Atrazine promotes biochemical changes and DNA damage in a Neotropical fish species
The effects of Atrazine, an herbicide used worldwide and considered as a potential contaminant in aquatic environments, were assessed on the Neotropical fish Prochilodus lineatus acutely (24 and 48h) exposed to 2 or 10μgL(-1) of atrazine by using a set of biochemical and genetic biomarkers. The following parameters were measured in the liver: activity of the biotransformation enzymes ethoxyresorufin-O-deethylase (EROD) and glutathione S transferase (GST), antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), content of reduced glutathione (GSH), generation of reactive oxygen species (ROS) and occurrence of lipid peroxidation (LPO); in brain and muscle the activity of acetylcholinesterase (AChE) and DNA damage (comet assay) on erythrocytes, gills and liver cells. A general decreasing trend on the biotransformation and antioxidant enzymes was observed in the liver of P. lineatus exposed to atrazine; except for GR, all the other antioxidant enzymes (SOD, CAT and GPx) and biotransformation enzymes (EROD and GST) showed inhibited activity. Changes in muscle or brain AChE were not detected. DNA damage was observed in the different cell types of fish exposed to the herbicide, and it was probably not from oxidative origin, since no increase in ROS generation and LPO was detected in the liver. These results show that atrazine behaves as enzyme inhibitor, impairing hepatic metabolism, and produces genotoxic damage to different cell types of P. lineatus.
Atrazine promotes biochemical changes and DNA damage in a Neotropical
Thais G. Santos, Cláudia B.R. Martinez
Department of Physiological Sciences, Londrina State University, P.B. 6001, 86051-990 Londrina, Paraná, Brazil
Atrazine effects were assessed in ﬁsh using biochemical and genetic biomarkers.
Atrazine promoted a decreasing trend on biotransformation and antioxidant enzymes.
DNA damage was observed in different cell types of ﬁsh exposed to atrazine.
Atrazine did not increase ROS generation and LPO in the liver.
Atrazine did not promote changes in muscle or brain AChE.
Received 28 August 2011
Received in revised form 26 April 2012
Accepted 29 May 2012
Available online 25 June 2012
The effects of Atrazine, an herbicide used worldwide and considered as a potential contaminant in aqua-
tic environments, were assessed on the Neotropical ﬁsh Prochilodus lineatus acutely (24 and 48 h)
exposed to 2 or 10
of atrazine by using a set of biochemical and genetic biomarkers. The following
parameters were measured in the liver: activity of the biotransformation enzymes ethoxyresoruﬁn-
O-deethylase (EROD) and glutathione S transferase (GST), antioxidant enzymes superoxide dismutase
(SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione reductase (GR), content of reduced glu-
tathione (GSH), generation of reactive oxygen species (ROS) and occurrence of lipid peroxidation (LPO); in
brain and muscle the activity of acetylcholinesterase (AChE) and DNA damage (comet assay) on erythro-
cytes, gills and liver cells. A general decreasing trend on the biotransformation and antioxidant enzymes
was observed in the liver of P. lineatus exposed to atrazine; except for GR, all the other antioxidant
enzymes (SOD, CAT and GPx) and biotransformation enzymes (EROD and GST) showed inhibited activity.
Changes in muscle or brain AChE were not detected. DNA damage was observed in the different cell types
of ﬁsh exposed to the herbicide, and it was probably not from oxidative origin, since no increase in ROS
generation and LPO was detected in the liver. These results show that atrazine behaves as enzyme inhib-
itor, impairing hepatic metabolism, and produces genotoxic damage to different cell types of P. lineatus .
Ó 2012 Elsevier Ltd. All rights reserved.
Atrazine is currently one of the most widely used herbicides
and several studies have already detected its presence in water
bodies at levels above the limits determined by local guidelines
(Konstantinou et al., 2006; Davis et al., 2007). Despite being classi-
ﬁed as moderately toxic, atrazine can promote toxic effects on
aquatic animals (Solomon et al., 2008; Ventura et al., 2008). Some
studies have been conducted on ﬁsh to evaluate the toxicity of
atrazine (Yang et al., 2010) and focused on several aspects such
as the biochemical (Dong et al., 2009), genetic (Ventura et al.,
2008), histopathological (Paulino et al., 2012a) and physiological
effects (Nieves-Puigdoller et al., 2007; Paulino et al., 2012b). How-
ever, the mechanisms of toxicity of this herbicide and its metabo-
lites are not yet fully understood (Graymore et al., 2001).
