Ecotoxicology (2008) 17:153163
DOI 10.1007/s10646-007-0178-5
Oxidative stress biomarkers and heart function in bullfrog
tadpoles exposed to Roundup Original1
Monica J. Costa Diana A. Monteiro
Abilio L. Oliveira-Neto Francisco T. Rantin
Ana L. Kalinin
Accepted: 15 October 2007 / Published online: 7 November 2007

Springer Science+Business Media, LLC 2007
Abstract Oxidative stress biomarkers, in vivo heart rate
(fH), and contraction dynamics of ventricle strips of bull-
frog (Lithobates catesbeiana) tadpoles were evaluated after
48 h of exposure to a sub-lethal concentration (1 ppm) of
the herbicide Roundup Original1 (glyphosate 41%). The
activities of the antioxidant enzymes superoxide dismutase
and catalase were increased in the liver and decreased in
muscle, while oxidative damage to lipids increased above
control values in both tissues, showing that the generation
of reactive oxygen species and oxidative stress are
involved in the toxicity induced by Roundup1. Addition-
ally, tadpoles hyperactivity was associated with
tachycardia in vivo, probably due to a stress-induced
adrenergic stimulation. Ventricle strips of Roundup1-
exposed tadpoles (R-group) presented a faster relaxation
and also a higher cardiac pumping capacity at the in vivo
contraction frequency, indicating that bullfrog tadpoles
were able to perform cardiac mechanistic adjustments to
face Roundup1-exposure. However, the lower maximal
in vitro contraction frequency of the R-group could limit
its in vivo cardiac performance, when the adrenergic-
stimulation is present. The association between the high
M. J. Costa (&)
Campus of Sorocaba, Federal University of Sao Carlos, Avenida
Darci Carvalho Dafferner 200, Sorocaba, SP 18043-970, Brazil
e-mail: monica@ufscar.br
D. A. Monteiro
F. T. Rantin
A. L. Kalinin
Department of Physiological Sciences, Federal University of Sao
Carlos, Via Washington Luiz, km 235, Sao Carlos,
SP 13565-905, Brazil
A. L. Oliveira-Neto
Center of High Technological Education, Campinas State
University, Rua Paschoal Marmo, 1888, Limeira,
SP 13484-970, Brazil
energetic cost to counteract the harmful effects of this
herbicide and the induction of oxidative stress suggest that
low and realistic concentrations of Roundup1 can have an
impact on tadpoles performance and success, jeopardizing
their survival and/or population establishment.
Keywords Roundup1
Oxidative stress biomarkers

Cardiac contractility
Bullfrog tadpoles

Lithobates catesbeiana
Introduction
The increased use of pesticides, alone or in association with
habitat loss, over harvesting, ultraviolet-B radiation, global
warming, and/or diseases, has been considered one of the
main factors underlying the decline in amphibian popula-
tions over recent decades (Collins and Storfer 2003; Relyea
2003). Pesticides are widely used in agriculture adversely
affecting nontarget organisms wherein amphibians are
major components of the wetland biota (Gurushankara et al.
2007). Amphibian species inhabiting agricultural sur-
roundings may be exposed to pesticides during their aquatic
phase as tadpoles are strongly susceptible to chemicals
(Johansson et al. 2006). Despite this, toxicological research
on amphibians has been rather scarce compared to that on
other vertebrates (Venturino et al. 2003).
Glyphosate, a broad-spectrum herbicide, is one of the
most frequently applied pesticides with agricultural purposes
in the world (Hultberg, 2007), and it is extensively used in the
aquatic environment to control aquatic weeds (Abdullah
et al. 1995; Tsui and Chu 2003). Roundup1 (Monsanto
Company, St. Louis, MO, USA) is one of the most common
glyphosate-based formulations, consisting of an isopropyl-
amine salt and the surfactant polyoxyethylene amine (POEA).
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154
This herbicide presents water solubility of 15,700 mg/l
and a half-life in pond water from 7 until 70 days (Giesy
et al. 2000). It is widely used in rice and soybean cultures in
Southern Brazil in concentrations ranging from 0.36 to
2.16 mg/l (Rodrigues and Almeida 2005). However, various
studies have shown that the lethal ingredient in Roundup1
was not the glyphosate itself, but rather the surfactant, that
allows the herbicide to penetrate plant cuticule (Perkins et al.
2000; Cox and Surgan 2006; Brausch and Smith 2007).
Due to its high water solubility and the extensive use in
the environment, the exposure of nontarget aquatic
organisms to this herbicide is a concern for ecotoxicolo-
gists (Cavas and Konen 2007).
At sublethal concentrations, Roundup1 induced toxic
effects in fish (Szarek et al. 2000; Terech-Majewska et al.
