Ecotoxicology (2008) 17:153–163 DOI 10.1007/s10646-007-0178-5
Oxidative stress biomarkers and heart function in bullfrog tadpoles exposed to Roundup Original1 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 (fH), and contraction dynamics of ventricle strips of bullfrog (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. Additionally, tadpoles’ hyperactivity was associated with tachycardia in vivo, probably due to a stress-induced adrenergic stimulation. Ventricle strips of Roundup1exposed 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 adrenergicstimulation 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:
[email protected] 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 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 populations 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 surroundings 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 isopropylamine 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 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 ecotoxicologists (C¸avas and Ko¨nen 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, microalgae, 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 AgroSciences) and found effects on behavior, survival, growth, and genotoxicity only at concentrations higher than those considered environmentally relevant. Although conventionally thought to be non-lethal to amphibians, Relyea (2005a) demonstrated that Roundup1 can induce extremely 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). Environmental contaminants such as herbicides, heavy metals and insecticides are known to modulate antioxidant defensive systems and to cause oxidative damage in aquatic organisms 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; Pen˜a-Llopis et al. 2003; Banudevi et al. 2006). Xenobiotic-induced ROS production and the corresponding 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 biomarkers 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 ([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 biochemical 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 exposure 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 catesbeiana. The choice of the North American bullfrog, a nonthreatened 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) tadpoles were obtained from a breeding colony at Ibate´, Sa˜o 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 dechlorinated 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 (35–40% protein), 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 CaCO3 48–56 mg l-1; alkalinity as CaCO3 40–43; DO2 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 Original1exposed (360 g/l glyphosate) at the sublethal concentration of 1 mg/l of the commercial formulation for 48 h (Rgroup; 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 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 Roundup1exposed tadpoles were maintained for 48 h in a static system. During this period, sublethal effects like level of
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activity and swimming performance were monitored. All procedures followed ASTM (2000) guidelines. After exposure, the heart rate (fH—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 -80C until the biochemical determinations were carried out.
Biochemical parameters All biochemical assays were measured spectrophotometrically (Spectronic Genesys 5, Milton Roy Co., NY, USA) at 25C. Samples of frozen liver and muscle were homogenized 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 tetrazolium (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 carbonate buffer (pH 10.2) with 2 mM EDTA. The reduction of NBT by superoxide anion to blue formazan was measured 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 absorbance 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 concentration 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 (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 FeSO4, 25 mM H2SO4, 0.1 mM xylenol orange and 4 mM butylated 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 (Wb—g). 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).
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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 subphysiological frequency of 0.2 Hz. Thereafter, pacing frequency 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 frequency was calculated. The cardiac pumping capacity (CPC—mN mm-2 min-1), the product of stimulation frequency and twitch force was calculated according to Matikainen and Vornanen (1992).
‘‘In vivo’’ heart rate fH measurements in bullfrog tadpoles were taken on individuals 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 caudal–cranial 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).
Statistical analysis
‘‘In vitro’’ experiments
Results
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 containing (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,
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 (Cgroup). 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 compared to the C-group.
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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.
