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Sistema de Información Científica
Red de Revistas Científicas de América Latina y el Caribe, España y Portugal
GENOTOXIC EFFECT OF AZINPHOS METHYL IN BACTERIA AND IN HUMAN
LYMPHOCYTE CULTURES AFTER PLANT ACTIVATION
Sandra GÓMEZ-ARROYO
1,*
, Liliana SÁNCHEZ-ESTRADA
1
, Selene ANDRADE-MORALES
1
,
Josefna CORTÉS-ESLAVA
1
and RaFael VILLALOBOS-PIETRINI
2
1
Laboratorio de Genotoxicología Ambiental,
2
Laboratorio de Mutagénesis Ambiental, Centro de Ciencias de
la Atmósfera, Universidad Nacional Autónoma de México, Ciudad Universitaria, Coyoacán, México, D.F.,
C.P. 04510
* Corresponding author slga@atmosfera.unam.mx
(Recibido agosto 2014; aceptado enero 2015)
Key words:
azinphos methyl,
Salmonella typhimurium
, human lymphocyte cultures, plant metabolism, sister
chromatid exchange
ABSTRACT
The evaluation of the potential risk of pesticides applied to crops consumed by humans
in Mexico is appropriate and necessary because plant pro-mutagenic transformation
in toxic metabolites and their subsequent incorporation involve a risk for health when
such crops are ingested. Plant metabolism oF agricultural insecticides produces com
-
pounds that could be introduced in the food chain, increasing the contamination and
poisoning risk by agrochemical metabolism. In this study we evaluated the eFFect oF
the organophosphorus insecticide azinphos methyl transFormed by S10 Fraction oF
broad bean (
Vicia faba
), using as indicator of mutagenic damage the reverse muta-
tion
of
Salmonella typhimurium
strains TA98 and TA100 and the sister chromatid
exchange (SCE) in human lymphocyte cultures. Results of mutagenicity showed that
when
Salmonella
TA98 and TA100 strains were treated directly with azinphos methyl,
negative response was obtained. The same occurred with human lymphocytes tested
directly with this insecticide. When
Vicia faba
S10 enzymatic mix was added, there
was a mutagenic response in both
Salmonella
strains. These results suggest that the
mechanisms to induce mutations by azinphos methyl were frameshift mutation (TA98
strain) as well as pair bases substitution (TA100 strain). Likewise, SCE production was
signifcant and dose-response relationship was observed in human lymphocyte cultures.
The cell kinetics (M1, M2 and M3 cells), the replication index and the mitotic index
are also analyzed. Only in the treatments with S10 Fraction the eFFects were observed.
At the highest concentration mitotic inhibition was produced.
Palabras clave: azinFos metílico,
Salmonella typhimurium
, cultivo de linfocitos humanos, metabolismo de
plantas, intercambio de cromátidas hermanas
RESUMEN
Es conveniente y necesaria la evaluación del riesgo potencial de la aplicación de pla-
guicidas a los cultivos de consumo humano en México debido a que las plantas a través
de la activación metabólica son capaces de transformar promutágenos en metabolitos
Rev. Int. Contam. Ambie. 31 (3) 227-236, 2015
S. Gómez-Arroyo
et al.
228
tóxicos y su subsecuente incorporación involucra un riesgo para la salud cuando tales
cosechas se ingieren. El metabolismo de insecticidas agrícolas produce compuestos
que pueden introducirse en la cadena alimenticia, con lo que se incrementa la conta-
minación y el riesgo de envenenamiento por la transformación de los agroquímicos.
En este estudio se evaluó el efecto del insecticida organofosforado azinfos metílico
transformado por la fracción S10 del haba (
Vicia faba
). Se utilizó como indicador de
daño la mutación reversa en las cepas TA98 y TA100 de
Salmonella typhimurium
y el
intercambio de cromátidas hermanas (ICH) en cultivo de linfocitos humanos. Los resul
-
tados de mutagenicidad mostraron que cuando las cepas TA98 y TA100 de
Salmonella
fueron tratadas directamente con azinfos metílico se obtuvo una respuesta negativa, lo
mismo ocurrió con los linfocitos humanos al aplicarles directamente este insecticida.
