESULTS
AND
DISCUSSION
There were no significant differences
in
the data obtained with diuron in
SCE
fre-
quencies
in
Vicia faba
(Table 1) as weii as in human lymphocytes (Table 111). These nega-
tive results were in agreement with those reported for mitotic gene conversion using the
loci ade
2
trp
5 of
Saccharomyces cerevisiae
(Siebert and Lemperle 1974), the mutants
rII
of the
Te
bacteriophage (Anderson
et
al. 1972), mutations
in
Salmoneiia typhimurium
and
Escherichia coli
(Moriya
et al.
1983) and micronuclei data from mice erythmcytes
(Seiler 1978). However, diuron was also reported to strongly inhibit testicular DNA
synthesis in mice (Seiler 1978), induce sex-linked recessive lethal mutations in
Drosophila
melaaqgaster
(Rodrfguez-Arnaiz
et al.
1989), mutation to resistance in
Anacystisnidulans,
mutation of
Salmonella typhimurium
in the presence of marnmah microsomes (Seiler
S. Gómez-Arroyo et
d.
TABLE
1.
SISTER CHROMATID EXCHANGES
(SCEs)
INDUCED
BY DIURON IN
Vicia fabat
Treatment
(PP~J
t value
t
n
=
50 metaphases
in
2 experiments
NS
not significant
TABLE 11. SISTER CHROMATID EXCHANGES
(SCEs)
INDUCED
BY LINURON IN
Vicia fabat
Treatmen t
S@s/cell
t value
(PP~)
X
f
SE
28.36 f
0.53
33.07 f 0.59
Celiular death
+n
=
50
metaphases in
2
experiments
*
p<
0.001
TABLE 111. SISTER CHROMATiD EXCHANGES
(SCEs)
INDUCED
- - - -
-
- - -
EíLI1IZIRONLN HUMAN LYMPHOCYTES
in vitrot
Trearmen t
SCEslcell
t value
(PP~)
XfSE
O
5.56 f 0.27
20
5.96 f 0.3
1
0.69
NS
40
7.10 f 0.43
2.19
NS
5 O
7.78 f 0.43
3.15
NS
7
5
7.48
+
0.38
2.93
NS
1 O 0
5.64 f 0.27
0.16
NS
150
6.24 f 0.39
1.02
NS
300
6.30 f 0.20
1.52
NS
f
n
=
50
metaphases in
2
experiments
NS
not significant
CYTOGENETIC EFFECTS PRODUCED BY UREIC HERBICIDES
TABLE IV. SISTER CHROMATID EXCHANGES
(SCEs)
INDUCED
BY LINURON IN HUMAN LYMPHOCYTES
in vitrot
Treatm en t
SQfs/cell
t vdue
(PP~)
X~SE'
6.78
f
0.34
6.88
f
0.3 1
Celiular death
p
n
=
SO
metaphases in 2 experiments
NS
not significant
1978, Colden and Hasekorn 1985), and mutation for resistance in
Saccharomyces.cere-
visiae
(Meunier and Colson 1989).
Linuron was tested from a low of 10 ppm up to 300 ppm, but it was found that 20
ppm was very toxic and induced celi death. In
Vicia faba,
10 ppm produced a significant
leve1 of
SCE
response (Table 11). However, the same concentration did not induce
SCE
in
human lymphocytes
in vitro.
Andersen
et al.
(1972), Marshail
et al.
(1976) and Moriya
et d.
(1983) found negative
results in
Saimoneíla typhimurium,
but on the other hand, Seiier (1978) described inhib-
ition of testicular DNA synthesis in mice, Wuu and Grant (1966) obsewed mutations in
Hotdeum vulgare
and Morgun (1982) induced chlorophyll mutations in maize.
The fact that linuron produced genetic alterations only in plants is possibly due to
promutagen activations caused by plant metabolic processes (Plewa
et al.
1988). This
could originate from the microsomal fraction
S-1
O d e s c r i b e d in
Vicia faba,
which acted in
a similar way to mammalian liver
S-9
(Takehisa and Kanaya 1983, Takehisa
et
al. 1988).
ACKNOWLEDGEMENTS
We thank Norma Noriega, Biol. Miguel Angel Meneses and M. en C. Ana Rosa Flores
for their wortwhile technical collaboration.
REFERENCES
Andenen K.J., Leighty E.G. and Takahashi M.T. (1972). Evaluation of herbicides for
possible mutagenic properties. J. Agnc. Food Chem. 20,649456.
Asthon F.M. and Alden S.C. (1973).
Mode
of
action of herbicide.
Wiley, New York. pp.
10-24,367-393.