Bullfrog tadpoles exposed to Roundup1
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C-group
R-group
P values
Liver SOD (U/mg protein) CAT (U.B./mg protein) LPO (nmol/g tissue)
5.25 ± 0.53
9.52 ± 1.26
0.011
2.06 ± 0.19 39.84 ± 4.54
5.97 ± 0.33 61.69 ± 7.73
0.001 0.031
23.60 ± 1.53
17.23 ± 2.50
0.040
1.35 ± 0.34
0.37 ± 0.05
0.030
21.90 ± 0.90
25.41 ± 1.01
0.019
Muscle SOD (U/mg protein) CAT (U.B./mg protein) LPO (nmol/g tissue)
The values are presented as means ± S.E
0.07 0.06 0.05 RVM (% Wb)
1.4
0.8 0.6 0.4 0.2
1.6
R-group
Fig. 1 Relative ventricular mass (RVM), given as a percentage of body mass (Wb) of control (C-group, n = 10) and Roundup1exposed (R-group, n = 10) bullfrog tadpoles. Mean values + S.E. No significant differences between groups were found (P [ 0.05)
C-group
R-group
*
*
1.2 1.0 0.8 0.6 0.4
0.0 0.0
C-group
R
1.4
0.03
0
C
0.0
0.2
0.01
*
1.0
0.04
0.02
*
1.2
CPC (mN.mm-2 .min-1)
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 fH (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
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
Fc (mN.mm-2)
Table 1 Antioxidant enzymes activities and lipid peroxidation levels in the liver and muscle of bullfrog tadpoles from control (C-group, n = 10) and Roundup1-exposed (R-group, n = 10) bullfrog tadpoles
C
0.2
0.4
R
0.6 0.8 1.0 Frequency (Hz)
1.2
1.4
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 Roundup1-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 Roundup1-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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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 (Cgroup: 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
TPT (ms)
400 300 R-group
C-group
200 100
C
R
0 500
THR (ms)
400
300
200
*
*
*
*
*
100 C 0 0,0
0,2
0,4
R
0,6 0,8 1,0 Frequency (Hz)
1,2
1,4
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 Roundup1-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 Roundup1-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 Original1. 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 (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 Roundup1 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 Original1 (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 intertidal 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, typically through unintentional and/or unavoidable aerial overspray (Relyea 2006) and/or carried by runoff (Relyea 2005b). Additionally, Roundup1 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 Eichhornia 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 considerable 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 Roundup1 has a negative impact on amphibians in aquatic
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. Ge´hin 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 oxidative stress was indicated by the increased levels of tissue lipid hydroperoxides, generated by oxidative attack on cell membrane phospholipids and circulating lipids. This indicates 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 glyphosate 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 ROSinduced 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 inducibility 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 xenobiotic (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.
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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 activities 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 depending 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 oxidative stress and stimulates lipid peroxidation (Halliwell and Gutteridge 1989; Dorval and Hontela 2003; Bagnyukovaa et al. 2005). Our results clearly indicated that Roundup1 can induce ROS generation, resulting in oxidative 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 tissuespecific 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, variation 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 acceleration 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 ventricular mass (RVM) in response to the acute exposure to Roundup1 is not surprising (Fig. 1) as a cardiac hypertrophy could occur only after more prolonged periods to this herbicide. It is well established that the sympathetic nervous system can exert significant cardio-respiratory and metabolic functions during stressful situations. Furthermore, catecholamines 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 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 Rgroup 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 22–24C, 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 values compared to the ones obtained in the present study could reflect methodological and/or inter-specific differences. 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 avoidance 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 forcefrequency 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 (Schwerte et al. 2002; Hedrik et al. 2005). Koyama et al. (1997) observed that the anionic surfactant LES (sodium polyoxyethylene laurylether sulfate) produced vasorelaxation in aortic ring segments of rats via nitric oxide synthase activation as LES caused a significant increase in NO production in cultured endothelial cells. Considering a possible nitrergic modulation on bullfrog tadpole cardiovascular system and the similarity between the non-ionic
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surfactant POEA (present in Roundup Original1 formulation) and LES, a POEA-mediated increase in NO production could be suggested. Additionally, Layland et al. (2002) observed that NO directly modulates cardiac contractility 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 contraction 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 maximal 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 stressinduced 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´, 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.