Cuando se agregó la mezcla enzimática S10 de
Vicia faba
junto con el azinfos metíli-
co, hubo respuesta positiva en ambas cepas. Este resultado sugiere que el mecanismo
que induce mutaciones, lo hace por corrimiento del marco de lectura (cepa TA98), así
como por sustitución de pares de bases (cepa TA100). Asimismo, la inducción de ICH
fue signiFcativa y se obtuvo una relación de concentración-respuesta en los linfocitos
humanos en cultivo. Adicionalmente, se analizó la cinética de proliferación celular
(células M1, M2 y M3), el índice de replicación y el índice mitótico. Únicamente en
los tratamientos con S10 se observaron efectos y en la concentración más alta hubo
inhibición de la mitosis.
INTRODUCTION
The fact that plants undergo enzymatic trans-
formation makes a deeper analysis necessary, since
persons are exposed to pesticides directly not only
when they are applied to vegetables, but also through
the metabolites that are stored in different structures
which may turn out to be more dangerous. Plants
can bio-concentrate these environmental agents and
convert pro-mutagens into toxic metabolites (Plewa
et al.
1988, 1993, Wagner
et al.
2003). This fact raises
the concern that plant systems might also activate
agrochemicals and environmental agents, thereby
introducing new mutagens into the human food chain
(Plewa 1978).
Peroxidases are among the most important en
-
zymes for oxidative metabolism in plants (Sandermann
et al.
1982, Wildeman and Nazar 1982). Peroxidases
are relatively stable enzymes and are abundant in
homogenates of plants. They are capable to catalyze
two categories of oxidative reactions in plant cells, the
classical peroxidative reaction that need H
2
O
2
, and
an oxidative reaction that requires molecular oxygen
(Lamoureux and ±rear 1979) originated free radical
intermediates that interacted with DNA and increased
the mutagenicity of some chemicals (Lamoureux and
±rear 1979, Donh and Krieger 1981, Plewa
et al.
1993). The hydrolysis is catalized by some hydrolases,
esterases, amidases, O-alkylhydrolases, etc. and repre-
sents a general degradation mechanism of xenobiotics
in animals, microorganisms and plants (Menn 1978,
Shimabukuro
et al.
1982).
In animals and plants the last step of molecular
biotransformation is conjugation (Higashi 1988).
The conjugates are excreted by animals, but in plants
there is not an excretory system, so plants polymerize
and incorporate these conjugates into their structural
components in a way that the initial products, the
reactive intermediates (and/or active oxygen species)
and the Fnal products may cause, Frst, damage to the
plant itself, and second, if conjugates are stored in
the same plant, they may be set free in the gastroin-
testinal tract or in the organs of animals when plants
are eaten. The last event could speciFcally occur as a
consequence of pesticide application to edible plants
(Sandermann 1988).
Vicia faba
is highly sensitive to pesticide effects
(Gómez-Arroyo and Villalobos-Pietrini 1995, Gómez-
Arroyo
et al
. 1995, Valencia-Quintana
et al
. 1998) and
is considered metabolically active because it contains
in the roots and in other tissues the S10 enzymatic
fraction, capable of metabolizing or activating different
compounds (
in vitro
activation) as well as the extracts
prepared from promutagen-treated roots (
in vivo
ac-
tivation; Takehisa and Kanaya 1983, Takehisa
et al
.
1988, Calderón-Segura
et al
. 1999, Gómez-Arroyo
et
al
. 2000a, ±lores-Maya
et al
. 2005).
The
Salmonella typhimurium
assay (
Salmonella
test, Ames test) is a widely accepted short-term
bacterial assay for identifying substances that can
produce genetic damage leading to gene mutations
(Mortelmans and Zeiger 2000).
The Ames test utilizes speciFc strains of the bacteria
Salmonella typhimurium
as tools to detect mutations.