2004) and other aquatic organisms such as bacteria, mic-
roalgae, protozoa and crustacea (Tsui and Chu 2003).
Saparling et al. (2006) exposed eggs of red-eared sliders
(Trachemys scripta elegans) to single applications of
another formulation of glyphosate (Glypro1, Dow Agro-
Sciences) and found effects on behavior, survival, growth,
and genotoxicity only at concentrations higher than those
considered environmentally relevant. Although conven-
tionally thought to be non-lethal to amphibians, Relyea
(2005a) demonstrated that Roundup1 can induce extre-
mely high rates of mortality to these animals which could
eventually lead to population declines. According to Howe
et al. (2004), amphibians are appropriate for examining the
toxicity of various glyphosate-based formulations in the
aquatic environment due to their dependence on aquatic
sites for reproduction and early development.
Oxidative stress develops when there is an imbalance
between prooxidants and antioxidants ratio, leading to the
generation of reactive oxygen species (ROS). Environ-
mental contaminants such as herbicides, heavy metals and
insecticides are known to modulate antioxidant defensive
systems and to cause oxidative damage in aquatic organ-
isms by ROS production (Risso-de Facerney et al. 2001;
Liu et al. 2006; Monteiro et al. 2006). ROS, such as
hydrogen peroxide (H2O2), superoxide anion (O- 2 ) and
hydroxyl radical ( OH), at supranormal levels can react
with biological macromolecules potentially leading to
enzyme inactivation, lipid peroxidation (LPO), DNA
damage and even cell death (Winston 1991; Pena-Llopis
et al. 2003; Banudevi et al. 2006).
Xenobiotic-induced ROS production and the corre-
sponding oxidative damage may be one of the major causes
of impairments in tadpole reproduction, development and
behavior that can be related to amphibian population
declines. Additionally, integrative approaches combining
the analysis of oxidative stress and physiological bio-
markers are crucial to understanding the mechanisms
underlying contaminant damage.
123
M. J. Costa et al.
When faced with adverse conditions, aquatic animals can
either try to escape from the stressful situation or activate
physiological adjustments that could counteract the imposed
stress. As a consequence, animals display a large variation in
cardiac function according to the mode of life and activity
level. The ability of the cardiac muscle to maintain pump
performance under different physiological conditions is one
of the most important characteristics that enable vertebrates
to survive under adverse conditions (Driedzic and Gesser
1994). Consequently, efficient adjustments of cardiac output
in response to xenobiotics, achieved by changes in stroke
volume and/or heart rate, are crucial.
Cardiac stroke volume is determined by the regulation
of myocardial contractility, which depends on the complex
regulation of intracellular calcium ([Ca2+]i) homeostasis on
a beat-to-beat basis (Lewatowski and Pytkowski 1987;
Bers 2001) while heart rate is under nervous and humoral
control (Farrell and Jones 1992).
The ability to predict the effects of pollutants on
organisms and to extrapolate toxicant effects from the
laboratory to population and community levels has become
a very important factor.
There is a need for additional physiological and bio-
chemical indicators of organism health and sublethal
toxicant effects. Biological indicators can help to identify
environmental problems before the health of aquatic systems
becomes seriously altered (Jimenez and Stegeman 1990).
Venturino et al. (2003) emphasized that the use of various
biomarkers with multiple endpoints is needed to link expo-
sure to response and to provide better predictive tools for the
environmental protection of endangered anuran species.
In this context, the goal of this work was to test the
effect of Roundup1 on the oxidative stress biomarkers and
on heart function of bullfrog tadpoles, Lithobates cates-
beiana. The choice of the North American bullfrog, a non-
threatened and widely farmed species, as a model for the
study of the effects of this pesticide minimizes the use of
endangered anuran in experiments.
Moreover, while several studies have observed the
negative aspects of pesticides on growth, development, and
behavior of anuran tadpoles (Bridges 2000; Christin et al.
2003; Broomhall 2005), this study focused on biochemical
(oxidative stress biomarkers) and physiological alterations
(cardiac function), which are much more specific and
sensitive biomarkers.
Methods
Chemicals
The commercial formulation of the herbicide glyphosate
(N-phosphonomethyl-glycine) Roundup Original1
Bullfrog tadpoles exposed to Roundup1
(glyphosate 41%, POEA % 15%Monsanto Company, St.
Louis, MO, USA) was used. It contains a 360 acid equivalent
per liter as the isopropylamine (ipa) salt. All other chemicals
and reagents were purchased from Sigma-Aldrich Chemical
Co.
Animal care
Newly hatched Lithobates catesbeiana (Shaw, 1802) tad-
poles were obtained from a breeding colony at Ibate, Sao
Paulo State, Southeast Brazil (21 o570 S, 47 o590 W).
