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Ecotoxicology (2008) 17:153–163 DOI 10.1007/s10646-007-0178-5

Oxidative stress biomarkers and heart function in bullfrog tadpoles exposed to Roundup Original 1

Monica J. Costa Æ Diana A. Monteiro Æ Abilio L. Oliveira-Neto Æ Francisco T. Rantin Æ 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 (f H ), 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 Original 1 (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 Roundup 1 . Addition- ally, tadpoles’ hyperactivity was associated with tachycardia in vivo, probably due to a stress-induced adrenergic stimulation. Ventricle strips of Roundup 1 - 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 Roundup 1 -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 Sa˜o 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 Sa˜o

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 Roundup 1 can have an impact on tadpoles’ performance and success, jeopardizing their survival and/or population establishment.

Keywords

Cardiac contractility Bullfrog tadpoles

Lithobates catesbeiana

Roundup 1 Oxidative stress biomarkers

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). Roundup 1 (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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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 Roundup 1 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 (C¸ avas and Ko¨nen 2007). At sublethal concentrations, Roundup 1 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 (Glypro 1 , 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 Roundup 1 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 (H 2 O 2 ), 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; Pen˜a-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.

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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 ([Ca 2+ ] 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 Roundup 1 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 Original 1

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(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´, Sa˜o Paulo State, Southeast Brazil (21 o 57 0 S, 47 o 59 0 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 ± 1 C) 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 (35–40% 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 7.1–7.3; hardness as CaCO 3 48–56 mg l -1 ; alkalinity as CaCO 3 40–43; DO 2 6.8–7.5 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 g—mean ± SE) and Roundup Original 1 - 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 g—mean ± 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 LC 50 16 d of 2.07 mg l -1 of the Roundup 1 active ingredient for bullfrog tadpoles while the LC 50 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 O 2 /l), with a controlled temperature (25 ± 1 C) on a 12:12 h light:dark cycle. Aquaria were dark-covered to prevent external disturbance. Control and Roundup 1 - exposed tadpoles were maintained for 48 h in a static system. During this period, sublethal effects like level of

activity and swimming performance were monitored. All procedures followed ASTM (2000) guidelines. After exposure, the heart rate (f H —bpm) 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 -80 C 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 25 C. 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 4 C 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 H 2 O 2 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 H 2 O 2 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 H 2 O 2 solution of any con- centration in 100 s at 25 C. According to Wilhelm Filho et al. (1993), 1 nmol CAT corresponds to 33 B.U. The xylenol orange assay for lipid hydroperoxide (FOX—ferrous oxidation-xylenol orange) was performed as described by Jiang et al. (1992). Tissue homogenates were prepared as described above for SOD, CAT and GPx

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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 FeSO 4 , 25 mM H 2 SO 4 , 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 Roundup 1 -exposed animals were sacrificed and the body mass was measured (W b —g). The heart was dissected and the ventricle carefully removed. Ventricles were weighed (W v ) and the ventricular mass was expressed as a percentage of body mass (relative ventricular mass, RVM—% of W b ).

‘‘In vivo’’ heart rate

f H 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 25 C was maintained throughout the chamber. The thoracic cavity was surgically opened in a caudal–cranial direction for pericardium exposure. The f H 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

Ventricle strips (diameter % 1 mm; mass = 1.6 ± 0.3 mg; length = 2.0 ± 0.3 mm—mean ± SE) were excised and transferred to a 30 ml water-jacketed organ bath con- taining (in mM): 115 NaCl, 5 KCl, 30 NaHCO 3 , 0,94 MgSO 4 , 2,5 CaCl 2 , and 5 glucose and bubbled throughout the experiment with a 2% carbogenic gas mixture (pH 7.4 at 25 C). 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,

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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 (Soluc¸a˜o 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 (TPT—ms) and time to half relaxation (THR—ms) 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 Roundup 1 on heart function in bullfrog tadpoles, the pumping capacity of the muscle at each stimulation fre- quency was calculated. The cardiac pumping capacity (CPC—mN 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.

Results

The activities of antioxidant enzymes and LPO levels in the liver and white muscle are shown in Table 1. The exposure to Roundup 1 (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, Roundup 1 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.

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Table 1 Antioxidant enzymes activities and lipid peroxidation levels in the liver and muscle of bullfrog tadpoles from control (C-group, n = 10) and Roundup 1 -exposed (R-group, n = 10) bullfrog tadpoles

 

C-group

R-group

P values

Liver

SOD (U/mg protein)

5.25 ± 0.53

9.52 ± 1.26

0.011

CAT (U.B./mg protein)

2.06 ± 0.19

5.97 ± 0.33

0.001

LPO (nmol/g tissue)

39.84 ± 4.54

61.69 ± 7.73

0.031

Muscle

SOD (U/mg protein)

23.60 ± 1.53

17.23 ± 2.50

0.040

CAT (U.B./mg protein)

1.35 ± 0.34

0.37 ± 0.05

0.030

LPO (nmol/g tissue)

21.90 ± 0.90

25.41 ± 1.01

0.019

The values are presented as means ± S.E

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

0.07 0.06 0.05 0.04 0.03 0.02 0.01 0 C-group R-group RVM (% W b )
0.07
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0.03
0.02
0.01
0
C-group
R-group
RVM (% W b )