The effects of atrazine can be evaluated by using biomarkers,
that are biochemical or cellular changes in body ﬂuids, cells, or tis-
sues that indicate the presence of contaminants, and can detect
more quickly the presence of toxic compounds, allowing earlier
identiﬁcation of change, before deleterious effects reach higher
organization levels (Monserrat et al., 2007). The effectiveness of
biomarkers has been demonstrated in several studies on the toxic-
ity of pesticides to ﬁsh (Oruç et al., 2004; Dorval et al., 2005;
Ramesh et al., 2009). However, the simultaneous use of several
biomarkers is recommended because the evaluation of a single
parameter can result in misinterpretation since not all of the dam-
age that a contaminant or impacted environment can cause to an
organism will necessarily be shown (Zorita et al., 2008).
0045-6535/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
Corresponding author. Tel.: +55 43 3371 4650; fax: +55 43 3371 4467.
E-mail address: firstname.lastname@example.org (C.B.R. Martinez).
Chemosphere 89 (2012) 1118–1125
Contents lists available at SciVerse ScienceDirect
journal homepage: www.elsevier.com/locate/chemosphere
Biochemical biomarkers can provide information about the pro-
cess of pesticide detoxiﬁcation. When in contact with an organism,
the toxic agent can be biotransformed by enzymes, which act to
make the xenobiotic substance a less toxic compound and facilitate
its excretion. In ﬁsh, the main enzymes that act on the biotransfor-
mation of xenobiotics are cytochrome P450 1A (CYP1A) and gluta-
thione S-transferase (GST) (Van der Oost et al., 2003). However,
these biotransformation processes can generate intermediates that
are more toxic than the contaminant itself: reactive oxygen species
(ROS), represented by the superoxide anion O
, the hydroxyl
OH) and hydrogen peroxide (H
)(Valavanidis et al.,
Organisms have both enzymatic and non-enzymatic antioxi-
dant defences against ROS (Lushchak, 2011). One of the ﬁrst en-
zymes that act in defence against ROS is superoxide dismutase
(SOD), that catalyses the conversion of reactive superoxide anions
to hydrogen peroxide (H
), which is subsequently detoxi-
ﬁed by catalase (CAT) and glutathione dependent peroxidase
(GPx). GPx catalyses the metabolism of H
to water, involving
a concomitant oxidation of reduced glutathione (GSH), one of the
most important non-enzymatic antioxidants in the cell (Van der
Oost et al., 2003). Another important enzyme in the antioxidant
defence of organisms is glutathione reductase (GR), responsible
for reducing oxidized glutathione (GSSG) and maintaining normal
levels of GSH (Hermes-Lima, 2004).
If the generation of ROS exceeds the antioxidant capacity of the
organism, oxidative stress occurs, which affects the normal func-
tioning of the cell and may cause damage to proteins, enzyme inac-
tivation, lipid peroxidation (LPO) and DNA strand breaks (Amado
et al., 2009). Mechanism of pesticide toxicity has been usually
associated with the increase of LPO (Singh et al., 2011) that is
one of the major tools used to assess the effects of a pollutant on
aquatic organisms (Valavanidis et al., 2006). On the other hand,
genotoxic parameters, such as DNA strand breaks, are currently
the most valuable biomarkers for environmental risk assessment
and there are many reports linking the DNA damage to subsequent
molecular, cellular and tissue level alteration of aquatic organisms
(Ohe et al., 2004).
The activity of acetylcholinesterase (AChE) is another biochem-
ical biomarker normally used to monitor aquatic environments
mainly contaminated by pesticides. This enzyme can be inhibited
by different types of agrochemicals, causing over-stimulation of
muscle ﬁbers and leading to paralysis, and even death, of animals
(Ferrari et al., 2007).
The species Prochilodus lineatus, popularly known as curimba, is
a Neotropical ﬁsh species of great economic importance and some
studies have already shown the sensitivity of this ﬁsh to various
types of pesticides (Cavalcante et al., 2008; Maduenho and Marti-
nez, 2008; Modesto and Martinez, 2010a, 2010b). The objective
of this work was to understand the effects of the herbicide atrazine
on this ﬁsh species by using a set of biochemical and genetic
biomarkers. To this end, it was measured the activity of hepatic
biotransformation enzymes, enzymatic and non-enzymatic antiox-
idant defences in liver, AChE activity in the brain and muscle, the
occurrence of LPO in liver and DNA damage in different tissues of
P. lineatus after acute exposure to two environmentally relevant
concentrations of atrazine.
2. Material and methods
2.1. Acclimation of animals and experimental design
Juveniles of P. lineatus (17.3 ± 7.9 g, 11.4 ± 1.8 cm, N = 168), sup-
plied by the Fish Hatchery Station of Londrina State University,
were acclimated for 7 d in 300 L tanks containing dechlorinated
water and with constant aeration. During this time they were fed
every 48 h with a commercial diet. They were not fed for 48 h be-
fore being exposed to the compound or during the toxicity tests.
The physical and chemical parameters of the water were moni-
tored throughout the acclimation period and remained constant
(pH: 7.18 ± 0.5, conductivity: 118.25 ± 8.7
, dissolved oxy-
gen: 8.49 ± 0.9 mg O
, temperature: 21.96 ± 2.7 °C).