References Abdullah MP, Daud J, Hong KS, Yew CH (1995) Improved method for the determination of glyphosate in water. J Chromatogr A 697:363–369
Bullfrog tadpoles exposed to Roundup1 Aebi H (1974) Catalase. In: Bergmayer HU (ed) Methods of enzymatic analysis. Academic Press, New York, pp 671–684 Ahmad I, Pacheco M, Santos MA (2006) Anguilla anguilla L. oxidative stress biomarkers: an in situ study of freshwater wetland ecosystem (Pateira de Fermentelos, Portugal). Chemosphere 65:952–962 Ahmad I, Ahmada I, Hamidb T, Fatimaa M, Chandc HS, Jainb SK, Athara M, Raisuddina S (2000) Induction of hepatic antioxidants in freshwater catfish (Channa punctatus Bloch) is a biomarker of paper mill effluent exposure. Biochim Biophys Acta 1519:37–48 American Veterinary Medical Association (2001) Report of the AVMA panel on euthanasia. J Am Vet Med Assoc 218:669–696 Andersen JB, Jensen FB, Wang T (2001) Effects of temperature and oxygen availability on circulating catecholamines in the toad Bufo marinus. Comp Biochem Physiol A 129:473–486 ASTM—American Society for Testing and Materials (2000) Standard guide for conducting acute toxicity tests on test materials with fishes, macroinvertebrates, and amphibians E 729–96. In: Annual Book of Standards, Vol 11.05, Section 11. Philadelphia, PA, pp 220–240 ¨ , Tu¨kel S, Canli M (2006) Response of catalase Atli G, Alptekin O activity to Ag+, Cd2+, Cr6+, Cu2+ and Zn2+ in five tissues of freshwater fish Oreochromis niloticus. Comp Biochem Physiol C 143:218–224 Avci A, Kac¸maz M, Durak I_ (2005) Peroxidation in muscle and liver tissues from fish in a contaminated river due to a petroleum refinery industry. Ecotoxicol Environ Saf 6:101–105 Bagnyukovaa TV, Vasylkiva OY, Storeyb KB, Lushchaka VI (2005) Catalase inhibition by amino triazole induces oxidative stress in goldfish brain. Brain Res 1052:180–186 Banudevi S, Krishnamoorthy G, Venkatataman P, Vignesh C, Aruldhas MM, Arunakaran J (2006) Role of a-tocopherol on antioxidant status in liver, lung and kidney of PCB exposed male albino rats. Food Chem Toxicol 44:2040–2046 Bers DM (2001) Excitation-contraction coupling and cardiac contractile force, 2nd edn. Dordrecht, Kluwer Academic Publishers, p 258 Boone MD, James SM (2003) Interactions of an insecticide, herbicide, and natural stressors in amphibian community mesocosms. Ecol Appl 13:829–841 Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254 Brausch JM, Smith PN (2007) Toxicity of three polyethoxylated tallowamine surfactant formulations to laboratory and field collected fairy shrimp, Thamnocephalus platyurus. Arch Environ Contam Toxicol 52(2):217–221 Bridges CM. (2000) Long-term effects of pesticide exposure at various life stages of the southern leopard frog (Rana sphenocephala). Arch Environ Contam Toxicol 39:91–96 Bridges CM (1997) Tadpole swimming performance and activity affected by acute exposure to sublethal levels of carbaryl. Env Toxicol Chem 16(9):1935–1939 Broomhall SD (2005) Measuring chemical impacts on amphibians: ecotoxicity and behavioural data in governmental regulation. Appl Herpetol 2:259–285 Burggren WW, Feder ME, Pinder A (1983) Temperature and the balance between aerial and aquatic respiration in larvae of Rana berlandieri and Rana catesbeiana. Physiol Zool 56:263–273 Calore EE, Perez NM, Herman MM (2007) Morphometric studies of cardiac myocytes of rats chronically treated with an organophosphate. Ecotoxicol Environ Saf 66(3):447–450 Carvalho FT, Velini ED, Negrisoli E, Rossi CVS (2005) Efica´cia do carfentrazone-ethyl no controle de plantas aqua´ticas latifoliadas em caixas-d’a´gua. Planta Daninha 23(2):305–310
