Toxicology Letters 230 (2014) 408–412
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Effect of atrazine and fenitrothion at no-observed-effect-levels (NOEL) on amphibian and mammalian corticosterone-binding-globulin (CBG) Sandra E. Hernández *, Conrad Sernia, Adrian J. Bradley School of Biomedical Sciences, The University of Queensland, Brisbane, 4072 Queensland, Australia
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
The study determines NOEL atrazine/ fenitrothion effect on amphibian/ mammal CBG. Atrazine/fenitrothion compete with B for binding CBG in cane toad and rat plasma. Atrazine NOEL would interfere with normal B-CBG interaction in amphibian. Displacement of B by the agrochemicals would affect total:free B in plasma. Increase of free B will lead to an indirect disruption to stress response.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2014 Received in revised form 13 August 2014 Accepted 14 August 2014 Available online 17 August 2014
This study determines the effect of atrazine and fenitrothion no-observed-effect-levels (NOEL) on the binding of corticosterone (B) to corticosterone-binding-globulin (CBG) in an amphibian and a mammal. Plasma from five cane toads and five Wistar rats was exposed to atrazine and fenitrothion at the NOEL approved for Australian fresh water residues and by the World Health Organization (WHO). The concentration required to displace 50% (IC50) of B binding to CBG was determined by a competitive microdialysis protein assay. Competition studies showed that both atrazine and fenitrothion at NOEL are able to compete with B for CBG binding sites in toad and rat plasma. The IC50 levels for atrazine in toads and rats were 0.004 nmol/l and 0.09 nmol/l respectively. In the case of fenitrothion the IC50 level found in toads was 0.007 nmol/l, and 0.025 nmol/l in rats. Plasma dilution curves showed parallelism with the curve of B, demonstrating that these agro-chemicals are competitively inhibiting binding to CBG. The displacement of B by atrazine and fenitrothion would affect the total:free ratio of B and consequently disrupt the normal stress response. This is the first time that the potential disruptive effect of atrazine and fenitrothion on B-CBG interaction at the NOELs has been demonstrated in amphibian and mammalian models. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: NOEL Atrazine Fenitrothion CBG Amphibian Rat
1. Introduction
* Corresponding author. Tel.: +61 7 33655178; fax: +61 7 33651299. E-mail addresses: [email protected], [email protected] (S.E. Hernández). http://dx.doi.org/10.1016/j.toxlet.2014.08.015 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Organisms can be affected by environmental contaminants in inconspicuous ways that can subsequently impact on populations and ecosystems. Developmental and metabolic systems are known
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to be adversely affected by both organic and inorganic contaminants. While toxicological studies of agrochemicals have been directed mostly toward their disruptive effect upon reproductive function, other physiological responses like the stress response have been overlooked. The stress response has an important role in maintaining homeostasis during times of emergency (Wingfield and Kitaysky, 2002). The organism may react to a wide variety of challenges or stressors by activating a complex of physiological regulatory networks to enable adaptation and permit survival. Among all these regulatory networks, the activation of the hypothalamic-pituitary-adrenal (HPA) axis has attracted most attention. The mediators operating on the HPA axis have been linked to important biological functions and, in particular, the glucocorticoids (GCs) cortisol (F), and corticosterone (B), are known to regulate metabolic, immunologic, and developmental functions in vertebrates (Sapolsky et al., 2000). Consequently any disruption of the HPA axis would be expected to adversely affect the biological responses to GCs (McEwen and Wingfield, 2003). In fact, there is already evidence that environmental pollutants are able to increase GCs levels in fish (Miller et al., 2009) and amphibia (Hopkins et al., 1997, 1999; Ward et al., 2007). There are several critical points in the HPA axis where environmental contaminants can act as disruptors. Studies in fish (Gravel et al., 2005; Hontela et al., 1992; Leblond et al., 2001), rats (Yamamoto et al., 1982) and amphibia (Goulet and Hontela, 2003) have demonstrated that heavy metals and pesticides are able to disrupt the production of GCs in steroidogenic cells. Pollutants have also been reported to act as stressors via a direct effect on the HPA axis. For example, degradation products of the pesticide fenitrothion increase concentrations of adreocorticotrophic hormone (ACTH) in rats (Li et al., 2007). Another critical point where pollutants may disrupt the HPA axis is by competition with GCs for binding to plasma globulins. Globulins work as carriers and reservoirs of hormones in plasma (Anderson, 1974; Rosner, 1991; Westphal, 1983). They are crucial in maintaining proper levels of free hormone in plasma as well as providing localized tissue delivery (Breuner and Orchinik, 2002). Several studies of sex steroid-binding globulins (SBG) have reported that environmental pollutants can compete for SBG in fish (Gale et al., 2004), green turtles (Ikonomopoulou et al., 2009), and humans (Dchaud et al., 1999). However, to our knowledge no