Distribution of 14C-Labelled Atrazine, Methoxychlor, Glyphosate, and Bisphenol-A in Goldfi Sh Studied by Whole-Body Autoradiography (WBARG)

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Distribution of 14C-labelled Atrazine, Methoxychlor, Glyphosate, and Bisphenol-A in Goldfi sh Studied by Whole-Body Autoradiography (WBARG)

Claude Rouleau1,2* and Jagmohan Kohli1

1 National Water Research Institute, 867 Lakeshore Road, P.O. Box 5050, Burlington, Ontario, Canada L7R 4A6 2 Present Address: Maurice-Lamontagne Institute, 850 route de la mer, C.P. 1000 Mont-Joli, Québec, Canada G5H 3Z4

Nonpersistent contaminants represent thousands of chemicals used as pesticides, pharmaceuticals, personal care products, additives, etc. Because of this diversity, the assessment of the environmental risks they may pose for the environment represents a formidable task. Identifi cation of target organs is key information needed to orient further research on newly- investigated organic xenobiotics. We used whole-body autoradiography to visualize the distribution of 14C-labelled atrazine, methoxychlor, glyphosate, and bisphenol-A in goldfi sh (Carassius auratus) and identify target organs. Fish were exposed for 2 days (glyphosate and bisphenol-A) and 7 days (atrazine and methoxychlor) to the radiolabelled compounds at a concentration of 15 nM. They were then frozen, embedded in carboxymethylcellulose gel, 20-Pm-thick cryosections were collected, freeze-dried, and exposed to phosphor screens to visualize the tissue distribution of radioactivity. Goldfi sh did not accumulate glyphosate. The three other compounds were accumulated, mostly in the gall bladder. Nevertheless, unforeseen accumulation sites were observed; atrazine accumulated in the uveal tract of the eye, high levels of radioactivity were found in the cerebrospinal fl uid of goldfi sh exposed to methoxychlor, and an important accumulation of bisphenol-A was seen in urine, oral mucosa, esophagus, and intestinal lumen. The potential toxicological consequences of the accumulation of these chemicals at very specifi c locations within the fi sh body are discussed and further research suggested.

Key words: atrazine, bisphenol-A, methoxychlor, tissue distribution, fi sh, whole-body autoradiography

Introduction complicated by the fact that very low levels of certain xenobiotic compounds, such as endocrine disrupters, There is increasing evidence that trace amounts of may perturb the normal biochemical balance of fi sh, many household and industrial chemicals, such as birds, and mammals (Crews et al. 1995), requiring the organophosphates, chlorinated pesticides, phenolic use of costly and time-consuming analytical chemistry. compounds, and synthetic estrogens, can perturb the Whole-body autoradiography (WBARG) (Ullberg endocrine system of aquatic organisms and may lead 1954; Ullberg et al. 1982) allows rapid and precise to reproductive failure (Islam and Tanaka 2004). This visualization of the distribution of a radiolabelled stresses the need to thoroughly and adequately assess the chemical in all the organs and tissues of a whole animal. environmental risks they may pose. In view of the very Though it is commonly used in the pharmaceutical large number of chemicals in use today and the fact that industry to study the distribution of new drugs (Solon hundreds of new ones are marketed every year, the task et al. 2002), its use in environmental studies is not of comprehensively assessing the links between chemical widespread. Nevertheless, WBARG has proven its value releases, environmental concentrations, target organism by enabling researchers to illuminate unsuspected routes exposures, tissue concentrations, and probability of of accumulation of metals, organometals, and organic adverse effects represents a formidable challenge to the chemicals in fi sh (Solbakken et al. 1984; Tjälve et al. scientifi c community (MacLeod et al. 2004). 1986, 1988; Bernhoft et al. 1994; Rouleau et al. 1999, One of the key parameters needed by scientists who 2003; Ruus et al. 2001; Scott et al. 2003). want to assess the environmental risks of a given chemical Here we present the results of a preliminary is the identifi cation of the preferential accumulation experiment aimed at identifying target organs and tissues site(s) in aquatic biota following exposure since it is the that accumulate chemicals (and/or their metabolites) in combination of a chemical’s selective accumulation (Tsai goldfi sh (Carassius auratus) upon exposure via water. and Liao 2006) at a specifi c target site and the mode of Goldfi sh has been extensively used as a biological model action at that site that determine the likelihood of toxic in neurophysiology (Finger 2008), and also to assess the effects (MacLeod et al. 2004). Identifi cation is often toxicity of chemicals in the aquatic environment (Chen et al. 2005; Teather and Parrot 2006; Liu et al. 2007; Yin et al. 2007). Chemicals examined were atrazine, a * Corresponding author: [email protected] triazine broadleaf herbicide (Solomon et al. 1996; Allran