Barberá C. (1976).
Pesticidas agrícolas.
Omega, Barcelona, pp. 399-412.
Boyd E.M. and Kmpa V. (1970). Protein deficient diet diuron toxicity. J. Agr. Food
Chem. 18, 1104-1 107.
Conger A.D. and Fairchild L.M. (1953). A quick-freeze method for making smear slides
permanent. Sta Technol. 28,28 1-283.
Golden S.S. and Haselkom R. (1985). Mutation to herbicide resistance maps within the
psbA
gene of
Anacystis nidulans
R2. Science 229, 1
104-1
107.
S.
Gómez-Arroyo
et
d.
Gbmez-Arroyo S., Noriega-Aldana N., Juárez-Rodrlguez D. and Villalobos-Pietrini R.
(1987). Sister chromatid exchanges induced by the organophosphorus insecticides
methyl parathion, dimethoate, phoxim and methyl azinfos in cultured human lym-
phocytes. Contam. Ambient. 3.63-70.
Gbmez-Arroyo S., Castiilo-Ruiz P
.,
Cortés-Eslava J. and Villalobos-Pietrini R. (1 988).
Vicia faba-sister chromatid exchanges of the organophosphorus insecticides methyl
parathion,dimethoate, oxydemeton methyl, azinphos methyl and phoxim. Cytologia
53,627634.
Grant W.F. (1973). Cytological effects of environmental mutagens-pesticides. Mutat. Res.
21,221-222.
~arshail
T.C., Dorough H.W. and Swim H.E. (1976). Screening of pesticides for mutagenic
potential using Salmonella fyphimunum mutants. J. Agric. Food Chem. 24, 560-563:
Metcalf L.R. (1971). The chemistry and biology of pesticides. In: Pesticides in the envi-
ronment. Vol. 1 part. 1, M. Dekker, New York. pp. 49-5 1.
Meunier B. and Colson A.M. (1989). Increased diuron resistance in the joint expression of
mutations located at the DZV2, DZV3 and DIV4 loci of Saccharomyces cerevisiae.
Curr. Genet. 15, 121-127.
Morgun V.V. (1982). Cytogenetic and genetic activity of herbicides: atrazin, simazin,
promet~
and linuron. Tsitol. Genet. 26.33-41.
Monya M., Ohta T., Watanabe K., Miyazawa T., Kato K. and Shirasu Y. (1983). Further
mutagenicity studies on pesticides in bacteria1 reversion assay systems. Mutat. Res.
116, 185-216.
Perry P. and Evans H.J. (1975). Cytological detection of mutagen-carcinogen exposure
by sister chromatid exchange. Nature 258, 121-124.
Plewa M.J., Wagner E.D. and Gentile J.M. (1988). The plant/microbe coincubation assay
for the analysis of plant-activated promutagens. Mutat. Res. 197, 207-219.
Rodriguez-Arnaiz R., Ramos Morales P., Gaytán Oyanun J.C., Rodriguez Ziifiiga D.L.
and Zimmering S. (1989). The herbicides daiapon and diuron tested for genotoxicity
in
Drosophila melanogaster. Rev. Int. Contam. Ambient. 5, 59-64.
Seiler J.P. (1978). Herbicidai phenylalkylureas as possible mutagens.
1.
Mutagenicity tests
with some urea herbicides. Mutat. Res. 58, 353-359.
Siebert D. and Lemperle E. (1974). Genetic effects of herbicides: induction of mitotic
gene conversion in Saccharomyces cerevisiae. Mutat. Res. 22, 1 1 1- 120.
Takehisa S. and Kanaya N. (1983). A comparison of Vicia faba-root S10 and rat-liver S9
activation of ethanol, maleic hydrazide and cyclophosphamide as measured by sister
chromatid exchanges induction in Chinese hamster ovary cells. Mutat. Res. 45, 263-
270.
Takehisa S., Kanaya N. and Rieger R. (1988). Promutagen activation by Vicia faba: an
assay based on the induction of sister-chromatid exchanges in Chinese hamster ovary
celis. Mutat. Res. 197, 195-205.
Tempelaar M.J., de Both M.T.J. and Versteegh
J.E.G.
(1982). Measurement of SCE
frequencies in plants: a simple Feulgen staining procedure for Vicia faba. Mutat. Res.
103,321-326.
Thomson W.T. (1975). Agricultura1 chemicals books. 11. Herbicides. Thomson Ed., New
York. pp. 157-180.
Wuu K.D. and Grant W.F. (1966). Morphological and somatic chromosomai aberrations
induced by pesticides in barley (Hordeum vulgare). Can. J. Genet. Cytol. 8,481-501.
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