study has reported the same effect for Corticosterone Binding Globulin (CBG). The agrochemicals used in this study were chosen based on previous reports of their disruptive effects on the endocrine system in amphibians and mammals. For example atrazine has been reported to induce hermaphrodism and reduce testosterone in male Xenopus laevis (Hayes et al., 2002) but on the other hand fenitrothion can interact with androgen receptors in vitro while no conclusive effect was found in vivo (Sohoni et al., 2001). The agrochemicals were also chosen on the likelihood of their presence in the environment at concentrations that could be detrimental to these species. Atrazine is a 2-chloro-s-triazine pesticide that inhibits photosynthesis in plants. It is one of the most widely used herbicides in the world, and one of the most common agrochemical contaminants found in ground and surface water bodies (Graymore et al., 2001; Murphy et al., 2006). Given its potential disruptive effect on reproductive function (Hayes et al., 2002; Stoker et al., 2000) and carcinogenic effects (McElroy et al., 2007), its use has been banned by the European Union (Sass and Colangelo, 2006). However its use is still allowed in the United States of America (U.S. Environmental Protection Agency, 2012) as well as in Australia (Agriculture and Resource Management Council of Australia and New Zealand, 2000; Australian Pesticides and Veterinary Medicines Authority, 2010). Fenitrothion is a dialkyl-aryl phosphorothioate insecticide that has a cholinergic action and is widely used for plant and forest
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protection (Miyamoto, 1969; Osicka-Koprowska et al., 1987). Doses higher than 10 ppm have been associated with deformities in the tadpole neural axis (Elliott-Feeley and Armstrong, 1981; Pawar and Katdare, 1983; Pawar and Katdare, 1984), and doses higher than 500 mg/kg (LD50) have been reported to be lethal in rats, mice and guinea pigs (Farmoz Pty Ltd, 2008). Its use in Australia is approved in horticultural and forage crops (Australian Pesticides and Veterinary Medicines Authority, 2012). The objective of this study was to determine the ability of atrazine and fenitrothion to bind to CBG in plasma of cane toads and rats, and to assess the significance of this binding. When the displacement of B from CBG by agrochemicals occurs in vitro, it is reasonable to hypothesise that such disruption might also occur in vivo. The displacement of B from CBG would affect the total:free ratio of B in plasma and consequently disrupt indirectly the normal stress response in both the cane toad and rat. Finally elevation of free B in plasma will also affect other physiological responses, including reproduction and immune function (Sapolsky et al., 2000). 2. Material and methods A total of five adult male cane toads (Rhinella marina) captured from The University of Queensland Lakes, St Lucia, SE Queensland and five adult male Wistar rats (Rattus norvegicus) sourced from The University of Queensland Biological Resources (UQBR) were selected to perform the tests. All animals were sampled in the laboratory on the day of capture or collection from the animal house. The animals selected for blood sampling were weighed and anesthetised before blood was drawn. Blood (600 ml) was collected via cardiac puncture with heparin treated syringes and placed into Eppendorf tubes and centrifuged at 1300 g for 5 min to separate plasma, which was stored at 20 C for later analysis. All procedures were approved by The University of Queensland Animal Ethics Committee (SBMS/ 437/09/URG/GOVTMEX/HSF/CFOC). 2.1. Microdialysis method and data analysis The range of concentrations for fenitrothion and atrazine used in this study was based upon the no-observed-effect level (NOEL) approved for Australian fresh water residues (Agriculture and Resource Management Council of Australia and New Zealand, 2000). Microdialysis was used to determine the competition of atrazine and fenitrothion for CBG in cane toad and rat plasma. The technique has been described previously by Ikonomopoulou et al. (2009) in the green turtle, and is carried out by exposing the plasma to radioactive and non-radioactive ligand under equilibrium conditions and determining the percentage bound by plasma globulin. After running trial dilutions to find the optimum dilution range, cane toad plasma (n = 5) and rat plasma (n = 5) were diluted 1:30 and 1:10 respectively in a solution of 0.2% dextran coated charcoal in PBS (0.05 M, pH 7.4). The dilute plasma was incubated for 30 min at 24 C followed by centrifugation at 1500 g for 5 min to strip endogenous hormones from the diluted plasma. Non-radioactive agrochemicals (5–0.5 ng/50 ml) and 30,000 dpm of tritiated corticosterone (Corticosterone [1,2,6,7-3H] 2.59 TBq/mmol, PerkinElmer, Australia) were added to the diluted plasma (100 ml) and allowed to equilibrate in a microdialysis chamber. Samples dialysed only with non-radioactive corticosterone (B) were used as controls. All agrochemicals (analytical grade) were donated by the Queensland Health Scientific Sciences (QHSS). The stock concentrations for both agrochemicals were 1 mg/l. Equilibrium was established after incubation of dialysis cells for 24 h at 4 C, and radioactivity was measured by removing 50 ml from the saline and plasma chambers of each microdialysis set of chambers. Radioactivity was counted in a liquid scintillation spectrometer (Beckman LS 6000 TA1).