Fig. 1 Relative ventricular mass (RVM), given as a percentage of body mass (Wb) of control (C-group, n = 10) and Roundup 1 - exposed (R-group, n = 10) bullfrog tadpoles. Mean values + S.E. No significant differences between groups were found (P [ 0.05)

constant ( % 1.0 mN mm -2 ) from 0.2 to 1.2 Hz, its highest sustained frequency. Despite the lower maximal sustained frequency presented by the R-group, the Fc developed above 0.8 Hz was % two times higher than that presented by the C-group (P = 0.002). According to Shiels and Farrell (1997) the peak of the pumping capacity versus frequency curve corresponds to an optimum frequency for pumping capacity, known as ‘power output’. The optimum frequency for pumping capacity in the C-group was % 0.6 Hz as the CPC was maintained constant ( % 0.6 mN mm -2 min -1 ) above this frequency. In contrast, the R-group reached the power output at 1.0 Hz (1.1 mN mm -2 min -1 ) considering that CPC values did not change significantly beyond this

1.4 1.2 * * 1.0 0.8 0.6 0.4 0.2 C R 0.0 Fc (mN.mm -2
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CPC (mN.mm -2 .min -1 )

Frequency (Hz)

Fig. 2 Twitch force (Fc—mN mm -2 , upper panel) and cardiac pumping capacity (CPC—mN mm -2 min -1 , lower panel) developed by ventricle strips during increases in stimulation frequency from control (C-group, n = 10) and Roundup 1 -exposed (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 Roundup 1 -exposed (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 indicate differences between experimental groups at the same frequency (P \ 0.05)

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(ms) TPT (ms)THR

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 relaxation—THR) 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

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100

0

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R-group C-group C R
R-group
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* * * * * C R 0,0 0,2 0,4 0,6 0,8 1,0 1,2 1,4
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*
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Frequency (Hz)

Fig. 3 Time to peak tension (TPT—ms, upper panel) and time to half relaxation (THR—ms, lower panel) developed by ventricle strips during increases in stimulation frequency from control (C-group, n = 10) and Roundup 1 -exposed (R-group, n = 10) bullfrog tad- poles. Mean values ± S.E. The arrows indicate the heart rate measured in vivo to control (C, n = 10)) and Roundup 1 -exposed (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 indicate differences between experimental groups at the same frequency (P \ 0.05)

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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- nal 1 . 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. Roundup 1 , should be evaluated (C¸ avas and Ko¨nen 2007). Many studies have addressed the impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities (Lambert 1997; Bridges 1997; Leonard et al. 1999; Smith 2001; Favari et al. 2002; Boone and James 2003). Particularly, the effect of Roundup 1 on aquatic amphibian communities has been a subject of

intense debate (e.g., Thompson et al. 2006 vs. Relyea

2006).

According to the label of Monsanto’s Roundup Origi- nal 1 (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- tidal areas below the mean high water mark’’. However, even though it is intended for terrestrial use, there is huge evidence that Roundup 1 gets into aquatic habitats, typi- cally through unintentional and/or unavoidable aerial

overspray (Relyea 2006) and/or carried by runoff (Relyea

2005b). Additionally, Roundup 1 is sprayed directly on water to control emergent aquatic plants in many countries (Giesy et al. 2000). In Brazil, besides the extensive use in

rice and soybean cultures (Rodrigues and Almeida 2005), it is also applied to control aquatic weeds including Eich- hornia crassipes, Pistia stratiotes and Salvinia auriculata (Carvalho et al. 2005). Moreover, the small wetlands occurring within the herbicide’s target sites are usually directly contaminated during aerial application, resulting in high potential exposure and effects for its constituent biota (Thompson et al. 2004). These small wetlands host con- siderable amphibian biodiversity and endemism as many species only breed and spend their larval stages in these overlooked habitats (Relyea 2006). Corroborating the statement from Relyea (2006) that Roundup 1 has a negative impact on amphibians in aquatic

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environments, the present results demonstrated that the exposure of bullfrog tadpoles to 1 ppm of Roundup 1 for 48 h has a high potential to induce oxidative stress and affects cardiac function. Ge´hin et al. (2006) and Hultberg (2007) reported that glyphosate, alone or as Roundup 1 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 Roundup 1 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 Ultra 1 360 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 Roundup 1 -induced ROS are not totally scavenged by this antioxidant system.

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 Roundup 1 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 Roundup 1 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 (Gu¨l 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).

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However, the absence of variation on relative ventricu- lar mass (RVM) in response to the acute exposure to Roundup 1 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; Gonza´lez et al. 2004; Kimmel 2004). In the present study, the f H values for C- and R-groups were % 43 bpm and % 54 bpm, respectively. Based on the findings described above, the higher f H 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 f H values per se, Jia and Burggren (1997) found mean values between 67 and 73 bpm in anesthetized tadpoles of L. catesbeiana at 22–24 C, with no significant differences between the studied stages. Feder (1983) recorded f H 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 Roundup 1 -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 Roundup 1 . 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

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surfactant POEA (present in Roundup Original 1 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 Roundup 1 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 Roundup 1 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 Roundup 1 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 Roundup 1 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 Roundup 1 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´, Sa˜o 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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