Tadpoles were housed in 500 l holding tanks equipped with
a continuous supply (1.2 l/h) of well-aerated and dechlo-
rinated water at a constant temperature (25 1C) under
natural photoperiod (%12 h light:dark cycle) until they
reached Gosner (1960) developmental stage 25 (%1 week)
at time of herbicide application. Animals were fed ad
libitum with commercial trout food flakes (3540% pro-
tein), which was withheld 48 h before intoxication.
Experimental design
The water was monitored daily to ensure that the physical
and chemical parameters were kept at acceptable levels
(pH 7.17.3; hardness as CaCO3 4856 mg l-1; alkalinity
as CaCO3 4043; DO2 6.87.5 mg l-1), as found in most
Brazilian inland waters.
Tadpoles were randomly divided into two replicated
experimental groups: control (C; n = 10, body mass =
10.3 1.7 gmean SE) and Roundup Original1-
exposed (360 g/l glyphosate) at the sublethal concentration
of 1 mg/l of the commercial formulation for 48 h (R-
group; n = 10; body mass = 11.0 2.7 gmean SE).
This concentration was chosen based on the concentration
range found in the water near agricultural areas in Brazil
(from 0.36 to 2.16 mg/l), as reported by Rodrigues and
Almeida (2005). Indeed, Relyea (2005b) found a LC50 16 d
of 2.07 mg l-1 of the Roundup1 active ingredient for
bullfrog tadpoles while the LC50 48 h for the glyphosate
isopropylamine salt estimated by Clements et al. (1997)
was 108 mg l-1. Moreover, we intended to observe the
effects of this pesticide in sublethal levels, as shown by the
lack of effect of 1 mg/l of glyphosate on the survival of
bullfrog tadpoles at Gosner stage 25 in a study carried out
by Relyea (2004).
Both experimental groups were placed in 30 l glass
aquaria filled with dechlorinated well-aerated water
([6.0 mg O2/l), with a controlled temperature (25 1C)
on a 12:12 h light:dark cycle. Aquaria were dark-covered
to prevent external disturbance. Control and Roundup1-
exposed tadpoles were maintained for 48 h in a static
system. During this period, sublethal effects like level of
155
activity and swimming performance were monitored. All
procedures followed ASTM (2000) guidelines.
After exposure, the heart rate (fHbpm) was monitored
and, subsequently, tadpoles were sacrificed by pithing
(American Veterinary Medical Association, 2001) to avoid
side effects on physiological parameters. Thereafter, the
heart, liver and tail muscle were carefully excised and
washed with cold physiological solution. The ventricles
were then immediately separated from the heart for the
in vitro experiments. The livers and muscle samples were
taken and immediately frozen into liquid nitrogen. Frozen
samples were stored at -80C until the biochemical
determinations were carried out.
Biochemical parameters
All biochemical assays were measured spectrophotometri-
cally (Spectronic Genesys 5, Milton Roy Co., NY, USA) at
25C. Samples of frozen liver and muscle were homoge-
nized in 0.1 M sodium phosphate buffer pH 7.0 at a ratio of
1:5 w/v using a Turratec TE 102 (Tecnal, SP, Brazil)
homogenizer at 18,000 rpm. Samples were centrifuged at
12,000 9 g for 30 min at 4C and the supernatant was used
directly for assay catalase (CAT) and superoxide dismutase
(SOD) activities and LPO determination according to the
methods described by Monteiro et al. (2006).
The SOD activity was determined based on the ability of
the enzyme to inhibit the reduction of nitro blue tetrazo-
lium (NBT) (Crouch et al. 1981), which was generated by
37.5 mM hydroxylamine in alkaline solution (Otero et al.
1983). The assay was performed in a 0.5 M sodium car-
bonate buffer (pH 10.2) with 2 mM EDTA. The reduction
of NBT by superoxide anion to blue formazan was mea-
sured at 560 nm. The rate of NBT reduction in the absence
of tissue was used as the reference rate. One unit of SOD
was defined as the amount of protein needed to decrease
the reference rate to 50% of maximum inhibition. The SOD
activity was expressed in units per mg protein.
The CAT activity was measured by decreasing the H2O2
concentration at 240 nm (Aebi 1974). Decays in absor-
bance were recorded during 17 s in a 50 mM sodium
phosphate buffer (pH 7.0) containing 15 mM H2O2 and the
enzyme extract. CAT values were expressed as Bergmeyer
units (B.U.) per mg protein. One unit of CAT (according to
Bergmeyer) is the amount of enzyme, which releases half
the peroxide oxygen from the H2O2 solution of any con-
centration in 100 s at 25C. According to Wilhelm Filho
et al. (1993), 1 nmol CAT corresponds to 33 B.U.