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A competitive protein binding technique (Bradley, 1987), was used to determine the concentration of agrochemical required to displace 50% of the radioligand bound to CBG in diluted plasma (IC50). For each curve the null hypothesis tested was that competition could be modeled as a one-site model. The IC50 binding parameters were then calculated by a one-site competition curve in Graph Pad 4 Prism program (GraphPad Software, Inc., 2009). A second assay was performed to determine whether the IC50 from the two agrochemicals would decrease binding of 3H-B to CBG in plasma, and compete for 3H-B binding CBG sites in parallel with the displacement curve for unlabelled B. For this experiment plasma from the five animals was pooled, and serial dilutions from 1:3 to 1:100 were performed. The IC50 was calculated from data obtained by equilibrium dialysis. Parallelism between the binding of agrochemicals and B was determined by plotting the binding percentage (log10 transformed) against the plasma dilution. All curves were generated and analysed in Graph Pad 4 Prism program (GraphPad Software, Inc., 2009). Parallelism between curves was determined by analysis of covariance (ANCOVA) (Zar, 1996).
CI = 0.10–0.65 pmol) for B; for atrazine it was 0.004 nmol (95% CI = 0.001–0.013 nmol); and for fenitrothion 0.007 nmol (95% CI = 0.002–0.03 nmol) (Fig. 1A). For rat CBG the displacement concentration of 3H-B was 1.07 pmol (95% CI = 0.37–3.02 pmol) for B; for atrazine it was 0.09 nmol (95% CI = 0.03–0.30 nmol); and for fenitrothion 0.025 nmol (95% CI = 0.01–0.06 nmol) (Fig. 1B). Significant differences were found between IC50 within atrazine and fenitrothion in toads (F(2,96) 4.26, p = 0.017) and rats (F(2,81)10.76, p < 0.0001). Fig. 2 shows the binding displacement curves for 3H-B from CBG in serial dilutions of toad (A) and rat (B) plasma. Linearization of the displacement percentage of 3H-B contained in serial dilutions of toad plasma by IC50-B (control) (F(1,3)397.2, p < 0.05) shows no significant differences (ANCOVA = F(2,9)3.92, p > 0.05) with the displacement slopes from IC50-atrazine (F(1,3)152.91, p < 0.05) and IC50-fenitrothion (F(1,3)238.9, p < 0.05) (Fig. 2A). In the case
3. Results Fig. 1 shows the curves obtained by competitive binding of CBG in toad (A) and rat (B) plasma stripped of endogenous steroid. The concentration required for 50% displacement (IC50) of 3HCorticosterone (3H-B), in cane toad CBG was 0.26 pmol (95%
Fig. 1. Displacement of corticosterone [1,2,6,7-3H] to CBG in the plasma of male cane toads (A) and male rats (B) by corticosterone (--), atrazine (.D.), and fenitrothion (–*–). Data are presented as mean SD (n = 5).
Fig. 2. Displacement of Corticosterone [1,2,6,7-3H] from CBG by IC50 concentrations for corticosterone [--] (toad = 0.26 pmol/50 ml, rat = 1.07 pmol/50 ml), atrazine [.D.] (toad = 0.004nmol/50 ml; rat = 0.09nmol/50 ml), and fenitrothion [–*–] (toad = 0.007 nmol/50 ml; rat = 0.025 nmol/50 ml) for serial dilutions of cane toad (A) and rat (B) pooled plasma (n = 5). Differences between slopes were analyzed by ANCOVA.
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of rat plasma, the slope obtained from the displacement percentage by IC50-B (control) (F(1,3)160.4, p < 0.05) was found to be significantly different (ANCOVA = F(2,9)6.35, p < 0.05) from the slopes obtained from IC50-atrazine (F(1,3)53.7, p < 0.05) and IC50fenitrothion (F(1,3)21.8, p < 0.05) (Fig. 2B). 4. Discussion This study demonstrated that atrazine and fenitrothion are able to compete with corticosterone by binding to CBG in cane toad and rat plasma. However the concentrations at which the agrochemicals bind to CBG were different between species. Agrochemicals were able to displace 3H-B from CBG at lower levels in toad plasma than in rat plasma. The differences between species can be attributed to the binding properties of CBG. Comparative studies between amphibians and mammals have reported that B binding affinities at 4 C in amphibia are higher (Ka < 109 M 1) (Martin and Ozon, 1975) than in mammals (107 M 1–108 M 1) (Breuner and Orchinik, 2002; Westphal, 1983). The binding capacities of B to amphibian CBG vary between 109 M 1 and 107 M 1 and are lower than the mammalian (106 M 1–107 M 1) (Breuner and Orchinik, 2002). The determination of parallelism besides the competitive properties of atrazine and fenitrothion for CBG in plasma samples, illustrates differences in the interaction between different ligands and a specific receptor. These studies are fundamental to determining the sensitivity of a system in a model animal, and establish a proper dose response to test chemical disruptors and determine controls (vom Saal et al., 2010). The parallel displacement of the curves obtained by IC50-atrazine and IC50-fenitrothion in toad plasma shows that these chemicals are inhibiting 3H-B binding with an effect that is similar to that of corticosterone. In contrast, rat plasma did not show parallelism between the B binding curve when tested with either agrochemical. However the competitive binding assay showed that these two compounds are able to bind to CBG in rat plasma, and therefore have ligand characteristics similar to