After this period, groups of eight ﬁsh were transferred to 100 L
glass aquaria where they were subjected to static acute toxicity
tests for 24 and 48 h. Experiments for each experimental period
were run separately, and for each period (24 or 48 h) one group
of ﬁsh was exposed to dechlorinated water only (control group)
and two groups were exposed to water containing atrazine at con-
centrations of 2
(ATZ 2 and ATZ 10, respec-
tively). The concentration of 2
corresponds to the
maximum concentration of atrazine allowed by the Brazilian
Guidelines (CONAMA Resolution 357, 2005) for continental waters
and the concentration of 10
corresponds to 40% of the con-
centration of atrazine used on crops (Ventura et al., 2008).
After the exposure periods the ﬁsh were removed from the
aquarium and anesthetized in benzocaine (0.1 g L
) and blood
samples were collected from the caudal vein. Following this the
animals were killed by medullar section and the gills, liver, brain
and muscle samples were removed. These procedures followed
the standard protocols approved by the Committee for Animal
Experimentation of Londrina State University. Immediately after
excision the gills and liver were carefully washed with PBS (NaCl
126.6 mM, KCL 4.8 mM, CaCl
1.5 mM, NaHCO
3.7 mM, Na
8.9 mM, NaH
2.9 mM) and gill ﬁlaments and part of the liver
were transferred to microtubes for the cellular dissociation to be
used in the comet assay. The remaining organs were stored in an
ultrafreezer (80 °C) for biochemical analysis.
2.3. Biochemical analysis in liver
Liver samples were homogenized (10 volume) in potassium
phosphate buffer (0.1 M, pH 7.0) and centrifuged (20 min,
13000 g, 4 °C) for biochemical analysis.
2.3.1. Biotransformation enzymes
The catalytic activity of CYP1A was determined by ethoxyres-
oruﬁn-O-deethylase (EROD) activity through the conversion of 7-
ethoxyresoruﬁn, provided in the reaction medium (potassium
phosphate buffer 0.1 M, pH 7.6, NADPH 2 mM, 7-ethoxyresoruﬁn
0.1 mM), to resoruﬁn, according to Eggens et al. (1992). The pro-
gressive increase in ﬂuorescence resulting from the formation of
resoruﬁn was measured at 1-min intervals for 10 min (excitation:
530 nm; emission: 590 nm). The initial linear portion of the curve
was used to evaluate the reaction rate and the EROD activity was
expressed in pmol of resoruﬁn min
mg of protein
, based on a
resoruﬁn standard curve.
The activity of GST was determined by the complexation of GSH
with 1-chloro-2,4-dinitrobenzene (CDNB) in a spectrophotometer
at 340 nm (Keen et al., 1976) and expressed as nmol of conjugated
2.3.2. Non-enzymatic antioxidant
Reduced GSH levels were estimated according to Beutler et al.
(1963), using 5,5
-dithiobis-2-nitrobenzoic acid (DTNB) following
Simonato et al. (2011). Supernatants of the acid extracts (1:1 v/v
with 12%TCA) were added to 0.25 mM DTNB in 0.1 M potassium
phosphate buffer, pH 8.0, and thiolate anion formation was
determined at 412 nm against a GSH standard curve
T.G. Santos, C.B.R. Martinez / Chemosphere 89 (2012) 1118–1125
mol of GSH). The GSH content was expressed in
GSH mg protein
2.3.3. Antioxidant enzymes
The activity of copper–zinc SOD was estimated by the inhibition
of the reduction rate of cytochrome c by the superoxide radical, at
550 nm and 25 °C, and expressed as U SOD mg of protein
U represents the amount of SOD that promoted the inhibition of
50% of the reduction rate of cytochrome c (McCord and Fridovich,
The activity of CAT was determined by the rate of decomposi-
tion of H
by the enzyme, which was evaluated by the decrease
in absorbance at 240 nm (Beutler, 1975) and expressed in
. The determination of selenium-
dependent GPx activity was basedon NADPH oxidation in the pres-
ence of GSH (0.95 mM) and H
at 340 nm (Hopkins and Tudhope,
1973). GPx activity was expressed in
mol oxidized NADPH min
. The activity of GR was determined indirectly based
on the reduction of NADPH in the presence of oxidized GSH at
340 nm (Carlberg and Mannervik, 1975). GR activity was expressed
2.3.4. Generation of reactive oxygen species (ROS)
Measurements of ROS were conducted according to the method
described by Ferreira-Cravo et al. (2007), which is based on the fact
that non-ﬂuorescent compound 2
DCF-DA) is oxidized by ROS present in the samples to the ﬂuo-
rescent compound dichloro-ﬂuorescein (DCF). In order to run the
assay samples of fresh liver were homogenized (1:10 – w/v) in a
Tris–HCl buffer (10 mM, pH 7.75) plus EDTA (2 mM) and MgCl
(5 mM) and centrifuged (10000 g, 20 min, 4 °C). Supernatant was
added to a reaction medium (HEPES 30 mM, KCl 200 mM, MgCl
1 mM, H
M, pH 7.2) in a microplate and the volume
in each well was adjusted to 1 mg protein mL
. The ﬂuorescence
intensity was determined during 15 min, at 25 °C, using a ﬂuorom-
eter, with excitation and emission wavelength of 485 and 520 nm,
respectively. Background ﬂuorescence was determined previous to
DCF-DA addition. The results were expressed as ﬂuorescence
units (FUs) per mg of protein.