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and Karasov 2000, 2001), methoxychlor, a chlorinated Biochemicals. Radiochemical purity was at least 95% pesticide used as a substitute to DDT (Johnson and Finley and the radiolabelled chemicals were used without 1980; Magliulo et al. 2002; ATSDR 2004; Versonnen et al. further purifi cation. Goldfi sh (10- to 15-g body 2004), glyphosate, perhaps the most important herbicide weight) were bought from Aleongs International Inc. ever developed (EXTOXNET 1996; Baylis 2000), and (Mississauga, Ontario). They were kept in 20-L aquaria bisphenol-A, used in the production of epoxy resins fi lled with city of Burlington (Ontario) tap water that and polycarbonate plastics and a potent estrogen mimic was previously dechlorinated with activated charcoal (Routledge and Sumpter 1996; Hing-Biu and Peart 2000; and continuously aerated. The aquaria were maintained Belfroid et al. 2002; Pait and Nelson 2003; Stoker et al. at 20 ± 1oC and under a 12 h-light:12 h-dark photoperiod 2003; vom Saal and Hughes 2005). Molecular structure, in a temperature-controlled room at the National Water

water solubility, KOW, and some toxicological data are Research Institute in Burlington. Fish were acclimated shown in Fig. 1 and Table 1. to experimental conditions for one week before the beginning of the experiment, with the water changed Material and Methods three times a week. Fish were not fed during this period nor during the experiment. Atrazine-[ring-14C(U)] was purchased from Sigma- Water in exposure aquaria was spiked with the Aldrich, methoxychlor-[ring-14C(U)], and glyphosate- radiolabelled compounds to a concentration of 15 nM. [glycine-2-14C] from American Radiolabeled Chemicals, The resulting 14C levels varied with the specifi c activity and bisphenol-A-[propyl-2-14C] from Moravek (Table 1) of the labelled compounds. A group of four fi sh

Fig. 1. Molecular structure of atrazine, methoxychlor, glyphosate, and bisphenol-A. * shows the location of the 14C-label.

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Fig. 2. Radioactivity levels in exposure water, expressed as percentage relative to the radioactivity measured at time 0, as a function of time.

was introduced into each aquarium 30 min after spiking. of hexane and dry ice. They were then quickly embedded Since the specifi c activity of radiolabelled atrazine in a carboxymethylcellulose gel on a microtome stage, and methoxychlor was lower than for the two other and the assembly was frozen in the same way. Fifteen to compounds, fi sh were exposed to these two chemicals 20 pairs of 20-Pm-thick cryosections were taken at -20oC for 7 days. Fish in aquaria containing glyphosate at different levels in the body of each fi sh with a specially and bisphenol-A were exposed for 2 days. During the designed cryomicrotome (Leica CM3600). The sections exposure period, aquaria were protected from light to were then freeze-dried and applied to fl exible storage prevent hydrolysis (especially for glyphosate), water was phosphor screens (Perkin-Elmer) for one to two weeks. not changed (temperature = 20oC), and radioactivity in After exposure, the phosphor screens were scanned 2-mL water samples was quantifi ed by liquid scintillation with a Cyclone Storage Phosphor Imager (Perkin- spectrometry using a Packard Tri-Carb Liquid Scintillation Elmer). Autoradiograms obtained were visualized, and Counter Model 2300 TR (see Fig. 2 for sampling radioactivity in tissues of interest was semiquantifi ed frequency). At the end of the exposure period, fi sh were using the software Optiquant (Packard Biosciences) (Fig. submitted to lethal anaesthesia (MS-222, 100 mg/L), 3). briefl y rinsed in clean water, and fl ash-frozen in a slurry

Fig. 3. Autoradiogram showing some of the regions of interest selected for quantifi cation. Numbers between brackets in Fig. 4 to 7 are radioactivity concentrations expressed as digital light units (DLU) per mm2 of section surface. Detection limit was set as 3 times the standard deviation of the average background measured in 10 blank areas on the same phosphor screen. Detection of radioactivity with phosphor screens had a dynamic range extending over 5 orders of magnitude, whereas shades of grey that can be visually distinguished range over some 2 orders of magnitude. Thus, black areas seen in some autoradiograms are not saturated but simply results from the output range used for prints, which was chosen to show as much details as possible. See Table 2 for abbreviations.

