Atrazine promotes biochemical changes and DNA damage in a Neotropical fish species

Article (PDF Available)inChemosphere 89(9):1118-25 · June 2012with61 Reads
DOI: 10.1016/j.chemosphere.2012.05.096 · Source: PubMed
Abstract
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
fish species
Thais G. Santos, Cláudia B.R. Martinez
Department of Physiological Sciences, Londrina State University, P.B. 6001, 86051-990 Londrina, Paraná, Brazil
highlights
"
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.
article info
Article history:
Received 28 August 2011
Received in revised form 26 April 2012
Accepted 29 May 2012
Available online 25 June 2012
Keywords:
Acetylcholinesterase
Antioxidants
Biotransformation
Comet assay
Oxidative stress
Prochilodus lineatus
abstract
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
l
gL
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 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.
1. Introduction
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.
http://dx.doi.org/10.1016/j.chemosphere.2012.05.096
Corresponding author. Tel.: +55 43 3371 4650; fax: +55 43 3371 4467.
E-mail address: cbueno@uel.br (C.B.R. Martinez).
Chemosphere 89 (2012) 1118–1125
Contents lists available at SciVerse ScienceDirect
Chemosphere
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

2

, the hydroxyl
radical (
OH) and hydrogen peroxide (H
2
O
2
)(Valavanidis et al.,
2006).
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
O

2

to hydrogen peroxide (H
2
O
2
), which is subsequently detoxi-
fied by catalase (CAT) and glutathione dependent peroxidase
(GPx). GPx catalyses the metabolism of H
2
O
2
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
l
Scm
1
, dissolved oxy-
gen: 8.49 ± 0.9 mg O
2
L
1
, 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
l
gL
1
and 10
l
gL
1
(ATZ 2 and ATZ 10, respec-
tively). The concentration of 2
l
gL
1
corresponds to the
maximum concentration of atrazine allowed by the Brazilian
Guidelines (CONAMA Resolution 357, 2005) for continental waters
and the concentration of 10
l
gL
1
corresponds to 40% of the con-
centration of atrazine used on crops (Ventura et al., 2008).
2.2. Sampling
After the exposure periods the fish were removed from the
aquarium and anesthetized in benzocaine (0.1 g L
1
) 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
2
1.5 mM, NaHCO
3
3.7 mM, Na
2
HPO
4
8.9 mM, NaH
2
PO
4
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
1
mg of protein
1
, 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
CDNB min
1
mg protein
1
.
2.3.2. Non-enzymatic antioxidant
Reduced GSH levels were estimated according to Beutler et al.
(1963), using 5,5
0
-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
1119
(100–200
l
mol of GSH). The GSH content was expressed in
l
gof
GSH mg protein
1
.
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
1
, where
U represents the amount of SOD that promoted the inhibition of
50% of the reduction rate of cytochrome c (McCord and Fridovich,
1969).
The activity of CAT was determined by the rate of decomposi-
tion of H
2
O
2
by the enzyme, which was evaluated by the decrease
in absorbance at 240 nm (Beutler, 1975) and expressed in
l
mol
H
2
O
2
min
1
mg protein
1
. The determination of selenium-
dependent GPx activity was basedon NADPH oxidation in the pres-
ence of GSH (0.95 mM) and H
2
O
2
at 340 nm (Hopkins and Tudhope,
1973). GPx activity was expressed in
l
mol oxidized NADPH min
1
mg protein
1
. 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
in
l
mol min
1
mg protein
1
.
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
0
,7
0
-dichlorofluorescin diacetate
(H