2.3.5. Lipid peroxidation
Lipid peroxidation was estimated from the production of mal-
ondialdehyde (MDA), which is one of the ﬁnal products of lipid
peroxidation. The MDA content was determined by the TBARS as-
say, which measures the thiobarbituric acid (TBA) reactive sub-
stances, at 530 nm, following the methodology described by
Federici et al. (2007). Lipid peroxidation was expressed in equiva-
lents of MDA as
mol MDA mg protein
2.4. Brain and muscle acetylcholinesterase (AChE)
Brain and muscle samples were homogenized (10 volume) in
potassium phosphate buffer (0.1 M, pH 7.5), centrifuged (20 min,
13000 g, 4 °C), and the supernatant was removed for analysis of
AChE. Enzyme activity was determined based on the colorimetric
method of Ellman et al. (1961) adapted for reading on microplates,
according to Alves Costa et al. (2007). The ﬁnal concentration of the
acetylthiocholine iodide substrate employed was 9 mM, while that
of the DTNB color reagent was 0.5 mM for both tissues. Absorbance
was determined in a microplate reader at 415 nm and the enzyme
activity was expressed in nmol DTNB min
2.5. Protein concentration
The total protein concentration in the samples used in the bio-
chemical assays was measured using the method of Lowry et al.
(1951), based on a standard curve of bovine serum albumin
(BSA) at 700 nm. The method of Bradford (1976), which uses a
BSA reference curve at 595 nm, was used to determine protein con-
centration in the samples used for estimating the amount of reac-
tive oxygen species.
2.6. Genetic analysis – comet assay
The Comet assay was performed using the alkaline (pH > 13)
version developed by Singh et al. (1988), with the modiﬁcations
detailed by Cavalcante et al. (2008), using three cell types: erythro-
cytes, liver and gill cells. For tissue dissociation gill ﬁlaments and
liver tissue were sectioned and the pieces were transferred to
microtubes, incubated for 15 min at 30 °C in 0.05% trypsin (diluted
in PBS Ca
free) and homogenized by periodic manual
inversion at room temperature. After that, the solution was ﬁltered
m mesh size) into a tube containing a fetal calf serum 10% to
halt the enzymatic digestion. The resultant solution was centri-
fuged (10 min, 1000 g) and the pellet was resuspended in PBS to
be used in the comet assay. Only blood, gill and liver samples with
cellular viability greater than 80%, determined by the Trypan blue
exclusion test, were used in the comet assay.
The basic steps of the comet assay for the three cell types were
executed as follows: (a) lysis: 1 h, at 4 °C, protected from light, in a
lysis buffer (NaCl 2.5 M, EDTA 100 mM, Tris 10 mM, DMSO 10%,
Triton X-100 1 mL, pH 10.0); (b) DNA unwinding: 30 min, in the
dark, in an electrophoresis buffer (NaOH 0.3 N, EDTA 1 mM,
pH > 13); (c) electrophoresis: 20 min, 300 mA, 25 V, 1 V cm
neutralization: three washes for 5 min each in buffer (Tris 0.4 M,
pH 7.5). Slides were ﬁxed with absolute ethanol for 10 min and
kept under refrigeration until cytological analyses. The slides,
stained with gelRed were analyzed with a Leica microscopy (DM
2500) adapted for ﬂuorescence/epiﬂuorescence, equipped with a
blue excitation ﬁlter (450–490 nm) and a barrier ﬁlter of 515 nm
at 1000 magniﬁcation. All slides were blind-reviewed. The extent
of DNA damage was quantiﬁed by the length of DNA migration,
which was visually determined in 100 randomly select and non-
overlapping cells per ﬁsh. According to Kobayashi et al. (1995),
DNA damage was classiﬁed in four classes 0: no visible damage;
(1) a short tail smaller than the diameter of the nucleus; (2) a tail
length 1–2 times the diameter of the nucleus; (3) a tail length > 2
times the diameter of the nucleus. The score of DNA damage for
100 comets was obtained by multiplying the number of cells in
each class by the damage class, and ranged from 0 (all undamaged)
to 300 (all maximally damaged). Results for DNA damage in each
cell type were expressed as the mean score of DNA damage for
each treatment group, for each exposure period.