161 C¸avas T, Konen S (2007) Detection of cytogenetic and DNA damage in peripheral erythrocytes of goldfish (Carassius auratus) exposed to a glyphosate formulation using the micronucleus test and the comet assay. Mutagenesis 22(4):263–268 Christin MS, Gendron AD, Brousseau P, Menard L, Marcogliese DJ, Cyr D, Ruby S, Fournier M (2003) Effects of agricultural pesticides on the immune system of Rana pipiens and on its resistance to parasitic infection. Environ Toxicol Chem 22:1127–1133 Clements C, Ralph S, Petras M (1997) Genotoxicity of select herbicides in Rana catesbeiana tadpoles using the alkaline single-cell gel DNA electrophoresis (Comet) assay. Environ Mol Mutagen 29(3):277–288 Collins JP, Storfer A (2003) Global amphibian declines: sorting the hypotheses. Divers Distrib 9:89–98 Comporti M (1985) Lipid peroxidation and cellular damage in toxic liver injury. Lab Investig 523:599–623 Cox C, Surgan M (2006) Unidentified inert ingredients in pesticides: implications for human and environmental health. Environ Health Persp 114(12):1803–1806 Crouch RK, Gandy SC, Kinsey G (1981) The inhibition of islet superoxide dismutase by diabetogenic drugs. Diabetes 30:235– 241 Di Giulio RT, Washiburn PC, Wenning RJ, Winston GW, Jewell CS (1989) Biochemical responses in aquatic animals: a review of determinants of oxidative stress. Environ Toxocol Chem 8:1103–1123 Dorval J, Hontela A (2003) Role of glutathione redox cycle and catalase in defense against oxidative stress induced by endosulfan in adrenocortical cells of rainbow trout (Oncorhynchus mykiss). Toxicol Appl Pharmacol 192:191–200 Driedzic WR, Gesser H (1994) Energy metabolism and contractility in ectothermic vertebrate hearts: hypoxia, acidosis, and low temperature. Physiol Rev 74(1): 221–258 Erlij D, Cetrangolo R, Valadez R (1965) Adrenotropic receptors in the frog. J Pharmacol Exp Ther 149:65–70 Farrell AP, Jones DR (1992) The Heart. In: Hoar WS, Randall DJ, Farrell AP (eds) Fish physiology: the cardiovascular system v 12A. Academic Press Inc., New York, pp 1–88 Favari L, Lopez E, Martinez-Tabche L, Diaz-Pardo E (2002) Effect of insecticides on plankton and fish of Ignacio Ramirez reservoir (Mexico): a biochemical and biomagnification study. Ecotoxicol Environ Saf 51:177–186 Feder ME (1983) Responses to acute aquatic hypoxia in larvae of the frog Rana beriandieri. J Exp Biol 104:79–95 Fulle S, Protasi F, Di Tano G, Pietrangelo T, Beltramin A, Boncompagni S, Vecchiet L, Fano G (2004) The contribution of reactive oxygen species to sarcopenia and muscle ageing. Exp Gerontol 39:17–24 Gehin A, Guyon C, Nicod L (2006) Glyphosate-induced antioxidant imbalance in HaCaT: the protective effect of vitamins C and E. Environ Toxicol Pharmacol 22:27–34 Georgieva NV (2005) Oxidative stress as a factor of disrupted ecological oxidative balance in biological systems – a review. Bulg J Vet Med 8(1):1–11 Giesy JP, Dobson S, Solomon KR (2000) Ecotoxicological risk assessment for Roundup herbicide. Rev Contam Toxicol 167:35– 120 Gonza´lez A, Marı´n O, Tuinhof R, Smeets WJAJ (2004) Ontogeny of catecholamine systems in the central nervous system of anuran amphibians: an immunohistochemical study with antibodies against tyrosine hydroxylase and dopamine. J Comp Neurol 346(1):63–79 Gu¨l S, Belge-Kurutas E, Yildiz E, Sahan A, Doran F (2004) Pollution correlated modifications of liver antioxidant systems and