GENOTOXIC EFFECT OF AZINPHOS METHYL
229
These strains are known as auxotrophs because they
cannot synthesize the amino acid histidine and thus
they will not grow unless the nutrient is supplied in
growth media. This test determines the ability of a
particular substance to cause a reverse mutation of
these auxotrophs to the original prototrophic state.
The mutant colonies that can synthesize histidine are
called revertants and the test is often referred to as
a “reversion assay” (Mortelmans and Zeiger 2000).
Several strains of the
S. typhimurium
may be used for
testing. Among the most frequently used are TA98,
which identifes FrameshiFt mutagens, and TA100,
which detects mutagens that can cause the substitu-
tion of pair bases. Each of these mutations is designed
to be responsive to mutagens that act by different
mechanisms (Maron and Ames 1983).
The sister chromatid exchange (SCE) assay is a
sensitive biomarker to detect DNA damage (Alptekin
et al
. 2006). It represents the symmetric interchange
between homologous
loci
of replication products
(Wolff 1982). SCE occur without either loss of DNA
or changes in the chromosomal morphology, and it
is possible to detect them in metaphase. The assay
is based on the incorporation of the thymidine DNA
base analog 5-bromo 2´-deoxyuridine (BrdU) into the
DNA oF cells that replicated twice (Latt 1979, Latt
et al
. 1981). In addition to SCE analysis, the BrdU
differential staining technique can be used to assess
the effects of pesticides in cell replication through
the cell proliFeration kinetics (CPK; Gómez-Arroyo
et al
. 2000a).
Organophosphorus pesticides are common com-
pounds involved in poisoning. They act by inhibiting
the hydrolysis reaction performed by acetylcholin-
esterase, an enzyme that is essential for the central
nervous system function in insects and humans. Such
inhibition leads to accumulation of the neurotrans-
mitter acetylcholine, causing interruption of nervous
impulses in the synapses (Eyer 2003). The routes oF
exposure to insecticides are absorption through skin,
inhalation and ingestion of contaminated water or
vegetables.
Organophosphorus insecticides are widely used
in Mexico (Gómez-Arroyo
et al
. 2000b, González
et al
. 2002, Martínez-Valenzuela
et al
. 2009). Some
of these insecticides, such as gusathion or azinphos
methyl, are classifed by the World Health Organiza
-
tion (WHO 2010) as IB class (“highly hazardous”)
and by the PAN International List oF Highly Hazard
-
ous Pesticides (2014) as Groups 1 (acute toxicity) and
3 (environmental toxicity). These compounds can be
metabolized by plants. Metabolites or their residues
can then remain stored in edible plants and may be
ingested by humans, where the metabolic machinery
can transForm them into more toxic products (Plewa
et al
. 1988, 1993, Cortés-Eslava
et al
. 2001,
Gómez-
Arroyo
et al
. 2007).
The aim of the present study was to evaluate the
capacity of broad bean (
Vicia faba
) S10 Fraction
to metabolize the organophosphorus insecticide
azinphos methyl and the in±uence oF such metabo
-
lites in the induction of reverse mutation in
Salmo-
nella typhimurium
TA98 and TA100 strains and in
the SCE frequency in human lymphocyte cultures.
MATERIALS AND METHODS
Chemicals
The chemicals used were the Following: 4-nitro-
o
-phenylenediamine (NOP)-CAS registry number
N-99-56-9, 2-amino±uorene (2A²)-CAS number
108-45-1, bromodeoxy uridine (BrdU)-CAS num
-
ber 59-14-3. The latter was purchased From Sigma
Chemical Co., St. Louis, MO, USA. Gusathion or
azinphos methyl (O,O-dimethyl-S- [4-oxo-1, 2,
3-benzotriazin-3(4H)-yl] methyl phosphorodithioate
(CAS number 86-50-0, was bought From Bayer de
México), guaiacol (CAS number 90-05-1, was pur
-
chased from Reasol of México), hydrogen peroxide
(CAS number 7722-84-1 was purchased From J.T.
Baker oF México). The other reagents used were oF
analytical grade. Roswell Park Memorial Institute
(RPMI) medium 1640 with L-glutamine and phyto
-
hemagglutinin was purchased from Gibco.