The xylenol orange assay for lipid hydroperoxide
(FOXferrous oxidation-xylenol orange) was performed
as described by Jiang et al. (1992). Tissue homogenates
were prepared as described above for SOD, CAT and GPx
123
156
assays. Lipid hydroperoxide was determined with 100 ll of
sample (previously deproteinised with 10% TCA) and
900 ll of reaction mixture containing 0.25 mM FeSO4,
25 mM H2SO4, 0.1 mM xylenol orange and 4 mM butyl-
ated hydroxytoluene in 90% (v/v) methanol. The mixtures
were incubated for 30 min at room temperature prior to
measurements at 560 nm. The molar extinction coefficient
of 4.3 104 M-1 cm-1 for cumene hydroperoxide (Jiang
et al. 1991) was used. Lipid hydroperoxide levels were
expressed as nmol per milligram protein.
Total protein contents were determined according to the
Bradford method with Coomassie Brilliant Blue G-250
(Bradford 1976) adapted to a microplate reader (Dynex
Technologies Ltd., UK) as described by Kruger (1994),
using bovine serum albumin as a standard.
Determination of Relative Ventricular Mass (RVM)
Control and Roundup1-exposed animals were sacrificed
and the body mass was measured (Wbg). The heart was
dissected and the ventricle carefully removed. Ventricles
were weighed (Wv) and the ventricular mass was expressed
as a percentage of body mass (relative ventricular mass,
RVM% of Wb).
In vivo heart rate
fH measurements in bullfrog tadpoles were taken on indi-
viduals placed in water-filled holding chambers made from
Petri dishes. A continuous flow of well-aerated water at
25C was maintained throughout the chamber. The thoracic
cavity was surgically opened in a caudalcranial direction
for pericardium exposure. The fH was determined visually
and expressed as beats per minute (bpm). This procedure
does not seem to be more traumatic to the tadpoles than the
implantation of subcutaneous ECG electrodes (Wassersug
et al. 1981; Burggren et al. 1983; Feder 1983).
In vitro experiments Results
Ventricle strips (diameter % 1 mm; mass = 1.6 0.3
mg; length = 2.0 0.3 mmmean SE) were excised
and transferred to a 30 ml water-jacketed organ bath con-
taining (in mM): 115 NaCl, 5 KCl, 30 NaHCO3, 0,94
MgSO4, 2,5 CaCl2, and 5 glucose and bubbled throughout
the experiment with a 2% carbogenic gas mixture (pH 7.4
at 25C).
Preparations were suspended using surgical silk to have
one end attached to a platinum chain which hung from a
LETICA isometric force transducer (Letica Corporation,
123
M. J. Costa et al.
USA), and the other end was tied around a platinum
electrode. This electrode and one placed in the bath were
connected to an AVS 100D stimulator (Solucao Integrada
Ltda., Brazil) sending electrical square pulses and having a
duration of 8 ms and a voltage 50% above the threshold in
order to provide a security margin and assuring maximal
stimulation throughout the experiment. Preparations were
stretched to obtain a twitch tension at the maximum of the
length-twitch tension relation. Twitch tension was then
allowed to stabilize for at least 40 min at 0.2 Hz (12 bpm)
before each protocol (see below).
After the stabilization period, the twitch force (Fc
mN mm-2), time to peak tension (TPTms) and time to
half relaxation (THRms) were measured at the sub-
physiological frequency of 0.2 Hz. Thereafter, pacing fre-
quency was increased in 0.2 Hz increments until the
frequency in which the muscle failed to show regular
contractions. The maximal in vitro stimulation frequency
was considered the frequency at which at least 80% of the
strips were still able to contract regularly.
To obtain a more integrative perspective on the effects
of Roundup1 on heart function in bullfrog tadpoles, the
pumping capacity of the muscle at each stimulation fre-
quency was calculated. The cardiac pumping capacity
(CPCmN mm-2 min-1), the product of stimulation fre-
quency and twitch force was calculated according to
Matikainen and Vornanen (1992).
Statistical analysis
Results are presented as means 1 S.E.M. For comparisons
between two groups, t-tests (parametric) or Mann-Whitney
U-tests (non-parametric) were applied. The Kolmogorov and
Smirnov method was applied to evaluate normality of
the samples and the F-test was applied for homogeneity
of variances (GraphPad Instat version 3.00, GraphPad
Software, USA). Differences between means at a 5%
(P \ 0.05) level were considered significant.
The activities of antioxidant enzymes and LPO levels in the
liver and white muscle are shown in Table 1. The exposure
to Roundup1 (R-group) increased the hepatic activities of
SOD, CAT and LPO levels (81%, 189% and 55%,
respectively) when compared to control tadpoles (C-
group). In contrast, Roundup1 induced a 27% decrease in
SOD activity and a 73% decrease in CAT activity of
muscle. Concomitantly, increases in the LPO levels (16%)
were observed in the muscle of the R-group when com-
pared to the C-group.