corticosterone. The displacement of 3H-B from CBG by atrazine and fenitrothion demonstrate its potential disruptive effect on B-CBG interactions, and therefore in the total:free GC ratio in blood. The exposure to these agrochemicals will result in an increase in the free GC fraction that, in consequence, will indirectly affect other physiological responses, like reproduction and immune function (Sapolsky et al., 2000). Moreover, previous studies have reported elevation in B levels by atrazine and fenitrothion in the rat supporting our assumption. For example, doses of atrazine higher than 50 mg/kg of body weight increase total B in plasma (Fraites et al., 2009; Laws et al., 2009). Rats given fenitrothion orally at 14.5 mg/kg for 28 days, develop adrenal hypertrophy and increase B levels in plasma (Yamamoto et al., 1982). Another study in rats found similar effects in rats exposed to 13 mg/kg of fenitrothion for 14 days (Osicka-Koprowska et al., 1987). This study also reported that these rats showed a reduction in 4-14C-corticosterone in pituitary, adrenals, hypothalamus and blood, demonstrating that fenitrothion displaces the radioactive marker from the tissue (Osicka-Koprowska et al., 1987). This study supports our findings, demonstrating that fenitrothion is able to compete for GC receptors in tissues. To our knowledge there are no studies of the effect of either of these two compounds on adrenal activity in amphibia. However, there is a report of changes in total:free GC ratio in Bufo terrestris exposed to polluted areas (Ward et al., 2007). The results found in the present study also have ecological implications. The plasma was exposed to a range of concentrations of agrochemicals based in the NOEL limits approved for Australian fresh water residues that provide 80% of protection in aquatic organisms (Agriculture and Resource Management Council of
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Australia and New Zealand, 2000). The limit approved for atrazine is 0.69 mmol/l (i.e., 0.034 nmol/50 ml) and for fenitrothion it is 0.0014 mmol/l (i.e., 0.07 pmol/50 ml). At these approved limits, our data suggest that fenitrothion should not lead to major physiological disruption. However, this is not the case for atrazine, where the approved water limits exceed the IC50 measured in our experiments (0.004 nmol/50 ml for cane toad). Therefore we predict that significant disruption of B-CBG interaction will occur in the cane toad at the approved atrazine limits in water. Furthermore, based on reports for other amphibian species showing similar CBG properties to those for the cane toad, it seems likely that our results are valid for Rana temporaria,Discoglossus pictus, Salamandra salamandra and Ambystoma tigrinum (Breuner and Orchinik, 2002; Martin and Ozon, 1975), and X. laevis (Jolivet and Leloup, 1986). It should also be noted that toxicokinetic studies with atrazine have demonstrated that it is easily absorbed and bioavailable. For example in rats after 2 h of oral administration of 14C-atrazine, half of the total dose can be detected in the bloodstream (Timchalk et al., 1990). In the amphibian Xenopus, whole body autoradiography has shown that 50% of the initial concentration of 14C atrazine in water was absorbed in the first 8 h of exposure (Edginton and Rouleau, 2005) 5. Conclusion Atrazine and fenitrothion NOEL compete with B for binding to CBG in cane toad and rat plasma. The resulting displacement of B would affect the total:free ratio of B and consequently disrupt the normal stress response and downstream physiological processes, including reproduction. It was also found that at approved limits, atrazine would interfere significantly with normal B-CBG interaction in both cane toads and rats. The in vivo consequences should be determined in future studies. Finally these studies should be extended to include bio-transformed products of agrochemicals. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements We thank Dr. Mary Hodge and Dr. Ron Cheng, from the Department of Organic Chemistry, Queensland Health Scientific Services (QHSS) for their suggestions and guidance in the development of this study. We also thank the Queensland Health Scientific Sciences (QHSS) for the donation of the agrochemicals. SEH thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico for providing the PhD scholarship. References Agriculture Resource Management Council of Australia, New Zealand, 2000. Australian and New Zealand guidelines for fresh and marine water quality. In: Agriculture Resource Management Council of Australia, New Zealand (Ed.), National Water Quality Management Strategy, vol. 1. Agriculture and Resource Management Council of Australia and New Zealand, Camberra, Australia. Anderson, D.C., 1974. Sex-hormone-binding globulin. Clin. Endocrinol. (Oxf.) 3, 69– 96. Australian Pesticides and Veterinary Medicines Authority, 2010. Atrazine Toxicity Analysis of Potential Modes of Action. Australian Pesticides and Veterinary Medicines Authority, Canberra, Australia. Australian Pesticides Veterinary Medicines Authority, 2012. Maximum Residue Limits in Food and Animal Feedstuff December 2012. Australian Pesticides and Veterinary Medicines Authority, Canberra, Australia.