Preparation of the bacterial suspension
The
Salmonella
tester strains TA98 and TA100
were maintained Frozen at –80 ºC as recommended by
Maron and Ames (1983). Cells were incubated in Luria
broth (LB) medium For proliFeration, and master plates
were prepared. Strain genetic markers were tested in
all mutagenic experiments according to the method of
Zeiger
et al
. (1981). The range of spontaneous number
oF revertant colonies per plate in TA98 was From 27 to
49 and in TA100 was From 148 to 173.
Mutagenic activity of azinphos methyl was de-
termined through the S10
Vicia faba
fraction (plant
metabolism) using NOP and 2A² as positive controls.
Bacteria were grown overnight in Oxoid Nutrient
Broth No. 2 at 37 ºC with shaking in darkness For
16 h, washed in 100 mM oF potassium phosphate
buFFer pH 7, and the bacterial suspension title was
determined spectrophotometrically and adjusted
to 1×10
10
colony-forming units/mL in phosphate
buffer. The experiments were performed under
S. Gómez-Arroyo
et al.
230
yellow light in order to avoid photo-oxidation of
mutagens (Nishi and Nishioka 1982).
Vicia faba
S10 mix preparation
Vicia faba
(var. minor) seeds were germinated
between two cotton layers soaked in tap water.
When the primary roots reached a length of 3-5 cm
were rinsed with distilled water and cut at approxi-
mately 2 cm from the primary tips. The roots were
then macerated and homogenized at 4 ºC in 0.1 M
sodium-phosphate buffer, pH 7.4. The ratio of the
buffer solution to the fresh weight of the root cuttings
in grams was 1:1 v/w (Takehisa
et al
. 1988, Gómez-
Arroyo
et al
. 2000a). The homogenized roots were
centrifuged for 15 min at 10 000 g at 4 ºC. The su
-
pernatant was sterilized by Fltration using Millipore
membranes (0.45 μm pore size). The total protein
concentration in these extracts was determined us-
ing the Bio-Rad method (Bradford 1976). Protein
concentration was fairly constant from experiment
to experiment, with values from 3.34 to 3.52.
The metabolic activation system with the S10
mixture was prepared from the microsomal S10 frac
-
tion at a 1:9 ratio (v/v) with the following compounds:
8 mM MgCl
2
, 3.3 mM KCl, 5 mM glucose-6 phos
-
phate, 4 mM NADP and NAD, and 0.1 M Na
2
HPO
4
-
NaH
2
PO
4
buffer at pH 7.4.
Mutagenicity assays
In test tubes, increasing concentrations of the
insecticide azinphos methyl (20, 40, 80, 100 and
200 µg/µL) and 100 µL of the bacterial suspension of
TA98 or TA100 were added to the direct treatments
(without plant metabolism). In the indirect treatments
(with metabolic activation), 500 µL of S10 mix were
incubated with the corresponding insecticide con-
centration plus 100 µL of bacterial suspension. In
both cases (direct and indirect treatments) phosphate
buffer was added making a Fnal volume of 2.5 mL.
The tubes were shaken (12 rpm) for 1.5 h at 28 ºC
in darkness. ±inally triplicate 0.25 mL aliquots of
incubation mix were added to 2 mL of molten top agar
supplemented with 0.5 M histidine/biotin, vortexed,
poured into Vogel-Bonner (V-B) minimal medium
agar plates and incubated for 48 h at 37 ºC (Maron
and Ames 1983).
Salmonella
revertant
his
+
colonies
were scored with a digital counter (New Brunswick
ScientiFc C-110). Three parallel plates were made
for each concentration tested, and each experiment
was repeated three times.
The insecticide concentrations were based on pre-
liminary assays as the best for obtaining a signiFcant
activation without affecting bacterial viability, which
was determined by observing the background lawn
of bacterial growth (De Flora
et al
. 1992).