123
162 histopathology of fish (Cyprinidae) living in Seyhan Dan Lake. Turkey Environ Int 30:605–609 Gurushankara HP, Krishnamurthy SV, Vasudev V (2007) Effect of malathion on survival, growth, and food consumption of Indian cricket frog (Limnonectus limnocharis) tadpoles. Arch Environ Contam Toxicol 52:251–256 Halliwell B, Gutteridge JMC (1987) Oxygen toxicity, oxygen radicals, transition metals and disease. J Biochem 219:1–14 Halliwell B, Gutteridge JMC (1989) Free radicals in biology and medicine, 2nd edn. Claredon Press, Oxford, p 469 Hazarika A, Sarkar SN, Hajare S, Kataria M, Malik JK (2003) Influence of malathion pretreatment on the toxicity of anilofos in male rats: a biochemical interaction study. Toxicology 185:1–8 Hedrick MS, Chen AK, Jessop KL (2005) Nitric oxide changes its role as a modulator of respiratory motor activity during development in the bullfrog (Rana catesbeiana). Comp Biochem Physiol A 142:231–240 Herman CA, Sandoval EJ (1983) Catecholamine effects on blood pressure and heart rate in the American bullfrog, Rana catesbeiana. Gen Comp Endocrinol 52:142–148 Hillman SS (1976) Cardiovascular correlates of maximal oxygen consumption rates in anuran amphibians. J Com Physiol 109:199–207 Hillman SS, Withers PC, Hedrick MS, Kimmel PB (1985) The effects of erythrocythemia on blood viscosity, maximal systemic oxygen transport capacity and maximal rates of oxygen consumption in an amphibian. J Com Physiol 155:577–581 Howe CM, Berrill M, Pauli BD, Helbing CC, Werry K, Veldhoen N (2004) Toxicity of glyphosate-based pesticides to four. North American Frog Species Environ Toxicol Chem 23(8):1928–1938 Hultberg M (2007) Cysteine turnover in human cell liones is influenced by glyphosate. Environ Toxicol Pharmacol 23:19–22 Jia XX, Burggren WW (1997) Developmental changes in chemoreceptive control of gill ventilation in larval bullfrogs (Rana catesbeiana). II. Sites of O2-sensitive chemoreceptors. J Exp Biol 200:2237–2248 Jiang ZY, Woollard ACS, Wolff SP (1991) Lipid hydroperoxide measurement by oxidation of Fe2+ in the presence of xylenol orange: comparison with the TBA assay and iodometric method. Lipids 26:777–860 Jiang ZY, Hunt JV, Wolff SP (1992) Ferrous ion oxidation in the presence of xylenol orange for detection of lipid hydroperoxide in low-density lipoprotein. Anal Biochem 202(2):384–389 Jimenez BD, Stegeman JJ (1990) Detoxication enzymes as indicators of environmental stress on fish. In: Adams SM (ed) Biological indicators of stress in fish, vol 8. American Fisheries Symposium. Bethesda, Maryland, pp 67–79 Johansson M, Piha H, Kylin H, Merila J (2006) Toxicity of six pesticides to common frog (Rana temporaria) tadpoles. Environ Toxicol Chem 25(12):3164–3170 Kavitha P, Rao V (2007) Oxidative stress and locomotor behaviour response as biomarkers for assessing recovery status of mosquito Wsh, Gambusia aYnis after lethal eVect of an organophosphate pesticide, monocrotophos. Pest Biochem Physiol 87:182–188 Kehrer JP (1993) Free radicals as mediators of tissue injury and disease. Crit Rev Toxicol 34:21–48 Kimmel KB (2004) Adrenergic receptors and the regulation of vascular resistance in bullfrog tadpoles (Rana catesbeiana). J Comp Physiol B 162(5):455–462 Kirby S, Burnstock G (1969) Pharmacological studies of the cardiovascular system in the anaesthetized sleepy lizard (Tiliqua rugosa) and toad (Bufo marinus). Comp Biochem Physiol 28:321–331 Kloberg AJ, Fritsche R (2002) Catecholamines are present in larval Xenopus laevis: a potential source for cardiac control. J Exp Zool 292(3):293–303