2.7. Statistical analysis
The results of each parameter obtained at the same exposure
period (24 or 48 h) were compared among treatment groups
(CTR X ATZ2 X ATZ10) using the parametric one-way analysis of
variance (ANOVA) or the non-parametric Kruskal–Wallis test,
according to data distribution (normality and homogeneity of
variance). Differences were analyzed by a post hoc Student–
Newman-Keuls test (after ANOVA) or Dunn’s test (after Kruskal–
Wallis). Statistical signiﬁcance was designated as P < 0.05.
3.1. Hepatic activity of the biotransformation enzymes
In the ﬁsh exposed to 2
of herbicide for 24 h, the activity
of EROD was signiﬁcantly decreased when compared to the
1120 T.G. Santos, C.B.R. Martinez / Chemosphere 89 (2012) 1118–1125
control. After 48 h of exposure to both concentrations of atrazine,
the activity of EROD was signiﬁcantly lower in comparison to the
control (Fig. 1A). The ﬁsh exposed to the higher concentration of
atrazine for 48 h also showed a signiﬁcant reduction in GST activity
3.2. Generation of ROS
The results showed that in animals exposed to 2 and 10
of atrazine for 24 h there was a signiﬁcant decrease in ROS gener-
ation in comparison to the animals of the CTR group. In 48 h, only
ﬁsh exposed to ATZ 10 showed a signiﬁcant reduction in ROS gen-
eration in relation to the CTR group (Fig. 2).
3.3. Hepatic activity of the antioxidant enzymes
The hepatic activities of the antioxidant enzymes SOD, CAT and
GPx were signiﬁcantly reduced in ﬁsh exposed to both atrazine
concentrations, for 24 and 48 h, in relation to their respective con-
trols (Table 1). On the other hand, the liver activity of GR signiﬁ-
cantly increased in ﬁsh exposed to ATZ2 and ATZ10, in both
experimental periods, when compared to respective controls
3.4. The hepatic content of GSH
Exposure to atrazine for 24 h did not lead to a signiﬁcant alter-
ation in the hepatic content of GSH. However, after 48 h exposure,
atrazine promoted a reduction in the hepatic content of GSH that
was signiﬁcant only in ﬁsh exposed to the lower concentration of
the herbicide (2
) in comparison to the respective control
3.5. Lipid peroxidation
Fish exposed to atrazine showed a reduction in the hepatic
content of MDA compared to the respective control group. This
reduction was signiﬁcant in animals exposed to both concentra-
tions of the herbicide for 24 h and also in those exposed to the low-
er concentration of atrazine for 48 h (Fig. 3).
3.6. DNA damage – the comet assay
Erythrocytes of ﬁsh exposed to atrazine for 24 and 48 h showed
a concentration-dependent increase in the occurrence of DNA
damage, this increase was signiﬁcant only at the higher concentra-
tion of the herbicide (Table 2). For the liver cells a concentration-
dependent increase in the occurrence of DNA damage was also ob-
served after 48 h exposure, and this increase was signiﬁcant only in
ﬁsh exposed to ATZ10 (Table 2). In the case of gill cells, only ﬁsh
exposed to the lower concentration of the herbicide for 48 h
showed a damage score signiﬁcantly higher than that of the control
Fig. 1. Hepatic activity of (A) Ethoxyresoruﬁn-O-deethylase (EROD) and (B) gluta-
thione S-transferase (GST) in P. lineatus exposed to 2 and 10
of atrazine (ATZ
2 and ATZ 10) or only water (CTR) for 24 and 48 h. The columns represent the
means and the vertical lines the standard errors. For each experimental period, the
different letters indicate signiﬁcant differences between the groups (P 6 0.05).
Fig. 2. Generation of reactive oxygen species (ROS) in the liver of P. lineatus exposed
2 and 10
of the herbicide atrazine (ATZ 2 and ATZ 10) or only water (CTR) for
24 and 48 h. The columns represent the means and the vertical lines the standard
errors. For each experimental period, the different letters indicate signiﬁcant
differences between the groups (P 6 0.05).
Hepatic activity of antioxidant enzymes superoxide dismutase (SOD, U mg protein
), catalase (CAT,
), glutathione peroxidase (GPx,
), glutathione reductase (GR, nmol NADPH. min
) and hepatic content of reduced glutathione (GSH,
g GSH mg protein
P. lineatus exposed to 2 and 10
of atrazine (ATZ 2 and ATZ 10, respectively) or only water (CTR), for 24 and 48 h.