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necessity to have enough radioactivity in fi sh to be able to visualize distribution by autoradiography. Fish were not fed to keep ammonia excretion low and to avoid ingestion of food particles or faeces to which radiolabelled chemicals may have adsorbed. Autoradiograms presented (Figs. 4 to 7) show the distribution of both the radioactive parent compound and its metabolites. Features described are typical of those seen in all fi sh of a given exposure group. None of the fi sh died during the experiment and they did not exhibit any behavioural signs of stress. Figure 2 shows the level of 14C radioactivity in water, expressed as a percentage of the activity measured at time 0. Radioactivity level in the water containing atrazine and glyphosate showed little variation (<5%), whereas it decreased by 20% in the water containing bisphenol-A. These tendencies likely refl ect the different uptake of the chemicals by fi sh; no radioactivity could be detected in the fi sh exposed to glyphosate, labelling of atrazine- exposed fi sh was very low, except for the gall bladder, and fi sh exposed to bisphenol-A exhibited a much stronger labelling (see below). In the case of methoxychlor, 14C activity in water decreased by almost 70% during the fi rst 24 h of exposure and increased thereafter to a level representing 65% of the activity measure at time 0. It has been shown that methoxychlor is effi ciently transformed to its monodemethylated and bisdemethylated metabolites by fi sh liver microsomes (Schlenk et al. 1997, 1998). The variation in the 14C activity in water to which Results and Discussion methoxychlor was added may have been caused by the fast uptake of methoxychlor followed by the release of Exposure conditions were a compromise between realistic water-soluble metabolites, conjugated or not. environmental conditions (e.g., concentration in water), Glyphosate did not bioaccumulate in goldfi sh, as minimization of fi sh stress (length of exposure), and the the autoradiograms were completely blank (not shown).

B A

Fig. 4. Autoradiograms and corresponding tissue sections of goldfi sh exposed to 14C-atrazine. Black areas contain the high- est concentration of radioactivity. Bar = 1 cm. Autoradiogram and tissue section pairs in A and B are from different fi sh. See Table 2 for abbreviations.

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our experiment and compared with the concentrations observed in well (Smith et al. 1996) and drainage waters (Vereecken 2005) following land application. The three other chemicals were accumulated to various extents. WBARGs and the corresponding tissue sections are presented in Fig. 4 to 7. In fi sh exposed to atrazine for 7 days, the radioactivity was mainly concentrated in the gall bladder (Fig. 4), indicating that atrazine was readily metabolized. A diffuse and weak labelling can be seen in all the other tissues. Labelling of organs such as the liver, intestine, kidney, and eye lens was somewhat higher. This is similar to the distribution of atrazine in the whitefi sh (Coregonus fera [Gunkel and Streit 1980]), carp (Cyprinus carpio [Gluth et al. 1985]), tilapia (Tilapia sparrmanii [du Preez and van Vuren 1992]), and larvae of the amphibian Xenopus laevis (Edginton and Rouleau 2005). An interesting observation is the labelling of the uveal tract of the eye (Fig. 5). This layer of the eye contains melanin, a polyanionic pigment which is well known to bind numerous substances via ionic interactions, especially positively charged organic amines and metals (Larsson 1993, and references therein; Leblanc et al. 1998; Bridelli et al. 2006). The potential impact of the binding and accumulation of xenobiotics in the eye is still subject to debate. There are indications that xenobiotic binding to melanin may be the main factor in the etiology of chronic lesions affecting melanin-containing tissues (Larsson 1993; Jaga and Dharmant 2006), though Leblanc et al. (1998) argued that melanin binding and retinal toxicity are separate phenomena that are not necessarily related. Nevertheless, the accumulation of atrazine in the uveal tract of goldfi sh raises the question of possible toxic effects on the retina due to the close proximity of these ocular tissues, a question that is certainly worthy of further investigation by environmental toxicologists. In the case of methoxychlor, our data (Fig. 6) show that the gall bladder and intestine contain most of the radioactivity, indicating an effi cient metabolisation and excretion. Most other tissues showed a weak labelling. However, some muscle tissue types show a higher level of radioactivity. The red muscle layer under the skin and the bundle of red muscle fi bres forming the ventral infracarilanis (VI) contained more radioactivity than the white muscle (Fig. 6B). Labelling of the muscles belonging Fig. 5. Detail from autoradiogram and tissue section shown to the adductor mandibulae (AM) complex was also in Fig. 4B. Bar = 0.25 cm. See Table 2 for abbreviations. higher (Fig. 6A, 6B); this may be related to their higher vascularization and different metabolism (Ostrander 2000) compared with white muscle. But the most This may be due to the short exposure time. However, striking feature of methoxychlor disposition in goldfi sh in view of the high water solubility and ionic character was the high labelling of the cerebrospinal fl uid around of glyphosate at environmentally relevant pH (pKa the brain, which was only surpassed by that of the gall = < 2, 2.6, and 5.6) (Mamy et al. 2005), it was not bladder, whereas the labelling of the brain itself was very expected to bioaccumulate or represent a toxic threat low. This indicated that methoxychlor or a metabolite (WHO 1994 and references therein). Adverse biochemical can cross the blood-cerebrospinal fl uid barrier (BCSFB) and histopathological effects of glyphosate have been but not the blood-brain barrier (BBB). Previous work has observed in fi sh (Szarek et al. 2000; Jiraungkoorskul et shown that some metals (Hg, Cd, Mn, Zn) can reach the al. 2003; Glusczak et al. 2006), but at concentrations brain via axonal transport after uptake in water-exposed in water that were 102 to 105 higher compared with sensory organs, thus circumventing the BBB (Rouleau et