123
M. J. Costa et al. Koyama Ka., Koyama Ky, Goto K (1997) Cardiovascular effects of a herbicide containing glufosinate and a surfactant: in vitro and in vivo analyses in rats. Toxicol Appl Pharmacol 145:409–414 Kruger NJ (1994) The Bradford method for protein quantification. Meth Mol Biol 32:9–15 Lambert MRK (1997) Effect of pesticides on amphibians and reptiles in sub-Saharan Africa. Rev Environ Contam Toxicol 150:31–73 Layland J, Li JM, Shah AM (2002) Role of cyclic GMP-dependent protein kinase in the contractile response to exogenous nitric oxide in rat cardiac myocytes. J Physiol 540:457–467 Leonard AWR, Hyne V, Lim RP, Chapman JC (1999) Effect of endosulfan runoff from cotton fields on macroinvertebrates in the Namoi River. Ecotoxicol Environ Saf 42:125–134 Lewatowski B, Pytkowski B (1987) Cellular mechanisms of the relationship between myocardial force and frequency of contractions. Prog Biophys Mol Biol 50:97–120 Lillo RS (1979) Autonomic cardiovascular control during submergence and emergence in bullfrogs. Am J Physiol 237:R210– R216 Liu Y, Zhang Y, Liu J, Huang D (2006) The role of reactive oxygen species in the herbicide acetochlor-induced DNA damage on Bufo raddei tadpole liver. Aquatic Toxicol 78:21–26 Matikainen N, Vornanen M (1992) Effect of season and temperature acclimation on the function of crucian carp (Carassius carassius) heart. J Exp Biol 167:203–220 Monsanto Company (1991) Roundup herbicide, material safety data sheet. Monsanto Canada Streetsville, ON, Canada Monteiro DA, Almeida JA, Rantin FT, Kalinin AL (2006) Oxidative stress biomarkers in the freshwater characid fish, Brycon cephalus, exposed to organophosphorus insecticide Folisuper 600 (methyl parathion). Comp Biochem Physiol 143:141–149 Oruc¸ EO, Usta D (2007) Evaluation of oxidative stress responses and neurotoxicity potential of diazinon in different tissues of Cyprinus carpio. Environ Toxicol Pharmacol 23:48–55 Otero J, Toni P, Garcia-Morato YV (1983) Superoxo dismutase: me´todo para su determinacio´n. Rev Iberoam Invest Clin 2:121– 127 Pen˜a-Llopis S, Ferrando MD, Pen˜a JB (2003) Fish tolerance to organophosphate-induced oxidative stress is dependent on the glutathione metabolism and enhanced by N-acetylcysteine. Aquat Toxicol 65:337–360 Perkins PJ, Herman J, Boermans HJ, Stephenson GR (2000) Toxicity of glyphosate and triclopyr using the frog embryo teratogenesis assay-Xenopus. Environ Toxicol Chem 19(4):940–945 Pieniazek D, Bukowska B, Duda W (2004) Comparison of the effect of Roundup Ultra 360 SL pesticide and its active compound glyphosate on human erythrocytes. Pest Biochem Physiol 79:58– 63 Relyea RA (2003) Predator cues and pesticides: A double dose of danger for amphibians. Ecol Appl 13(6):1515–1521 Relyea RA (2004) Growth and survival of five amphibian species exposed to combinations of pesticides. Environ Toxicol Chem 23(7):1737–1742 Relyea RA (2005a) The lethal impact of Roundup on aquatic and terrestrial amphibians. Ecol Appl 15:1118–112 Relyea RA (2005b) The impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol Appl 15(2):618–627 Relyea RA (2006) Letters to the editor: the impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities: response. Ecol Appl 16(5):2027–2034 Risso-de-Faverney C, Devaux A, Lafaurie M, Girard JP, Bailly B, Rahmani R (2001) Cadmium induces apoptosis and genotoxicity in rainbow trout hepatocytes through generation of reactive oxygen species. Aquat Toxicol 53:65–76