Peroxidase activity determination
To evaluate peroxidase activity, we measured the
oxidation of guaiacol to tetraguaiacol by observing the
change in absorbance at 470 nm (Chance and Maehly
1955). Peroxidase activity was detected at 30-sec
intervals over a 5-min time period. The experiments
were repeated three times for each sample as described
by Gentile and Gentile (1991). The peroxidase activ-
ity rate was calculated as described by Gichner
et
al
. (1994), and the reaction rate of peroxidase was
obtained in nmol of tetraguaiacol/mg of protein/min.
For each treated group the following equation
was applied:
Peroxidase reaction rate =
min
t
p
l
A
u
u
U
±
²
²
³
´
µ
·
¸
¹
º
×
ν
ε
470
470
where A
470
is the absorbance at 470 nmol, ε
470
is the
extinction coefFcient of tetraguaiacol (26.6 mM/cm),
l
is the path length of the cuvette
,
n is the volume
of the reaction in liters,
p
is the protein content in
mg and
t
min
is the time in minutes. The peroxidase
activity of the cultures is expressed as a percentage
of the initial value.
Treatments with azinphos methyl applied directly
and using
in vitro
promutagen activation by
Vicia
faba
S10 mix in human lymphocyte cultures
Lymphocytes cultured for 48 h were exposed to
azinphos methyl 2, 4, 8, 10, 20, 30 and 40 μg/mL
(chosen in preliminary experiments) for 4 h (cultures
were stationary for the Frst two hours and shaking
for the second two hours) in the dark at 37 ºC, with
and without
metabolic activation. After treatment, the
cells were rinsed twice in 0.9 % sodium chloride and
incubated for 24 h in an RPMI medium containing
BrdU at a Fnal concentration of 5 μg/mL. Colchicine
(0.1 mL, 5 × 10
-6
M) was added 70 h after the start
of the culture.
The metabolic activation system with the S10
mixture was prepared from the microsomal S10
fraction at a 1:9 ratio (v/v) with the following com-
pounds: 8 mM MgCl
2
, 3.3 M KCl, 5 mM glucose-6
phosphate, 4 mM NADP and NAD, and 0.1 M Na
2
H-
PO
4
-NaH
2
PO
4
buffer at pH 7.4. The 48 h cultures
were incubated for 4 h at 37 ºC with 500 μL of the
activation system and the different concentrations of
the insecticide. Ethanol 0.1 M was used as a positive
control. Ethanol is a proved promutagen in
Vicia faba
GENOTOXIC EFFECT OF AZINPHOS METHYL
231
and increases the SCE frequency (Takehisa
et al
.
1988, Gómez-Arroyo
et al
. 1995, 2000a, Calderón-
Segura
et al
. 1999, Flores-Maya
et al
. 2005).
Metaphase cells were harvested by centrifuga-
tion, treated with 0.075 M of KCl for 20 min, and
Fxed in methanol-acetic acid (3:1). The slides were
prepared by the air-drying method, dropping the
cell suspension onto the wet slide, and stained using
the ±uorescence-plus-Giemsa technique (Perry and
Wolff 1974). In addition to the study of SCE, a BrdU
differential staining technique was used to assay the
effect of azinphos methyl on cell replication. For
evaluation of cytokinetics, the proportion of Frst
(M1), second (M2), and third (M3) metaphases were
obtained from 100 consecutive mitoses of each treat
-
ment, and the replication index (RI) was calculated
as follows: RI = 1M1 + 2M2 + 3M3/100 (Lamberti
et al
. 1983); the mitotic index was also determined.
The slides were blinded to avoid bias.
Statistical analysis
Differences were tested among the treated groups
within the experimental series through the analysis
of variance. When a signiFcant ² value (p < 0.0001)
was found, each treated group and its corresponding
negative control were tested for signiFcance using
a Student-Newman-Keuls multiple comparison test
at p < 0.001. A
chi
squared (
χ
2
) test was used for RI
and MI. The parts of the decomposed
χ
2
were used
to compare the values of M1, M2, and M3.