Exposure time Group SOD CAT GPx GR GSH
24 h CTR 36.6 ± 2.2
32.9 ± 3.6
153.6 ± 8.6
7.1 ± 1.1
7.7 ± 1.8
ATZ 2 16.1 ± 1.1
10.6 ± 1.1
70.8 ± 2.8
25.3 ± 1.4
3.1 ± 0.9
ATZ 10 19.2 ± 1.0
9.5 ± 3.8
91.3 ± 5.3
24.6 ± 2.5
3.4 ± 0.7
48 h CTR 47.8 ± 7.4
40.6 ± 2.3
45.3 ± 6.6
7.1 ± 0.8
35.5 ± 6.5
ATZ 2 13.7 ± 5.0
23.6 ± 1.9
21.5 ± 1.3
12.2 ± 1.4
14.3 ± 1.8
ATZ 10 28.5 ± 3.7
20.1 ± 1.9
20.6 ± 0.6
16.0 ± 3.4
20.3 ± 1.6
Data are means ± S.E., n = 8. Different letters indicate signiﬁcant differences between the groups at the same exposure time (P 6 0.05).
T.G. Santos, C.B.R. Martinez / Chemosphere 89 (2012) 1118–1125
The exposure to both atrazine concentrations, for 24 and 48 h,
did not affect the brain or muscle activity of acetylcholinesterase
in P. lineatus (Fig. 4A and B).
In the current work, a general decreasing trend on the biotrans-
formation and antioxidant enzymes was observed in the liver of P.
lineatus exposed to atrazine; except for GR, all the other antioxi-
dant enzymes (SOD, CAT and GPx) and biotransformation enzymes
(EROD and GST) showed inhibited activity. These results suggest
that atrazine, at the concentrations here applied behaves as en-
zyme inhibitor impairing hepatic metabolism.
Agrochemicals, in general, can be modiﬁed and become less
toxic to organisms via biotransformation pathways. It is known
that in several species of ﬁsh, the activity of the phase I biotrans-
formation enzyme, CYP1A, may change in the presence of pesti-
cides and other pollutants in the aquatic environment (Agradi
et al., 2000; Van Der Oost et al., 2003). In present study, atrazine
was responsible for the reduced hepatic activity of EROD (one of
the ways of measuring CYP1A activity) in P. lineatus after 24 and
48 h of exposure. This result could be related both to a reduction
in the amount of this enzyme in the liver. This idea is supported
by Salaberria et al. (2009), who found that atrazine at doses of 2
was able to decrease the gene expression of
CYP1A in Oncorhynchus mykiss.
This inhibition of CYP1A by atrazine may affect the metabo-
lism of endogenous compounds, such as sex hormones, thereby
inﬂuencing the endocrine homeostasis of the organism, and so
this herbicide may be considered an important endocrine disrup-
tor (Spanò et al., 2004; Salaberria et al., 2009). Atrazine metabo-
lism itself by cytochrome P-450-related enzymes may be of less
importance in ﬁsh, considering that the chemical structure of
atrazine allows direct conjugation to GSH, without activation by
phase I enzymes (Wiegand et al., 2001). Glutathione-S-transfer-
ase, a major phase II biotransformation enzyme, in this case,
would act on the metabolism of atrazine, compensating for the
inactivity of EROD. However, the GST activity in P. lineatus ex-
posed to atrazine was reduced, as well as the activity of EROD,
after 48 h exposure to 10
of atrazine. This reduction in
GST activity might be related with the low levels of GSH in the
liver of the ﬁsh, since GSH is the main substrate for the action
of GST in the metabolism of atrazine, and both were diminished,
probably due to the large demand for herbicide conjugation with
The biotransformation of atrazine in hydrophilic compounds to
facilitate its excretion is extremely important in order to restrict its
accumulation; according to Solomon et al. (2008) this herbicide
normally does not bioaccumulate since it is easily metabolized.
However, for P. lineatus, atrazine at very low concentrations (2
) inhibited the major pathways for its own metabo-
lism and then the herbicide could be accumulated within the hepa-
tocyte. This idea was corroborated by the results of the comet
assay, which showed an increase on DNA damage in the liver cells
of ﬁsh exposed to 10
of atrazine for 48 h, the same group of
ﬁsh that showed a signiﬁcant decrease on GST activity. Atrazine
has already been recognized in the literature as a potential geno-
toxic agent in various tissues of various organisms (Lin et al.,
2005; Singh et al., 2008; Ventura et al., 2008) and the present work
reinforces this idea, showing that atrazine caused DNA damage to
the hepatocytes of P. lineatus.
Another result that should be highlighted in order to better
understand the effects of atrazine on P. lineatus is the signiﬁcant
Fig. 3. Liver contents of malondialdehyde (MDA), a sub-product of lipid peroxida-
tion, in P. lineatus exposed to 2 and 10
of atrazine (ATZ 2 and ATZ 10) or only
water (CTR), for 24 and 48 h. The columns represent the means and the vertical
lines the standard errors. For each experimental period, the different letters indicate
signiﬁcant differences between the groups (P 6 0.05).