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Effect of atrazine and fenitrothion at no-observed-effect-levels (NOEL) on amphibian and mammalian corticosterone-binding-globulin (CBG) Sandra E. Hernández *, Conrad Sernia, Adrian J. Bradley School of Biomedical Sciences, The University of Queensland, Brisbane, 4072 Queensland, Australia
H I G H L I G H T S
G R A P H I C A L A B S T R A C T
The study determines NOEL atrazine/ fenitrothion effect on amphibian/ mammal CBG. Atrazine/fenitrothion compete with B for binding CBG in cane toad and rat plasma. Atrazine NOEL would interfere with normal B-CBG interaction in amphibian. Displacement of B by the agrochemicals would affect total:free B in plasma. Increase of free B will lead to an indirect disruption to stress response.
A R T I C L E I N F O
A B S T R A C T
Article history: Received 17 July 2014 Received in revised form 13 August 2014 Accepted 14 August 2014 Available online 17 August 2014
This study determines the effect of atrazine and fenitrothion no-observed-effect-levels (NOEL) on the binding of corticosterone (B) to corticosterone-binding-globulin (CBG) in an amphibian and a mammal. Plasma from five cane toads and five Wistar rats was exposed to atrazine and fenitrothion at the NOEL approved for Australian fresh water residues and by the World Health Organization (WHO). The concentration required to displace 50% (IC50) of B binding to CBG was determined by a competitive microdialysis protein assay. Competition studies showed that both atrazine and fenitrothion at NOEL are able to compete with B for CBG binding sites in toad and rat plasma. The IC50 levels for atrazine in toads and rats were 0.004 nmol/l and 0.09 nmol/l respectively. In the case of fenitrothion the IC50 level found in toads was 0.007 nmol/l, and 0.025 nmol/l in rats. Plasma dilution curves showed parallelism with the curve of B, demonstrating that these agro-chemicals are competitively inhibiting binding to CBG. The displacement of B by atrazine and fenitrothion would affect the total:free ratio of B and consequently disrupt the normal stress response. This is the first time that the potential disruptive effect of atrazine and fenitrothion on B-CBG interaction at the NOELs has been demonstrated in amphibian and mammalian models. ã 2014 Elsevier Ireland Ltd. All rights reserved.
Keywords: NOEL Atrazine Fenitrothion CBG Amphibian Rat
1. Introduction
* Corresponding author. Tel.: +61 7 33655178; fax: +61 7 33651299. E-mail addresses: [email protected], [email protected] (S.E. Hernández). http://dx.doi.org/10.1016/j.toxlet.2014.08.015 0378-4274/ ã 2014 Elsevier Ireland Ltd. All rights reserved.
Organisms can be affected by environmental contaminants in inconspicuous ways that can subsequently impact on populations and ecosystems. Developmental and metabolic systems are known
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to be adversely affected by both organic and inorganic contaminants. While toxicological studies of agrochemicals have been directed mostly toward their disruptive effect upon reproductive function, other physiological responses like the stress response have been overlooked. The stress response has an important role in maintaining homeostasis during times of emergency (Wingfield and Kitaysky, 2002). The organism may react to a wide variety of challenges or stressors by activating a complex of physiological regulatory networks to enable adaptation and permit survival. Among all these regulatory networks, the activation of the hypothalamic-pituitary-adrenal (HPA) axis has attracted most attention. The mediators operating on the HPA axis have been linked to important biological functions and, in particular, the glucocorticoids (GCs) cortisol (F), and corticosterone (B), are known to regulate metabolic, immunologic, and developmental functions in vertebrates (Sapolsky et al., 2000). Consequently any disruption of the HPA axis would be expected to adversely affect the biological responses to GCs (McEwen and Wingfield, 2003). In fact, there is already evidence that environmental pollutants are able to increase GCs levels in fish (Miller et al., 2009) and amphibia (Hopkins et al., 1997, 1999; Ward et al., 2007). There are several critical points in the HPA axis where environmental contaminants can act as disruptors. Studies in fish (Gravel et al., 2005; Hontela et al., 1992; Leblond et al., 2001), rats (Yamamoto et al., 1982) and amphibia (Goulet and Hontela, 2003) have demonstrated that heavy metals and pesticides are able to disrupt the production of GCs in steroidogenic cells. Pollutants have also been reported to act as stressors via a direct effect on the HPA axis. For example, degradation products of the pesticide fenitrothion increase concentrations of adreocorticotrophic hormone (ACTH) in rats (Li et al., 2007). Another critical point where pollutants may disrupt the HPA axis is by competition with GCs for binding to plasma globulins. Globulins work as carriers and reservoirs of hormones in plasma (Anderson, 1974; Rosner, 1991; Westphal, 1983). They are crucial in maintaining proper levels of free hormone in plasma as well as providing localized tissue delivery (Breuner and Orchinik, 2002). Several studies of sex steroid-binding globulins (SBG) have reported that environmental pollutants can compete for SBG in fish (Gale et al., 2004), green turtles (Ikonomopoulou et al., 2009), and humans (Dchaud et al., 1999). However, to our knowledge no