Bullfrog tadpoles exposed to Roundup1 157

Table 1 Antioxidant enzymes activities and lipid peroxidation levels constant (%1.0 mN mm-2) from 0.2 to 1.2 Hz, its highest
in the liver and muscle of bullfrog tadpoles from control (C-group, sustained frequency. Despite the lower maximal sustained
n = 10) and Roundup1-exposed (R-group, n = 10) bullfrog tadpoles
frequency presented by the R-group, the Fc developed
C-group R-group P values above 0.8 Hz was % two times higher than that presented
by the C-group (P = 0.002).
Liver
According to Shiels and Farrell (1997) the peak of the
SOD (U/mg protein) 5.25 0.53 9.52 1.26 0.011
pumping capacity versus frequency curve corresponds to
CAT (U.B./mg protein) 2.06 0.19 5.97 0.33 0.001
an optimum frequency for pumping capacity, known as
LPO (nmol/g tissue) 39.84 4.54 61.69 7.73 0.031
power output. The optimum frequency for pumping
Muscle
capacity in the C-group was %0.6 Hz as the CPC was
SOD (U/mg protein) 23.60 1.53 17.23 2.50 0.040
maintained constant (%0.6 mN mm-2 min-1) above this
CAT (U.B./mg protein) 1.35 0.34 0.37 0.05 0.030
frequency. In contrast, the R-group reached the power
LPO (nmol/g tissue) 21.90 0.90 25.41 1.01 0.019
output at 1.0 Hz (1.1 mN mm-2 min-1) considering that
The values are presented as means S.E CPC values did not change significantly beyond this

The R-group increased its swimming activity in relation 1.4

to the C-group, which tended to keep stationary at the
bottom of the aquarium or float without movement. The 1.2 * *
increased activity in the R-group was accompanied by an 1.0
increase (P = 0.004) of almost 28% of fH
(54.3 2.5 bpm) relative to the C-group Fc (mN.mm-2)
0.8
(42.8 2.9 bpm). However, both groups presented similar
values of relative ventricular mass (RVM % 0.05%; 0.6
Fig. 1).
0.4
Figure 2 shows the effects of increasing stimulation
frequency on force development (Fc) and cardiac pumping 0.2 C R
capacity (CPC) of the C- and R-groups. The C-group
maintained a constant Fc (%0.9 mN mm-2) from 0.2 to 0.0
0.8 Hz, decreasing significantly and progressively during
subsequent increases in frequency, reaching minimum 1.6
C-group R-group
values (0.4 mN mm-2; P = 0.001) at the highest sustained *
1.4
frequency (1.4 Hz). In the R-group, Fc was maintained
1.2 *
CPC (mN.mm-2 .min-1)

1.0
0.07
0.8
0.06
0.6
0.05
0.4
RVM (% Wb)

0.04 0.2 C R

0.03 0.0
0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4
Frequency (Hz)
0.02
Fig. 2 Twitch force (FcmN mm-2, upper panel) and cardiac
0.01 pumping capacity (CPCmN mm-2 min-1, lower panel) developed
by ventricle strips during increases in stimulation frequency from
0 control (C-group, n = 10) and Roundup1-exposed (R-group,
C-group R-group n = 10) bullfrog tadpoles. Mean values S.E. The arrows indicate
the heart rate measured in vivo to control (C, n = 10) and
Fig. 1 Relative ventricular mass (RVM), given as a percentage of Roundup1-exposed (R, n = 10) tadpoles. Open symbols denote a
body mass (Wb) of control (C-group, n = 10) and Roundup1- significant difference in relation to the values obtained at 0.2 Hz
exposed (R-group, n = 10) bullfrog tadpoles. Mean values + S.E. No (P \ 0.05), while asterisks indicate differences between experimental
significant differences between groups were found (P [ 0.05) groups at the same frequency (P \ 0.05)

123
158 M. J. Costa et al.

frequency. CPC values of the C-group were approximately
two times lower than those presented by the R-group at
1.0 Hz (P = 0.002) and 1.2 Hz (P = 0.004).
The time-dependent variables (time to peak tension
TPT, and time to half relaxationTHR) during increasing
stimulation frequency are shown in Fig. 3.
Both groups presented constant TPT values until 0.8 Hz
(C-group: %502 ms) and 1.0 Hz (R-group: %486 ms).
Above these frequencies, the TPT decreased progressively
and significantly, reaching minimum values at 1.4 Hz (C-
group: 318 ms; P = 0.001) and 1.2 Hz (R-group: 369 ms;
P = 0.011). There were no significant differences between
the TPT values of the experimental groups.