DNA damage score in blood, liver and gill cells of P. lineatus exposed to 2 and
of atrazine (ATZ 2 and ATZ 10, respectively) or only water (CTR), for 24 and
Exposure time Group Cell type
Blood Liver Gills
24 h CTR 34.6 ± 8.1
31.4 ± 6.5
83.0 ± 11.0
ATZ 2 45.6 ± 3.7
40.3 ± 6.5
105.2 ± 10.7
ATZ 10 83.9 ± 11.7
42.1 ± 5.5
112.2 ± 11.7
48 h CTR 51.4 ± 4.5
81.7 ± 3.9
64.3 ± 6.0
ATZ 2 123.0 ± 11.3
111.4 ± 14.1
117.2 ± 7.3
ATZ 10 140.7 ± 27.5
127.0 ± 14.4
72.0 ± 18.7
Data are means ± S.E., n = 8. Different letters indicate signiﬁcant differences
between the groups at the same exposure time (P 6 0.05).
Fig. 4. Acetylcholinesterase activity in (A) brain and (B) muscles of P. lineatus
exposed to 2 and 10
of atrazine (ATZ 2 and ATZ 10) or only water (CTR), for
24 and 48 h. The columns represent the means and the vertical lines the standard
errors. For each experimental period, the different letters indicate signiﬁcant
differences between the groups (P 6 0.05).
1122 T.G. Santos, C.B.R. Martinez / Chemosphere 89 (2012) 1118–1125
decrease in ROS generation and MDA levels in liver tissues. The
production of ROS occurs via the metabolism of endogenous and
exogenous compounds, from cellular respiration and other pro-
cesses, as a natural consequence of the cellular metabolism
(Hermes-Lima, 2004). This decrease in ROS generation indicates
that atrazine reduces the metabolism of liver cells, as clearly indi-
cated by the reduced liver activity of the biotransformation and
antioxidant enzymes in ﬁsh exposed to the herbicide. As it would
be expected MDA levels in the liver of ﬁsh exposed to atrazine
were also lower compared to MDA levels in the controls, thus there
was no indication of lipid peroxidation in consequence of atrazine
exposure. This hypothesis can be supported by Paulino et al.
(2012a, 2012b), which reported that ﬁsh of the same species ex-
posed to concentrations of 2, 10 and 25
of atrazine during
48 h and 14 d showed histological changes in the gills that would
restrict oxygen uptake into the body causing hypoxia to various or-
gans, including the liver.
In the liver of P. lineatus exposed to the two concentrations of
atrazine for both experimental periods, except for GR, all the other
antioxidant enzymes (SOD, CAT and GPx) showed reduced activity.
These enzymes can be inhibited by different causes. For example,
SOD can be inhibited by an excess of H
in the cell. In turn,
CAT activity can be inhibited when the levels of O
are high. In
other words, the excess of substrate of one enzyme inﬂuences
the activity of the other enzyme and vice versa. However, as in
the present study no increase in ROS generation was observed it
is very unlikely that the excess of H
had inhibited antiox-
idant enzymes activities. Another possibility is that atrazine would
have interfered on the synthesis of these enzymes. However, stud-
ies conducted by Jin et al. (2010) showed that the ﬁsh species Da-
nio rerio, also exposed to 10
of atrazine for 14 d, had
increased mRNA levels of SOD and CAT in the liver. Thus, the most
likely cause for the inhibition of these enzymes in P. lineatus ex-
posed to atrazine would be a general reduction in the liver cell
metabolism caused by the herbicide.
The ﬁsh exposed to atrazine showed a reduced hepatic content
of GSH and an increase in GR activity. GSH levels are mainly de-
pend on the balance between GSH synthesis rate, conjugation rate
(by GSTs), oxidation rate (non-enzymatically or by GPx), and oxi-
dized glutathione (GSSG) reduction to GSH, by GR (Peña-Llopis et
al., 2003). In the present study the increased activity of GR, in ﬁsh
exposed to both concentrations of atrazine, indicates an attempt to
maintain normal levels of GSH. The maintenance of GSH levels by
GR, in this case, might contribute for the reduction of atrazine tox-
icity through its association with the herbicide molecule (Wiegand
et al., 2001).
Damage to DNA can occur as a consequence of oxidative stress,
xenobiotic metabolites or the xenobiotic itself. In the case of P. line-
atus, atrazine itself was found to be genotoxic to the three tissues
analyzed (blood, gill and liver), since no evidence was obtained to
show the metabolism of this herbicide or the occurrence of oxida-
tive stress. The results obtained from the genetic material of hepa-
tocytes of ﬁsh exposed to 10
of herbicide for 48 h, as
discussed above, may be a consequence of the accumulation of
atrazine in the liver due to the reduction of GST activity. Assays
to assess levels of damage in the genetic material of liver cells
should be considered in toxicology studies, since the liver is the
main organ responsible for xenobiotic metabolism and therefore
is more susceptible to damage due to the presence of these toxic
compounds in its cells (Rajaguru et al., 2003).