study has reported the same effect for Corticosterone Binding Globulin (CBG). The agrochemicals used in this study were chosen based on previous reports of their disruptive effects on the endocrine system in amphibians and mammals. For example atrazine has been reported to induce hermaphrodism and reduce testosterone in male Xenopus laevis (Hayes et al., 2002) but on the other hand fenitrothion can interact with androgen receptors in vitro while no conclusive effect was found in vivo (Sohoni et al., 2001). The agrochemicals were also chosen on the likelihood of their presence in the environment at concentrations that could be detrimental to these species. Atrazine is a 2-chloro-s-triazine pesticide that inhibits photosynthesis in plants. It is one of the most widely used herbicides in the world, and one of the most common agrochemical contaminants found in ground and surface water bodies (Graymore et al., 2001; Murphy et al., 2006). Given its potential disruptive effect on reproductive function (Hayes et al., 2002; Stoker et al., 2000) and carcinogenic effects (McElroy et al., 2007), its use has been banned by the European Union (Sass and Colangelo, 2006). However its use is still allowed in the United States of America (U.S. Environmental Protection Agency, 2012) as well as in Australia (Agriculture and Resource Management Council of Australia and New Zealand, 2000; Australian Pesticides and Veterinary Medicines Authority, 2010). Fenitrothion is a dialkyl-aryl phosphorothioate insecticide that has a cholinergic action and is widely used for plant and forest
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protection (Miyamoto, 1969; Osicka-Koprowska et al., 1987). Doses higher than 10 ppm have been associated with deformities in the tadpole neural axis (Elliott-Feeley and Armstrong, 1981; Pawar and Katdare, 1983; Pawar and Katdare, 1984), and doses higher than 500 mg/kg (LD50) have been reported to be lethal in rats, mice and guinea pigs (Farmoz Pty Ltd, 2008). Its use in Australia is approved in horticultural and forage crops (Australian Pesticides and Veterinary Medicines Authority, 2012). The objective of this study was to determine the ability of atrazine and fenitrothion to bind to CBG in plasma of cane toads and rats, and to assess the significance of this binding. When the displacement of B from CBG by agrochemicals occurs in vitro, it is reasonable to hypothesise that such disruption might also occur in vivo. The displacement of B from CBG would affect the total:free ratio of B in plasma and consequently disrupt indirectly the normal stress response in both the cane toad and rat. Finally elevation of free B in plasma will also affect other physiological responses, including reproduction and immune function (Sapolsky et al., 2000). 2. Material and methods A total of five adult male cane toads (Rhinella marina) captured from The University of Queensland Lakes, St Lucia, SE Queensland and five adult male Wistar rats (Rattus norvegicus) sourced from The University of Queensland Biological Resources (UQBR) were selected to perform the tests. All animals were sampled in the laboratory on the day of capture or collection from the animal house. The animals selected for blood sampling were weighed and anesthetised before blood was drawn. Blood (600 ml) was collected via cardiac puncture with heparin treated syringes and placed into Eppendorf tubes and centrifuged at 1300 g for 5 min to separate plasma, which was stored at 20 C for later analysis. All procedures were approved by The University of Queensland Animal Ethics Committee (SBMS/ 437/09/URG/GOVTMEX/HSF/CFOC). 2.1. Microdialysis method and data analysis The range of concentrations for fenitrothion and atrazine used in this study was based upon the no-observed-effect level (NOEL) approved for Australian fresh water residues (Agriculture and Resource Management Council of Australia and New Zealand, 2000). Microdialysis was used to determine the competition of atrazine and fenitrothion for CBG in cane toad and rat plasma. The technique has been described previously by Ikonomopoulou et al. (2009) in the green turtle, and is carried out by exposing the plasma to radioactive and non-radioactive ligand under equilibrium conditions and determining the percentage bound by plasma globulin. After running trial dilutions to find the optimum dilution range, cane toad plasma (n = 5) and rat plasma (n = 5) were diluted 1:30 and 1:10 respectively in a solution of 0.2% dextran coated charcoal in PBS (0.05 M, pH 7.4). The dilute plasma was incubated for 30 min at 24 C followed by centrifugation at 1500 g for 5 min to strip endogenous hormones from the diluted plasma. Non-radioactive agrochemicals (5–0.5 ng/50 ml) and 30,000 dpm of tritiated corticosterone (Corticosterone [1,2,6,7-3H] 2.59 TBq/mmol, PerkinElmer, Australia) were added to the diluted plasma (100 ml) and allowed to equilibrate in a microdialysis chamber. Samples dialysed only with non-radioactive corticosterone (B) were used as controls. All agrochemicals (analytical grade) were donated by the Queensland Health Scientific Sciences (QHSS). The stock concentrations for both agrochemicals were 1 mg/l. Equilibrium was established after incubation of dialysis cells for 24 h at 4 C, and radioactivity was measured by removing 50 ml from the saline and plasma chambers of each microdialysis set of chambers. Radioactivity was counted in a liquid scintillation spectrometer (Beckman LS 6000 TA1).