RESULTS
Table I
shows the data from the mutagenic evalu-
ation of the insecticide applied to strains TA98 and
TA100, either directly or following
Vicia faba
S10
mix. Azinphos methyl applied directly was toxic for
the TA98 strain, which was demonstrated by the total
disappearance of the background lawn. This toxicity
decreased in the presence of plant metabolism, giving
the appearance of the background and of revertant
colonies in the TA98 strain at several insecticide con-
centrations up to 200 μg/µL. In the TA100 strain, the
insecticide directly applied was less toxic and its plant
metabolites also induced mutagenicity. In all cases, the
behavior of the positive and negative controls was in
agreement with the values described previously.
In the samples treated with azinphos methyl,
peroxidase activity increased after the treatment,
TABLE I.
MUTAGENIC EVALUATION O² AZINPHOS METHYL WITH
-
OUT AND WITH
PLANT
a
METABOLISM IN
S. typhimurium
TA98 AND TA100 STRAINS
b
Revertants/plate
TA98
TA100
Negative controls
b
Mean
–S10
27 ³ 4
+
S10
49 ³
4
–S10
148 ³ 14
+
S10
173 ³ 11
Positive controls
c
NOP
d
2-AF
659 ³ 74*
185 ³ 17*
1685 ³ 102*
1642 ³ 30*
274 ³ 9*
237 ³ 2
413 ³ 18*
1517 ³ 21*
Treatments
(µg/µL)
Azinphos methyl
20
40
80
100
200
19 ³ 5†
18 ³ 4†
19 ³ 3†
17 ³ 2†
18 ³ 3†
122 ³
3*
226 ³ 13*
494 ³ 24*
492 ³ 10*
9 ³
2†
79 ³ 5
167 ³ 4
83 ³ 13
165 ³ 2
114 ³ 8
144 ³ 6
193 ³ 3*
314 ³ 2*
312 ³ 21*
409 ³ 16*
* SigniFcant differences between the control group and each treated group
were obtained by analysis of variance for TA98, ² = 102.499 and for TA100,
² = 127.64 ² value is p < 0.0001, and therefore the Newman- Keuls multiple
comparison test was applied p < 0.001.
a
Vicia faba
S10 mix.
b
Mean of revertants obtained in three independent assays ³ S.E.
c
4-nitro-
o
-phenylenediamine ( 100 µg/µL).
d
2-amino±uorene (20 µg/µL)
† Values lower to the negative control produced by toxicity
S. Gómez-Arroyo
et al.
232
whereas in samples treated with NOP and 2AF, both
values remained similar (
Table II
).
Table III
shows that there is no difference be-
tween the SCE frequencies and those of the negative
control when azinphos methyl is applied directly. On
the other hand, when S10 mix was added, a concen
-
tration-response relationship was observed starting
at 8 μg/mL and a positive response was obtained (p
< 0.001). The cell kinetics and the replication index
are also listed in
Table III
. No signifcant di±±erences
were ±ound with 2 and 4 μg/mL. However, start
-
ing at 8 μg/mL M1 increased and M3 signifcantly
decreased, RI and MI diminished as the concentra
-
tion was increased up to 400 μg/L in which mitotic
inhibition was observed. As expected, signifcant
differences were observed in SCE
frequency, cell
kinetics and RI with the positive control ethanol. No
signifcant di±±erences were ±ound in the MI.
DISCUSSION
Azinphos methyl is not mutagenic in
Schizosac-
charomyces pombe
(Degraeve
et al
. 1980), however
Gilot-Delhalle
et al
. (1983) found a positive response
in the same yeast and Bianchi
et al
. (1994) in
Sac-
charomyces cerevisiae
. It does ±orm DNA adducts in
calf thymus in the presence of the S9 fraction (Shah
et al
. 1997). It does not induce SCE in human lym
-
phocyte cultures (Gómez-Arroyo
et al
. 1987) while
in plants it causes SCE in
Vicia faba
(Gómez-Arroyo
et al
. 1988).