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Fig. 6. Autoradiograms and corresponding tissue sections of goldfi sh exposed to 14C-methoxychlor. Bar = 1 cm. Autoradio- gram and tissue section pairs in A and B are from different fi sh. See Table 2 for abbreviations.

Fig. 7. Autoradiograms and corresponding tissue sections of goldfi sh exposed to 14C-bisphenol-A. Dotted box in A shows an enlargement of a section of the autoradiogram with output levels adjusted to reveal labelling differences in gall bladder, intestine, and urine. Bar = 1 cm. Autoradiogram and tissue section pairs in A and B are from different fi sh. See Table 2 for abbreviations.

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al. 1995, 1999; Persson et al. 2003; Scott et al. 2003). bisphenol-A. The phenomenon underlying the higher Tributyltin (TBT) has been shown to cross the BBB, but labelling of the oral mucosa and esophagus, as compared not the BCSFB, and is also taken up in the brain via with skin, remain unknown at present. Nevertheless, axonal transport (Rouleau et al. 2003). It is the fi rst time binding sites in these tissues appear to have quite a high that we observed a preferential uptake of a xenobiotic in affi nity for this compound. Further research is needed to the cerebrospinal fl uid (CSF) of a fi sh. characterize these binding sites and determine whether or The maintenance of a strict homeostasis in the not this may have an impact on fi sh health. central nervous system is a necessary requirement for the highly specialized brain cells to fulfi ll their physiological Conclusion functions. The role of the BBB and the BCSFB in the maintenance of this homeostasis has been extensively WBARG has allowed the identifi cation of unforeseen studied (Suzuki et al. 1997; Zheng et al. 2003; Strazielle accumulation sites in goldfi sh for atrazine (uveal tract), et al. 2004; Löscher and Potschka 2005). The CSF fulfi lls methoxychlor (CSF), and bisphenol-A (oral mucosa). many mechanical, transport, and buffering functions as These fi ndings will help us to orient and focus further well as neuroimmune regulation and transmission of research work on the fate and effects of these chemicals neuroactive compounds. Choroid plexuses, located in in fi sh on the accumulation sites observed. Work will brain ventricles, form the interface between blood and be continued to quantify the biokinetics (rates of CSF. The BCSFB is constituted by the tight junctions uptake, distribution, metabolism, elimination) of these between the epithelial cells that restrict the paracellular compounds in relation to their accumulation in target route, and thus the entry of polar compounds that are not tissues and determine if this can lead to physiological a substrate for transbarrier transporters (Strazielle et al. function impairment under realistic environmental 2004). However, lipophilic compounds of low to medium conditions. molecular weight can cross epithelial cell membranes via passive diffusion (Zheng et al. 2003). Acknowledgment Though methoxychlor has been shown to inhibit brain mitochondrial respiration and increase hydrogen The authors gratefully acknowledge the skillful technical peroxide production in rat and mice, both in vitro and assistance of G. Pacepavicius. in vivo (Schuh et al. 2005), the risk represented for fi sh brain functions is unknown. 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Received: 23 January 2008; accepted: 22 July 2008.

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