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
2
(5 mM) and centrifuged (10000 g, 20 min, 4 °C). Supernatant was
added to a reaction medium (HEPES 30 mM, KCl 200 mM, MgCl
2
1 mM, H
2
DCF-DA 40
l
M, pH 7.2) in a microplate and the volume
in each well was adjusted to 1 mg protein mL
1
. 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
H
2
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
l
mol MDA mg protein
1
.
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
1
mg protein
1
.
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
+2
and Mg
+2
free) and homogenized by periodic manual
inversion at room temperature. After that, the solution was filtered
(30
l
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
1
; (d)
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. Results
3.1. Hepatic activity of the biotransformation enzymes
In the fish exposed to 2
l
gL
1
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
(Fig. 1B).
3.2. Generation of ROS
The results showed that in animals exposed to 2 and 10
l
gL
1
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
(Table 1).
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
l
gL
1
) in comparison to the respective control
(Table 1).
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
(Table 2).
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
l
gL
1
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
l
gL
1
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).
Table 1
Hepatic activity of antioxidant enzymes superoxide dismutase (SOD, U mg protein
1
), catalase (CAT,
l
mol H
2
O
2
min
1
mg protein
1
), glutathione peroxidase (GPx,
l
mol NADPH
oxidized min
1
mg protein
1
), glutathione reductase (GR, nmol NADPH. min
1
mg protein
1
) and hepatic content of reduced glutathione (GSH,
l
g GSH mg protein
1
)in
P. lineatus exposed to 2 and 10
l
gL
1
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
a
32.9 ± 3.6
a
153.6 ± 8.6
a
7.1 ± 1.1
a
7.7 ± 1.8
a
ATZ 2 16.1 ± 1.1
b
10.6 ± 1.1
b
70.8 ± 2.8
b
25.3 ± 1.4
b
3.1 ± 0.9
a
ATZ 10 19.2 ± 1.0
b
9.5 ± 3.8
b
91.3 ± 5.3
b
24.6 ± 2.5
b
3.4 ± 0.7
a
48 h CTR 47.8 ± 7.4
a
40.6 ± 2.3
a
45.3 ± 6.6
a
7.1 ± 0.8
a
35.5 ± 6.5
a
ATZ 2 13.7 ± 5.0
b
23.6 ± 1.9
b
21.5 ± 1.3
b
12.2 ± 1.4
b
14.3 ± 1.8
b
ATZ 10 28.5 ± 3.7
b
20.1 ± 1.9
b
20.6 ± 0.6
b
16.0 ± 3.4
b
20.3 ± 1.6
ab
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
1121
3.7. Acetylcholinesterase
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).
4. Discussion
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
and 200
l
gkg
1
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
l
gL
1
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
GSH.
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
and 10
l
gL
1
) 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
l
gL
1
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
l
gL
1
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).
Table 2
DNA damage score in blood, liver and gill cells of P. lineatus exposed to 2 and
10
l
gL
1
of atrazine (ATZ 2 and ATZ 10, respectively) or only water (CTR), for 24 and
48 h.
Exposure time Group Cell type
Blood Liver Gills
24 h CTR 34.6 ± 8.1
a
31.4 ± 6.5
a
83.0 ± 11.0
a
ATZ 2 45.6 ± 3.7
ab
40.3 ± 6.5
a
105.2 ± 10.7
a
ATZ 10 83.9 ± 11.7
b
42.1 ± 5.5
a
112.2 ± 11.7
a
48 h CTR 51.4 ± 4.5
a
81.7 ± 3.9
a
64.3 ± 6.0
a
ATZ 2 123.0 ± 11.3
ab
111.4 ± 14.1
ab
117.2 ± 7.3
b
ATZ 10 140.7 ± 27.5
b
127.0 ± 14.4
b
72.0 ± 18.7
a
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
l
gL
1
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
l
gL
1
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
2
O
2
in the cell. In turn,
CAT activity can be inhibited when the levels of O