600
500
400
TPT (ms)
The THR remained unchanged for the R-group
(%242 ms) in all the tested frequencies while it decreased
progressively and significantly above 0.6 Hz (from 328 to
206 ms) for the C-group. Moreover, the THR values of the
R-group were significantly lower than those of the C-group
until 1.0 Hz (P = 0.049).
Discussion
According to the Environmental Protection Agency of the
USA (EPA), glyphosate acid and its salts are moderately
toxic compounds (toxicity class II) and are classified as
General Use Pesticides (GUP).
The herbicide glyphosate is sold under a variety of
commercial names worldwide, including Roundup Origi-
nal1. Because glyphosate is not applied in the field as a
pure active ingredient, but as its technical formulations, the
toxicity of the commercial form, i.e. Roundup1, should be
evaluated (Cavas and Konen 2007).
Many studies have addressed the impact of insecticides

300
and herbicides on the biodiversity and productivity of
aquatic communities (Lambert 1997; Bridges 1997;
R-group C-group Leonard et al. 1999; Smith 2001; Favari et al. 2002; Boone
200
and James 2003). Particularly, the effect of Roundup1 on
aquatic amphibian communities has been a subject of
100
C R intense debate (e.g., Thompson et al. 2006 vs. Relyea
2006).
0
According to the label of Monsantos Roundup Origi-
500 nal1 (1991), in its Environmental Hazards warning
section, this herbicide should not be applied directly to
water, to areas where surface water is present or to inter-
400 tidal areas below the mean high water mark. However,
even though it is intended for terrestrial use, there is huge
evidence that Roundup1 gets into aquatic habitats, typi-
THR (ms)

300
cally through unintentional and/or unavoidable aerial
overspray (Relyea 2006) and/or carried by runoff (Relyea
200 * * * * 2005b). Additionally, Roundup1 is sprayed directly on
*
water to control emergent aquatic plants in many countries
100 (Giesy et al. 2000). In Brazil, besides the extensive use in
C R rice and soybean cultures (Rodrigues and Almeida 2005), it
is also applied to control aquatic weeds including Eich-
0
0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4 hornia crassipes, Pistia stratiotes and Salvinia auriculata
Frequency (Hz) (Carvalho et al. 2005). Moreover, the small wetlands
occurring within the herbicides target sites are usually
Fig. 3 Time to peak tension (TPTms, upper panel) and time to half directly contaminated during aerial application, resulting in
relaxation (THRms, lower panel) developed by ventricle strips
during increases in stimulation frequency from control (C-group,
high potential exposure and effects for its constituent biota
n = 10) and Roundup1-exposed (R-group, n = 10) bullfrog tad- (Thompson et al. 2004). These small wetlands host con-
poles. Mean values S.E. The arrows indicate the heart rate siderable amphibian biodiversity and endemism as many
measured in vivo to control (C, n = 10)) and Roundup1-exposed species only breed and spend their larval stages in these
(R, n = 10) tadpoles. Open symbols denote a significant difference in
relation to the values obtained at 0.2 Hz (P \ 0.05), while asterisks
overlooked habitats (Relyea 2006).
indicate differences between experimental groups at the same Corroborating the statement from Relyea (2006) that
frequency (P \ 0.05) Roundup1 has a negative impact on amphibians in aquatic

123
Bullfrog tadpoles exposed to Roundup1
environments, the present results demonstrated that the
exposure of bullfrog tadpoles to 1 ppm of Roundup1 for
48 h has a high potential to induce oxidative stress and
affects cardiac function.
Gehin et al. (2006) and Hultberg (2007) reported that
glyphosate, alone or as Roundup1 formulation, can alter
the cellular antioxidant status. The key role in metabolite
detoxification and the high oxygen consumption make the
liver and the skeletal muscle, respectively, appropriate
organs to investigate pesticide-induced damage (Fulle et al.
2004; Banudevi et al. 2006). In our experiments, the oxi-
dative stress was indicated by the increased levels of tissue
lipid hydroperoxides, generated by oxidative attack on cell
membrane phospholipids and circulating lipids. This indi-
cates that ROS accumulated in liver and muscle of bullfrog
tadpoles after 48 h of exposure to 1 ppm of Roundup1 and
suggests that ROS-induced damage may be one of the main
toxic effects of this organophosphate. Organophosphates
may enhance lipid peroxidation by directly interacting with
the cellular plasma membrane (Hazarika et al. 2003). In
this context, Pieniazek et al (2004) demonstrated that
Roundup Ultra1 360 SL and its active compound gly-
phosate increased the levels of lipid peroxidation in human
erythrocytes.