The blood cells of P. lineatus also demonstrated the genotoxi-
city of atrazine, since ﬁsh exposed to the higher concentration
of atrazine for the two experimental periods showed increased
levels of DNA damage in erythrocytes. Ventura et al. (2008) tested
different concentrations of the same herbicide in Oreochromis
niloticus and they also observed the genotoxic effects of atrazine
on erythrocytes after 72 h of exposure. These data indicate that
atrazine can affect the genetic material in erythrocytes of differ-
ent ﬁsh species.
For the gill cells, a signiﬁcant increase in DNA damage was
found only in ﬁsh exposed to the lower concentration of atrazine
for 48 h. The concentration of 10
of atrazine was genotoxic
to the other tissues of P. lineatus and probably was also genotoxic
to gill cells. However this higher atrazine concentration may have
caused apoptosis of the gill cells and considering that apoptotic
cells are not considered for the calculation of DNA damage score
ﬁsh from ATZ 10 group showed a lower damage score less than
the ﬁsh from ATZ 2.
The damage in DNA observed in different cells of P. lineatus ex-
posed to the herbicide atrazine was probably not from oxidative
origin, since no increase in ROS generation was detected in the
liver and probably would not have been detected in the other or-
gans tested either. According to Oliveira-Brett and Silva (2002),
triazine herbicides, including atrazine, are able to directly bind
to DNA through the mechanisms of intercalation and adduct for-
mation between adenine and guanine. One of the main ways of
preventing DNA damage is the conjugation of the xenobiotic with
GSH, catalyzed by the action of GST (Chakraborty et al., 2009).
Thus, as discussed above, atrazine needs GST to conjugate with
GSH to become less toxic for the organism and its genotoxicity
possibly gets worse as the result of a decrease in GSH content
and GST activity.
Although atrazine exposure showed several effects on P. linea-
tus, the herbicide had no effect on brain or muscles acetylcholines-
terase activity in this ﬁsh species. Data about the effect of atrazine
on AChE activity in aquatic vertebrate species are scarce (Tyler
Mehler et al., 2008). In a work conducted by Xing et al. (2010) juve-
niles of common carp (Cyprinus carpio) showed inhibited AChE
activity in brain and muscle after 40 d exposure to different atra-
zine concentrations (4.28, 42.8 and 428
). This variation be-
tween the results obtained for P. lineatus and C. carpio may be
produced by the differences in exposure periods, the size and the
type of the ﬁsh. P. lineatus showed to be less sensitive to atrazine
effects on AChE, even though its cholinergic activity was found to
be inﬂuenced by other agrochemicals, for example, Diﬂubenzuron,
Roundup and Roundup Transorb (Maduenho and Martinez, 2008;
Modesto and Martinez, 2010a, 2010b).
In summary, the results obtained in this work show that atra-
zine was toxic to the Neotropical ﬁsh P. lineatus, even at low con-
centrations (2 and 10
), since it promoted signiﬁcant
alterations in the biochemical and genetic parameters evaluated.
One effect of atrazine was a reduction in the hepatic activity of
GST, which is primarily responsible for metabolism, and which
caused the accumulation of this herbicide in the hepatocytes of
the ﬁsh. The ﬁsh exposed to atrazine also showed inhibition of
the biotransformation pathways of phase I, as shown by a reduc-
tion in the activity of EROD, which directly contributes to a reduc-
tion in the generation of reactive oxygen species in the liver and a
consequent reduction in the activity of antioxidant liver enzymes.
Moreover, atrazine stimulated the action of GR, which is responsi-
ble for the maintenance of normal GSH levels, which were found to
be reduced in this work. Even with the reduction in antioxidant de-
fence, atrazine did not cause lipid peroxidation in the liver of P.
lineatus. In addition to the biochemical effects, atrazine caused
DNA damage in the three types of tissue studied, blood, liver and
gills, which did not have an oxidative origin since no increase in
the generation of reactive oxygen species was detected. These re-
sults show that the limit of 2
of atrazine in fresh water, al-
lowed by Brazilian legislation (CONAMA, 2005), is not a safe
concentration for the ﬁsh P. lineatus.
T.G. Santos, C.B.R. Martinez / Chemosphere 89 (2012) 1118–1125
The authors thank the Hatchery Station of the State University
of Londrina for supplying the ﬁsh for this research. This work is
part of the master thesis of T.G. Santos and was supported by
INCT-TA (CNPq: 573949/2008-5) and Fundação Araucária. C.B.R.
Martinez is research fellow from CNPq.
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