Bullfrog tadpoles exposed to Roundup1 Rodrigues BN, Almeida FLS (2005) Guia de Herbicidas, 5th edn. Londrina, Grafmark, p 592 Saygili EI, Konukoglu D, Papila S, Aksay T (2003) Levels of plasma vitamin E, vitamin C, TBARS and cholesterol in male patients with colorectal tumors. Biochemistry (Moscow) 68(3):325–328 Schwerte T, Printz E, Regina Fritsche R (2002) Vascular control in larval Xenopus laevis: the role of endothelial-derived factors. J Exp Biol 205:225–232 Shiels H, Farrell AP (1997) The effect of temperature and adrenaline on the relative importance of the sarcoplasmic reticulum in contributing Ca2+ to force development in isolated ventricular trabeculae from rainbow trout. J Exp Biol 1(200):1607–1621 Sk UH, Bhattacharya S (2006) Prevention of cadmium induced lipid peroxidation, depletion of some antioxidative enzymes and glutathione by series of novel organoselenocyanates Environ. Toxicol Pharmacol 22:298–308 Smith GR (2001) Effects of acute exposure to a commercial formulation of glyphosate on the tadpoles of two species of anurans. Bull Environ Contam Toxicol 67:483–488 Sparling DW, Matson C, Bickham J, Doelling-Brown P (2006) Toxicity of glyphosate as Glypro and LI700 to red-eared slider (Trachemys scripta elegans) embryos and early hatchlings. Environ Toxicol Chem 25(10):2768–2774 Sys SU, Pellegrino D, Mazza R, Gattuso A, Andries LJ, Tota B (1997) Endocardial endothelium in the avascular heart of the frog: Morphology and role of nitric oxide. J Exp Biol 200:3109– 3118 Szarek J, Siwicki A, Andrzejewska A, Terech-Majewska E, Banaszkiewicz T (2000) Effects of the herbicide Roundup on the ultrastructural pattern of hepatocytes in carp (Cyprinus carpio). Mar Environ Res 50(1–5):263–266
163 Terech-Majewska E, Siwicki AK, Szweda W (2004) Modulative influence of lysozyme dimer on defense mechanisms in the carp (Cyprinus carpio) and European sheatfish (Silurus glanis) after suppression induced by herbicide. Roundup Pol J Vet Sci 7(2):123–128 Thompson DG, Wojtaszek BF, Staznik B, Chartrand DT, Stephenson GR (2004) Chemical and biomonitoring to assess potential acute effects of Vision herbicide on native amphibian larvae in forest wetlands. Environ Toxicol Chem 23:843–849 Thompson DG, Solomon KR, Wojtaszek BF, Edginton AN, Stephenson GR (2006) Letters to the editor: the impact of insecticides and herbicides on the biodiversity and productivity of aquatic communities. Ecol Appl 16(5):2022–2027 Tsui MTK, Chu LM (2003) Aquatic toxicity of glyphosate-based formulations: comparison between different organisms and the effects of environmental factors. Chemosphere 52(7):1189–1197 Venturino A, Rosenbaum E, De Castro AC, Anguiano OL, Gauna L, De Schroeder TF, D’Angelo AMP (2003) Biomarkers of effect in toads and frogs. Biomarkers 8:167–186 Wassersug RJ, Paul RD, Feder ME (1981) Cardiorespiratory synchrony in anuran larvae (Xenopus laevis, Pachymedusa dacnicolor, Rana berlandieri). Comp Biochem Physiol 70A:329–334 Withers PC, Hillman SS (2001) Allometric and ecological relationships of ventricle and liver mass in anuran amphibians. Functional Ecol 15:60–69 Wilhelm-Filho D, Giulivi C, Boveris A (1993) Antioxidant defenses in marine fish – I. Teleosts Comp Biochem Physiol C 160:409– 413 Winston GW (1991) Oxidants and antioxidants in aquatic animals. Comp Biochem Physiol 100C:173–176
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