2
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
2
O
2
or O

2
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
l
gL
1
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
l
gL
1
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
l
gL
1
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
l
gL
1
). 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
l
gL
1
), 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
l
gL
1
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
1123
Acknowledgements
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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1125
    • "The activity of SOD was increased in groups 115 and 460 g L −1 in G. brasiliensis. One of the first enzymes that act on the defense against ROS is superoxide dismutase (SOD), which catalyze the conversion of reactive superoxide anions to hydrogen peroxide (H 2 O 2 ), which is subsequently detoxified by CAT and GPx [33]. Jin et al. [19] observed that SOD could be increased in liver of zebrafish exposed to herbicide atrazine in the concentration of 100 and 1000 g L −1 . "
    [Show abstract] [Hide abstract] ABSTRACT: Mesotrione is one of the new herbicides that have emerged as an alternative after the ban of atrazine in the European Union. To our knowledge, any work using genetic or biochemical biomarkers was performed in any kind of fish evaluating the toxicity of this compound. The impact of acute (96h) exposure to environmentally relevant mesotrione concentrations (1.8, 7, 30, 115 e 460μgL-1) were evaluated on the liver of Oreochorimis niloticus and Geophagus brasiliensis by assessing the activity of superoxide dismutase (SOD), glutathione peroxidase (GPx) and glutathione-S- transferase (GST), the levels of reduced glutathione (GSH), carbonyl assays (PCO) and lipid peroxide (LPO) as well as the DNA damage to erithrocytes, liver and gills through the comet assay. We observed an increase in the concentration of GSH and the GPx activity in O. niloticus, and the GST and SOD activity in G. brasiliensis. We found significant increase in DNA damage in all tissues in both species. The results indicated that the acute exposure to mesotrione can induce oxidative stress and DNA damage in both species.
    Full-text · Article · Dec 2015
    • "For this reason, atrazine has been banned in European Union (Bethsass and Colangelo, 2006). Many studies have indicated the deleterious effects of atrazine on animal and plant species in aquatic ecosystems (Dalton and Boutin, 2010; Brain et al., 2012; Santos and Martinez, 2012; Flores et al., 2013), as well as on soil microbiota (Chen et al., 2015) and human health (Sathiakumar et al., 2011). Even in non-target tolerant plant species, atrazine accumulation has been shown to cause toxic responses, inducing oxidative stress and negatively affecting crop growth and productivity (Alla and Hassan, 2006; Li et al., 2012). "
    [Show abstract] [Hide abstract] ABSTRACT: Poly(epsilon-caprolactone) (PCL) nanocapsules have been used as a carrier system for the herbicide atrazine, which is commonly applied to maize. We demonstrated previously that these atrazine containing polymeric nanocapsules were 10-fold more effective in the control of mustard plants (a target species), as compared to a commercial atrazine formulation. Since atrazine can have adverse effects on non-target crops, here we analyzed the effect of encapsulated atrazine on growth, physiological and oxidative stress parameters of soil-grown maize plants (Zea mays L.). One day after the post-emergence treatment with PCL nanocapsules containing atrazine (1 mg mL(-1)), maize plants presented 15 and 21% decreases in maximum quantum yield of photosystem II (PSII) and in net CO2 assimilation rate, respectively, as compared to water-sprayed plants. The same treatment led to a 1.8-fold increase in leaf lipid peroxidation in comparison with control plants. However, all of these parameters were unaffected 4 and 8 days after the application of encapsulated atrazine. These results suggested that the negative effects of atrazine were transient, probably due to the ability of maize plants to detoxify the herbicide. When encapsulated atrazine was applied at a 10-fold lower concentration (0.1 mg mL(-1)), a dosage that is still effective for weed control, no effects were detected even shortly after application. Regardless of the herbicide concentration, neither pre- nor post-emergence treatment with the PCL nanocapsules carrying atrazine resulted in the development of any macroscopic symptoms in maize leaves, and there were no impacts on shoot growth. Additionally, no effects were observed when plants were sprayed with PCL nanocapsules without atrazine. Overall, these results suggested that the use of PCL nanocapsules containing atrazine did not lead to persistent side effects in maize plants, and that the technique could offer a safe tool for weed control without affecting crop growth.
    Full-text · Article · Nov 2015
    • "Some pesticides detected in the streams in this study can also lead to genotoxic effects, even when tested alone. For example, the genotoxic effects of atrazine to fish were observed in several species, such as P. lineatus (Santos and Martinez, 2012), Carassius auratus (Çavas, 2011), Oreochromis niloticus (Ventura et al., 2008) and Channa punctatus (Nwani et al., 2011). In addition to this herbicide, an increase in the frequency of ENAs, MNs and DNA breaks has also been reported in fish exposed to endosulfan (Neuparth et al., 2006; Pandey et al., 2006). "
    [Show abstract] [Hide abstract] ABSTRACT: In order to assess the quality of streams susceptible to contamination by pesticides we apply biochemical and genotoxic biomarkers in the Neotropical fish Prochilodus lineatus submitted to in situ tests. Fish were caged, for 96 h, in two streams located in areas with intensive use of pesticides, the Apertados (AP) and the Jacutinga (JC), and in a small stream(Godoy stream-GD) found inside a forest fragment adjacent to a State Park. Biochemical parameters, such as biotransformation enzymes 7-ethoxyresorufin-O-deethylase (EROD) and glutathione-Stransferase (GST), non-protein thiols (NPSH), lipoperoxidation (LPO), protein carbonylation (PCO) and acetylcholinesterase (AChE) were evaluated in various fish organs, as well as genotoxic biomarkers (damage to DNA and occurrence of micronuclei and erythrocyte nuclear abnormalities). Samples ofwater and sedimentwere collected for analysis ofmetals (Cu, Cr, Pb,Ni,Mn, Cd and Zn), organochloride pesticides, and triazine and glyphosate herbicides.We observed an increase in liver GST activity in fish at AP and gill GST activity in fish at JC. An increase in liver LPOwas also observed in fish exposed to AP and JC. The same animals also exhibited increased DNA damage and erythrocyte nuclear abnormalities (ENAs) compared to the fish kept in GD. A number of compounds showed concentrations higher than the permitted levels, in particular, dichlorodiphenyltrichloroethane (DDT), its metabolites dichlorodiphenyldichloroethylene (DDE) and dichlorodiphenyldichloroethane (DDD), hexachlorocyclohexanes (HCH), heptachloride, diclofluanid and aldrins. These pesticideswere detected at higher concentrations in water and sediment samples from AP, followed by JC and GD. The Integrated Biomarker Response Index (IBR) indicated that AP and JC (AP: 21.7 N JC: 18.5 N GD: 12.6) have the worst environmental quality. Integrated biomarker analysis revealed that the alterations observed related well with the levels of environmental contaminants, demonstrating the effectiveness of this biomonitoring approach.
    Full-text · Article · Oct 2015
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