Due to their easy oxidation, lipids are the preferred
substrate to free radical damage via LPO, resulting in
phospholipid degradation, membrane injury, and saturated
hydrocarbon formation such as lipid hydroperoxides
(Comporti 1985; Georgieva 2005; Oruc and Usta 2007).
LPO is among the best predictors of the level of ROS-
induced systemic biological damage (Saygili et al. 2003;
Georgieva 2005) and it is one of the molecular mechanisms
involved in pesticide toxicity (Kehrer 1993; Kavitha and
Rao 2007).
On the other hand, the key antioxidant enzymes for ROS
detoxification in all organisms are SOD, which catalyzes
the dismutation of superoxide anion to hydrogen peroxide,
and CAT that removes hydrogen peroxide. An important
feature of these ROS-scavenging enzymes is their induc-
ibility under oxidative stress, providing an important
adaptation to this condition. Furthermore, these systems
may also be inhibited; a process that can lead to antioxidant
mediated toxicity (Di Giulio et al. 1989; Oruc and Usta
2007).
In hepatic tissue, SOD and CAT are the major enzymes
in eliminating ROS formed during bioactivation of xeno-
biotic (Sk and Bhattacharya 2006) and the induction of the
SOD/CAT system provides a first line defense against
ROS. In the present study, the increased activity of these
hepatic antioxidant enzymes in the R-group was a response
towards increased ROS generation. However, the enhanced
hepatic LPO shows that the Roundup1-induced ROS are
not totally scavenged by this antioxidant system.
159
According to Hazarika et al. (2003) and Kavitha and Rao
(2007) LPO may be the first step of cell membrane damage
by organophosphates.
Conversely, significant decreases in the specific activi-
ties of SOD and CAT were observed in the skeletal muscle
of the R-group. The antioxidant enzymes activities may be
increased or inhibited under xenobiotic exposure depend-
ing on the intensity and the duration of the stress applied as
well as the susceptibility of the exposed species (Oruc and
Usta 2007).
SOD and CAT are easily inactivated by lipid peroxide or
ROS (Halliwell and Gutteridge 1987). The enhanced LPO
by Roundup1 exposure could be a consequence of the
decreased SOD and CAT activities in skeletal muscle. The
antioxidant enzyme CAT prevents the SOD inactivation by
hydrogen peroxide. Reciprocally, SOD prevents CAT
inhibition by superoxide anion (Banudevi et al. 2006). As a
result, there is an accumulation of superoxide anion radical
and other ROS, thereby inducing the oxidative damage
shown by increased LPO levels in the R-group skeletal
muscle. CAT and SOD depletion typically leads to oxi-
dative stress and stimulates lipid peroxidation (Halliwell
and Gutteridge 1989; Dorval and Hontela 2003; Bag-
nyukovaa et al. 2005). Our results clearly indicated that
Roundup1 can induce ROS generation, resulting in oxi-
dative stress in liver and skeletal muscle of bullfrog
tadpoles, demonstrating a tissue-specific antioxidant
modulation.
The liver is a site of multiple oxidative reactions and
maximal free radical generation (Gul et al. 2004; Avci
et al. 2005; Atli et al. 2006). Consequently, this tissue
showed a stronger antioxidant potential than skeletal
muscle due to its higher antioxidant enzymatic activities.
The increased CAT activity in liver was a trend toward the
reduced CAT activity in R-group skeletal muscle showing
that muscle antioxidant enzymes are less efficient than liver
ones, increasing its vulnerability towards ROS. This tissue-
specific response can be related to its anatomic position
which determines the exposure route and distribution of
pollutants, and its antioxidant potential and defensive
capacity (Ahmad et al, 2000, 2006).
Cardiac output is the product of stroke volume and heart
rate (Whiters and Hillman 2001). As a consequence, vari-
ation in cardiac output can be determined by differences in
stroke volume (Hillman et al. 1985), which is in partly
reflected by differences in ventricle mass (Hillman 1976).
When increases in cardiac performance are required in
response to xenobiotics, acute exposure leads to accelera-
tion in cross-bridges cycle, and therefore, in the heart rate,
while chronic exposure causes cardiac hypertrophy (Calore
et al. 2007). The former (i.e., positive chronotropism) has a
higher energetic cost than the latter (i.e., increased stroke
volume).
123
160
However, the absence of variation on relative ventricu-
lar mass (RVM) in response to the acute exposure to
Roundup1 is not surprising (Fig. 1) as a cardiac hyper-
trophy could occur only after more prolonged periods to
this herbicide.