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A competitive protein binding technique (Bradley, 1987), was used to determine the concentration of agrochemical required to displace 50% of the radioligand bound to CBG in diluted plasma (IC50). For each curve the null hypothesis tested was that competition could be modeled as a one-site model. The IC50 binding parameters were then calculated by a one-site competition curve in Graph Pad 4 Prism program (GraphPad Software, Inc., 2009). A second assay was performed to determine whether the IC50 from the two agrochemicals would decrease binding of 3H-B to CBG in plasma, and compete for 3H-B binding CBG sites in parallel with the displacement curve for unlabelled B. For this experiment plasma from the five animals was pooled, and serial dilutions from 1:3 to 1:100 were performed. The IC50 was calculated from data obtained by equilibrium dialysis. Parallelism between the binding of agrochemicals and B was determined by plotting the binding percentage (log10 transformed) against the plasma dilution. All curves were generated and analysed in Graph Pad 4 Prism program (GraphPad Software, Inc., 2009). Parallelism between curves was determined by analysis of covariance (ANCOVA) (Zar, 1996).
CI = 0.10–0.65 pmol) for B; for atrazine it was 0.004 nmol (95% CI = 0.001–0.013 nmol); and for fenitrothion 0.007 nmol (95% CI = 0.002–0.03 nmol) (Fig. 1A). For rat CBG the displacement concentration of 3H-B was 1.07 pmol (95% CI = 0.37–3.02 pmol) for B; for atrazine it was 0.09 nmol (95% CI = 0.03–0.30 nmol); and for fenitrothion 0.025 nmol (95% CI = 0.01–0.06 nmol) (Fig. 1B). Significant differences were found between IC50 within atrazine and fenitrothion in toads (F(2,96) 4.26, p = 0.017) and rats (F(2,81)10.76, p < 0.0001). Fig. 2 shows the binding displacement curves for 3H-B from CBG in serial dilutions of toad (A) and rat (B) plasma. Linearization of the displacement percentage of 3H-B contained in serial dilutions of toad plasma by IC50-B (control) (F(1,3)397.2, p < 0.05) shows no significant differences (ANCOVA = F(2,9)3.92, p > 0.05) with the displacement slopes from IC50-atrazine (F(1,3)152.91, p < 0.05) and IC50-fenitrothion (F(1,3)238.9, p < 0.05) (Fig. 2A). In the case
3. Results Fig. 1 shows the curves obtained by competitive binding of CBG in toad (A) and rat (B) plasma stripped of endogenous steroid. The concentration required for 50% displacement (IC50) of 3HCorticosterone (3H-B), in cane toad CBG was 0.26 pmol (95%
Fig. 1. Displacement of corticosterone [1,2,6,7-3H] to CBG in the plasma of male cane toads (A) and male rats (B) by corticosterone (--), atrazine (.D.), and fenitrothion (–*–). Data are presented as mean SD (n = 5).
Fig. 2. Displacement of Corticosterone [1,2,6,7-3H] from CBG by IC50 concentrations for corticosterone [--] (toad = 0.26 pmol/50 ml, rat = 1.07 pmol/50 ml), atrazine [.D.] (toad = 0.004nmol/50 ml; rat = 0.09nmol/50 ml), and fenitrothion [–*–] (toad = 0.007 nmol/50 ml; rat = 0.025 nmol/50 ml) for serial dilutions of cane toad (A) and rat (B) pooled plasma (n = 5). Differences between slopes were analyzed by ANCOVA.
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of rat plasma, the slope obtained from the displacement percentage by IC50-B (control) (F(1,3)160.4, p < 0.05) was found to be significantly different (ANCOVA = F(2,9)6.35, p < 0.05) from the slopes obtained from IC50-atrazine (F(1,3)53.7, p < 0.05) and IC50fenitrothion (F(1,3)21.8, p < 0.05) (Fig. 2B). 4. Discussion This study demonstrated that atrazine and fenitrothion are able to compete with corticosterone by binding to CBG in cane toad and rat plasma. However the concentrations at which the agrochemicals bind to CBG were different between species. Agrochemicals were able to displace 3H-B from CBG at lower levels in toad plasma than in rat plasma. The differences between species can be attributed to the binding properties of CBG. Comparative studies between amphibians and mammals have reported that B binding affinities at 4 C in amphibia are higher (Ka < 109 M 1) (Martin and Ozon, 1975) than in mammals (107 M 1–108 M 1) (Breuner and Orchinik, 2002; Westphal, 1983). The binding capacities of B to amphibian CBG vary between 109 M 1 and 107 M 1 and are lower than the mammalian (106 M 1–107 M 1) (Breuner and Orchinik, 2002). The determination of parallelism besides the competitive properties of atrazine and fenitrothion for CBG in plasma samples, illustrates differences in the interaction between different ligands and a specific receptor. These studies are fundamental to determining the sensitivity of a system in a model animal, and establish a proper dose response to test chemical disruptors and determine controls (vom Saal et al., 2010). The parallel displacement of the curves obtained by IC50-atrazine and IC50-fenitrothion in toad plasma shows that these chemicals are inhibiting 3H-B binding with an effect that is similar to that of corticosterone. In contrast, rat plasma did not show parallelism between the B binding curve when tested with either agrochemical. However the competitive binding assay showed that these two compounds are able to bind to CBG in rat plasma, and therefore have ligand characteristics similar to corticosterone. The displacement of 3H-B from CBG