The results obtained in the Ames assay in which
several
Salmonella typhimurium
strains were exposed
to azinphos methyl without and with S9 metabolic
activation have been contradictory because in some
studies the response is negative (Simmon
et al
. 1976,
Carere
et al
. 1978, Garret
et al
. 1986), in another it is
weak positive (Zeiger
et al
. 1987), and when the plant
cell/microbe coincubation assay with tobacco cells
is applied, a positive response is observed (Gómez-
Arroyo
et al
. 2007).
In the present study, the direct application o±
azinphos methyl did not induce mutagenesis but
prevented background lawn growth in all concentra-
tions. According to De Flora
et al
. (1992), toxicity
to bacteria is indicated by the disappearance of the
background lawn.
It is important to choose care±ully the source ±or
plant homogenates, since the presence and activity
of an enzymatic system may vary in different plant
tissues and at different developmental stages (Callen
1982). In this study we investigated the activation o±
azinphos methyl by cell-free extracts of
Vicia faba
roots, because not all the plant species can activate the
same promutagen. Azinphos methyl was previously
tested by Gómez-Arroyo
et al
. (2007) with the plant
cell/microbe coincubation assay using
Salmonella
typhimurium
TA98 and TA100 strains as a the muta
-
genic indicator organism and
Nicotiana tabacum
cells
as a promutagen activator, and by Cortés-Eslava
et al
.
(2014) with
Coriandrum sativum
cells. In both cases
they found that the peroxidase activity decreases in
treatments when the insecticide is metabolized. How-
ever, our study, in which a remarkable increment of
peroxidase enzymes by azinphos methyl was observed,
is in disagreement with the results mentioned above.
For instance, in
Vicia faba
S10 ±raction other mecha
-
nisms could be involved, possibly through the forma-
tion of DNA adducts due to the action of alkylating
groups. In this way, Shah
et al
. (1997) demonstrated
that azinphos methyl form DNA adducts in calf thymus
in the presence of the S9 fraction. Stakes
et al
. (1995)
found similar results in urine of applicators, were the
short-term exposure to the pesticide was validated by
the presence of dimethyl thiophosphate, a metabolite
of azinphos methyl.
TABLE II.
PEROXIDASE ACTIVITY IN S10
Vicia faba
FRACTION BEFORE AND AFTER 48 HOUR
TREATMENT WITH NOP, 2-AF AND THE IN
-
SECTICIDE AZINPHOS METHYL
Peroxidase activity
a
(nM of tetraguaiacol/min/
µ
g of protein)
Be±ore treatment
After treatment
S10 mix
0.065 ² 0.08
0.056 ² 0.04
Treatments
(µg/µ
L)
NOP
b
100
0.230 ² 0.01 N.S. 0.228 ² 0.06 N.S.
2AF
C
20
0.225 ² 0.03 N.S. 0.232 ² 0.09 N.S.
Azinphos methyl
5
40
100
0.061 ² 0.08 N.S.
0.054 ² 0.07 N.S.
0.061 ² 0.04 N.S.
0.126 ² 0.09*
0.178 ² 0.06*
0.149 ² 0.09*
N.S.: No signifcant di±±erences between the control and each
treated group were obtained by analysis of variance.
* Signifcant di±±erences between the control and each treated
group were obtained by analysis o± variance, F = 60.567, F
value is p < 0.0001, and there±ore the Newman- Keuls multiple
comparison test was applied p < 0.001.
a
Mean o± three assays ² S.E.
b
4-nitro-
o-
phenylenediamine.
c
2-amino³uorene.
GENOTOXIC EFFECT OF AZINPHOS METHYL
233
Plewa
et al
. (1984) suggested that some chemicals
may be activated in plants by similar enzymatic sys-
tems as those of animals. Takehisa
et al
. (1988) found
that ethanol induced SCE after
Vicia
S10 activation
as well as after rat-liver S9 activation. The strength
in activating ethanol was stronger with Vicia S10
than with rat-liver S9. For this reason the activation
capacity specifc For
Vicia faba
could be also found
for azinphos methyl.