It is well established that the sympathetic nervous sys-
tem can exert significant cardio-respiratory and metabolic
functions during stressful situations. Furthermore, cate-
cholamines affect cardiac function and vascular resistance
in amphibians (e.g. Erlij et al. 1965; Kirby and Burnstock
1969; Lillo 1979; Herman and Sandoval 1983; Andersen
et al. 2001) even during larval stages (Kloberg and Fritsche
2002; Gonzalez et al. 2004; Kimmel 2004). In the present
study, the fH values for C- and R-groups were %43 bpm
and %54 bpm, respectively. Based on the findings
described above, the higher fH values recorded for the R-
group could be attributed to a stress-induced catecholamine
release as circulating catecholamines are involved in
adrenergic cardiac control.
Regarding the fH values per se, Jia and Burggren (1997)
found mean values between 67 and 73 bpm in anesthetized
tadpoles of L. catesbeiana at 2224C, with no significant
differences between the studied stages. Feder (1983)
recorded fH values of about 85 bpm in tadpoles of Rana.
beriandieri under normoxia and 25 C. These higher val-
ues compared to the ones obtained in the present study
could reflect methodological and/or inter-specific differ-
ences. Furthermore, the in vivo heart rates were 49% and
25% lower than the maximal frequencies obtained in vitro
for C- and R-groups, respectively (Fig. 2).
The increased heart rate of R-group was associated with
hyperactivity, which may represent an unfavorable avoid-
ance response suggesting that Roundup1-exposure shifts a
considerable amount of energy from the morphogenetic
processes to counteract the negative effects of this herbicide.
Interestingly, R-group ventricle strips were able to
maintain a constant twitch force at all tested stimulation
frequencies, while the C-group presented a negative force-
frequency relationship above 0.8 Hz (Fig. 2). A plausible
explanation could be a nitric oxide (NO) modulation of
contractile force induced by Roundup1. Sys et al. (1997)
demonstrated that the nitric oxide synthase could exert a
direct effect on myocardial contractility of the frog Rana
esculenta. Moreover, there is strong evidence that nitric
oxide influences tadpoles cardiovascular function (Schw-
erte et al. 2002; Hedrik et al. 2005). Koyama et al. (1997)
observed that the anionic surfactant LES (sodium poly-
oxyethylene laurylether sulfate) produced vasorelaxation in
aortic ring segments of rats via nitric oxide synthase acti-
vation as LES caused a significant increase in NO
production in cultured endothelial cells. Considering a
possible nitrergic modulation on bullfrog tadpole cardio-
vascular system and the similarity between the non-ionic
123
M. J. Costa et al.
surfactant POEA (present in Roundup Original1 formula-
tion) and LES, a POEA-mediated increase in NO
production could be suggested. Additionally, Layland et al.
(2002) observed that NO directly modulates cardiac con-
tractility by accelerating relaxation in rat cardiac myocytes.
Therefore, the lower THR values observed in the R-group
(Fig. 3) could be related to a surfactant-mediated increase
in the NO production.
For both experimental groups, the optimum frequency
for pumping capacity calculated from our study falls within
the in vivo contraction frequencies. However, due to the
constant Fc presented by the R-group during increases in
stimulation frequency, its CPC values were higher
(1.0 mN mm-2 min-1) than those in the C-group
(0.6 mN mm-2 min-1) at their respective in vivo con-
traction frequencies (Fig. 2). These results indicate that
bullfrog tadpoles were able to perform cardiac mechanistic
adjustments to face the acute exposure to sublethal
Roundup1 concentrations. Nevertheless, it is important to
emphasize that the R-group was unable to reach the max-
imal contraction frequency achieved by the C-group
(1.4 Hz). This Roundup1 effect could limit the in vivo
cardiac performance, as the maximum in vitro frequency of
the R-group was only 25% higher than that obtained in
vivo under rest conditions. Considering the potential stress-
induced catecholamine release (discussed above) during
Roundup1 exposure, one could expect that the heart rate
should be even higher under the hyperactivity observed for
R-group tadpoles. Moreover, our results were obtained
after 48 h exposure and longer exposure periods could
exacerbate these responses.
Taken together, the Roundup1 induced ROS generation
and the consequent oxidative stress, the increased energetic
expenditure to maintain the hyperactivity and tachycardia
observed under low and realistic herbicide concentrations
could have a negative impact on tadpoles performance and
success, jeopardizing their survival and/or population
establishment. Nonetheless, a chronic exposure to
Roundup1 should be performed to observe if the species is
able to develop adaptive strategies to counteract the
harmful xenobiotic effects or if the long term exposure
results in even worse conditions.
Acknowledgments Bullfrog tadpoles were kindly provided by the
Estrela breeding colony at Ibate, Sao Paulo State, Brazil. The authors
are thankful to the field technician Mr. Angelo Carnelosi for caring
for the tadpoles in the laboratory. All the experiments were performed
complying with the Brazilian laws.
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