by atrazine and fenitrothion demonstrate its potential disruptive effect on B-CBG interactions, and therefore in the total:free GC ratio in blood. The exposure to these agrochemicals will result in an increase in the free GC fraction that, in consequence, will indirectly affect other physiological responses, like reproduction and immune function (Sapolsky et al., 2000). Moreover, previous studies have reported elevation in B levels by atrazine and fenitrothion in the rat supporting our assumption. For example, doses of atrazine higher than 50 mg/kg of body weight increase total B in plasma (Fraites et al., 2009; Laws et al., 2009). Rats given fenitrothion orally at 14.5 mg/kg for 28 days, develop adrenal hypertrophy and increase B levels in plasma (Yamamoto et al., 1982). Another study in rats found similar effects in rats exposed to 13 mg/kg of fenitrothion for 14 days (Osicka-Koprowska et al., 1987). This study also reported that these rats showed a reduction in 4-14C-corticosterone in pituitary, adrenals, hypothalamus and blood, demonstrating that fenitrothion displaces the radioactive marker from the tissue (Osicka-Koprowska et al., 1987). This study supports our findings, demonstrating that fenitrothion is able to compete for GC receptors in tissues. To our knowledge there are no studies of the effect of either of these two compounds on adrenal activity in amphibia. However, there is a report of changes in total:free GC ratio in Bufo terrestris exposed to polluted areas (Ward et al., 2007). The results found in the present study also have ecological implications. The plasma was exposed to a range of concentrations of agrochemicals based in the NOEL limits approved for Australian fresh water residues that provide 80% of protection in aquatic organisms (Agriculture and Resource Management Council of
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Australia and New Zealand, 2000). The limit approved for atrazine is 0.69 mmol/l (i.e., 0.034 nmol/50 ml) and for fenitrothion it is 0.0014 mmol/l (i.e., 0.07 pmol/50 ml). At these approved limits, our data suggest that fenitrothion should not lead to major physiological disruption. However, this is not the case for atrazine, where the approved water limits exceed the IC50 measured in our experiments (0.004 nmol/50 ml for cane toad). Therefore we predict that significant disruption of B-CBG interaction will occur in the cane toad at the approved atrazine limits in water. Furthermore, based on reports for other amphibian species showing similar CBG properties to those for the cane toad, it seems likely that our results are valid for Rana temporaria,Discoglossus pictus, Salamandra salamandra and Ambystoma tigrinum (Breuner and Orchinik, 2002; Martin and Ozon, 1975), and X. laevis (Jolivet and Leloup, 1986). It should also be noted that toxicokinetic studies with atrazine have demonstrated that it is easily absorbed and bioavailable. For example in rats after 2 h of oral administration of 14C-atrazine, half of the total dose can be detected in the bloodstream (Timchalk et al., 1990). In the amphibian Xenopus, whole body autoradiography has shown that 50% of the initial concentration of 14C atrazine in water was absorbed in the first 8 h of exposure (Edginton and Rouleau, 2005) 5. Conclusion Atrazine and fenitrothion NOEL compete with B for binding to CBG in cane toad and rat plasma. The resulting displacement of B would affect the total:free ratio of B and consequently disrupt the normal stress response and downstream physiological processes, including reproduction. It was also found that at approved limits, atrazine would interfere significantly with normal B-CBG interaction in both cane toads and rats. The in vivo consequences should be determined in future studies. Finally these studies should be extended to include bio-transformed products of agrochemicals. Conflict of interest The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements We thank Dr. Mary Hodge and Dr. Ron Cheng, from the Department of Organic Chemistry, Queensland Health Scientific Services (QHSS) for their suggestions and guidance in the development of this study. We also thank the Queensland Health Scientific Sciences (QHSS) for the donation of the agrochemicals. SEH thanks the Consejo Nacional de Ciencia y Tecnología (CONACYT), Mexico for providing the PhD scholarship. References Agriculture Resource Management Council of Australia, New Zealand, 2000. Australian and New Zealand guidelines for fresh and marine water quality. In: Agriculture Resource Management Council of Australia, New Zealand (Ed.), National Water Quality Management Strategy, vol. 1. Agriculture and Resource Management Council of Australia and New Zealand, Camberra, Australia. Anderson, D.C., 1974. Sex-hormone-binding globulin. Clin. Endocrinol. (Oxf.) 3, 69– 96. Australian Pesticides and Veterinary Medicines Authority, 2010. Atrazine Toxicity Analysis of Potential Modes of Action. Australian Pesticides and Veterinary Medicines Authority, Canberra, Australia. Australian Pesticides Veterinary Medicines Authority, 2012. Maximum Residue Limits in Food and Animal Feedstuff December 2012. Australian Pesticides and Veterinary Medicines Authority, Canberra, Australia.
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