The Vicia Faba products obtained From the in vitro
promutagen activation of azinphos methyl applied to
Salmonella typhimurium
and to lymphocyte cultures
were capable of increasing the reverse mutations
in TA98 and TA100 strains and SCE Frequency,
respectively, which means that this compound acted
indirectly. However, toxicity diminished with plant
metabolism, possibly indicating that some detoxifca
-
tion mechanisms were involved. The same detoxifca
-
tion was observed with the thiocarbamate herbicides
molinate and butylate in the presence oF the S10
fraction (Calderón-Segura
et al
. 1999).
CONCLUSIONS
The products obtained from the
in vitro
Vicia faba
promutagen activation of azinphos methyl applied to
Salmonella typhimurium
were capable of increasing
the reverse mutations in TA98 and TA100 strains.
These results indicate that the mechanism to induce
mutations by the insecticide azinphos methyl was
frameshift mutation (in TA98 strain) as well as pair
base substitution (in TA100 strain). However, toxic
-
ity diminished with plant metabolism, suggesting
that some detoxifcation mechanisms were involved.
Also, the SCE frequency in human lymphocyte
TABLE III.
SISTER CHROMATID EXCHANGES AND E±±ECTS ON CELL KINETICS
(M1, M2 AND M3 CELLS), REPLICATION INDEX (RI) AND MITOTIC INDEX
(MI) IN HUMAN LYMPHOCYTE CULTURES INDUCED BY AZINPHOS
METHYL WITHOUT AND WITH
Vicia faba
METABOLIC ±RACTION (S10)
SCE X
a
² S.E.
M1
M2
M3
% RI
b
MI
Negative controls
Lymphocytes
4.82 ² 0.21
32
34
21
2.01
3.75
S10
5.28 ² 0.20
28
33
39
2.11
3.70
Ethanol
c
- S10
4.56 ² 0.23
33
38
29
1.96
3.15
Positive control
Ethanol
c
+
S10
9.12 ² 0.35*
17*
38
45
2.28
3.35
Treatments
Azinphos methyl
e
S10
2
4
8
10
20
30
40
4.50 ² 0.20
4.80 ² 0.18
4.92 ² 0.28
4.88 ² 0.32
4.26 ² 0.45
4.62 ² 0.57
4.86 ² 0.26
25
27
25
29
31
34
29
33
39
39
40
43
41
43
42
34
36
31
26
25
31
2.17
2.13
2.11
2.02
1.95
1.91
2.08
3.50
3.12
3.62
3.35
3.43
3.24
3.14
Azinphos methyl
e
+
S10
2
4
8
10
20
30
40
5.64 ² 0.23
5.16 ² 0.17
7.16 ² 0.31*
7.18 ² 0.31*
8.70 ² 0.26*
9.04 ² 0.38*
f
36
43
46**
49**
51**
57**
f
28
33
30
30
28**
25**
f
36
22**
24**
21**
21**
18**
f
2.00
1.79
1.78
1.70**
1.70**
1.61**
f
3.10
2.55
1.95
1.95
1.45**
1.30**
f
a
Mean oF 50 metaphase cells in two independent assays ² S. E.
b
Replication index, n = 200 consecutive metaphases.
c
Ethanol concentration (0.1 M).
e
Azinphos methyl to μg/mL.
f
Mitotic inhibition (stimulated cells were not observed).
*
Signifcant diFFerences among controls and each treated group were obtained by analysis oF
variance ± = 31.102 ± value is < 0.0001, and thereFore the Newman - Keuls multiple comparison
test was applied p < 0.001.
** Signifcant with
X
2
, p < 0.05.
S. Gómez-Arroyo
et al.
234
cultures was signifcant and a dose-response relation
-
ship was observed, which means that gusathion or
azinphos methyl acted indirectly in both biological
systems.
ACKNOWLEDGMENTS
The authors would like to thank the Programa de
Apoyo a Proyectos de Investigación e Innovación Tec
-
nológica (PAPITT-DGAPA), Universidad Nacional
Autónoma de México (UNAM) For fnancial support
through the project IN227305. The authors thank Ana
Rosa Flores Márquez for her technical assistance and
Claudio Amescua For his scientifc editing.
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