Neurochemical alterations in lemon shark (Negaprion brevirostris) brains in association with brevetoxin exposure

Aquatic Toxicology 99 (2010) 351–359

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Neurochemical alterations in lemon shark (Negaprion brevirostris) brains in association with brevetoxin exposure Dong-Ha Nam a , Douglas H. Adams b , Leanne J. Flewelling c , Niladri Basu a,∗ a b c

Department of Environmental Health Sciences, University of Michigan School of Public Health, Ann Arbor, MI 48109, USA Florida Fish & Wildlife Conservation Commission, Fish & Wildlife Research Institute, 1220 Prospect Avenue #285, Melbourne, FL 32901, USA Florida Fish & Wildlife Conservation Commission, Fish & Wildlife Research Institute, 100 8th Ave. SE. St. Petersburg, FL 33701, USA

a r t i c l e

i n f o

Article history: Received 29 March 2010 Received in revised form 13 May 2010 Accepted 18 May 2010 Keywords: Neurochemical biomarkers Biotoxins Algal blooms Neurotoxicology

a b s t r a c t Brevetoxins are persistent, bioaccumulative, lipophilic polyether neurotoxins synthesized by Karenia brevis, a harmful algal bloom (HAB) dinoflagellate. Although some marine organisms accumulate potentially harmful levels of brevetoxins, little is known about neurotoxic effects in wild populations. Here, tissue (i.e., liver, kidney, muscle, intestine, gill, brain) brevetoxin levels (as ng PbTx-3 eq/g) and four neurochemical biomarkers (monoamine oxidase, MAO; cholinesterase, ChE; muscarinic cholinergic receptor, mAChR; N-methyl-d-aspartic acid receptor, NMDAR) were compared between eleven lemon sharks collected during a K. brevis bloom and eighteen lemon sharks not exposed to a bloom (controls) in a case–control manner. Brevetoxin levels in tissues were significantly higher in HAB-exposed sharks when compared to controls, and tissue levels (e.g., 277–3112 ng/g in livers, 429–2833 ng/g in gills) in HAB-exposed sharks were comparable to levels detected in a shark (e.g., 1223 ng/g in liver, 930 ng/g in gill) that died presumably of toxin exposure. Further, there were significant correlations between brain brevetoxin levels and ChE activity (r = −0.41; p < 0.05), MAO activity (r = −0.37; p < 0.05), mAChR levels (r = 0.55; p < 0.01), and NMDAR levels (r = −0.49; p < 0.01). There were no relationships between neurochemical biomarkers and metals (total mercury, methylmercury, selenium). Overall, these results in tissues from free-ranging lemon sharks indicate that ecologically relevant exposures to brevetoxins may cause significant changes in brain neurochemistry. As disruptions to neurochemistry precede structural and functional damage to the nervous system, these results suggest that relevant exposures to HABs may be causing sub-clinical effects in lemon sharks and raise further questions about the ecological and physiological impacts of HABs on marine biota. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Brevetoxins (PbTx) are potent lipid-soluble polyether neurotoxins synthesized by Karenia brevis, a harmful algal bloom (HAB) dinoflagellate responsible for red tide events in the Gulf of Mexico and Atlantic waters of the southeastern U.S. (Baden et al., 2005; Steidinger et al., 1998b). Exposures to PbTxs are of ecological concern because they have been attributed mortalities of marine animals including invertebrates, fish, turtles, birds, and mammals (Landsberg et al., 2009). Humans are also susceptible to PbTxs and are generally exposed via inhalation of ocean aerosols (Backer et al., 2005; Fleming et al., 2009; Kirkpatrick et al., 2008; Milian et al., 2007) or consumption of PbTx-contaminated shellfish (Kalaitzis et al., 2009). Studies on animals in both the laboratory and in the field have shown that PbTxs cause neurotoxicity (e.g., ataxia, behav-

∗ Corresponding author. Tel.: +1 734 764 9490; fax: +1 734 936 7283. E-mail address: [email protected] (N. Basu). 0166-445X/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.aquatox.2010.05.014

ioral changes, muscular contractions) (Baden and Mende, 1982; Poli et al., 1990; Risk et al., 1979b; Risk et al., 1979a; Templeton et al., 1989a; Wu et al., 1985), depression of pulmonary (e.g., bronchoconstriction, asthmatics) (Abraham et al., 2005; Baden et al., 2005, 1982; Borison et al., 1980; Cheng et al., 2005; Fleming et al., 2005; Kirkpatrick et al., 2006; Pierce et al., 2005) and cardiac function (e.g., cardiac arrhythmias) (Poli et al., 1990; Templeton et al., 1989a), impairment of immune system (e.g., decreased phagocytosis and lymphocyte proliferation, apoptosis) (Bossart et al., 1998; Sudarsanam et al., 1992; Walsh et al., 2008, 2005), and possible genotoxicity (e.g., DNA adducts, chromosomal aberration) (Radwan and Ramsdell, 2008; Sayer et al., 2005, 2006). The primary mechanism by which PbTxs cause neurotoxicity is related to its interaction with voltage-gated sodium channels (Baden and Mende, 1982; Catterall and Gainer, 1985; Gawley et al., 1995; Jeglitsch et al., 1998; LePage et al., 2003; Risk et al., 1979b; Stuart and Baden, 1988; Wu et al., 1985; Yuhi et al., 1994). The binding of PbTxs to site 5 on the ␣-subunit of voltage-gated sodium channels augments sodium (Na+ ) influx by inhibiting channel inac-

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tivation thus prolonging the length of time a channel remains open and causing hyperpolarization of cells and excitotoxicity in the nervous system (LePage et al., 2003; Massensini et al., 2003; Poli et al., 1986). The interaction of PbTx with voltage-gated sodium channels may also result in a series of downstream neurochemical effects, including activation of NMDA receptors as a result of enhanced glutamate release and calcium (Ca2+ ) influx (Berman and Murray, 2000; Dravid et al., 2005; George et al., 2009; Risk et al., 1982). Almost annually, HABs caused by K. brevis impact the eastern Gulf of Mexico and southwest Florida (Landsberg et al., 2009; Steidinger, 2009; Tester and Steidinger, 1997). Blooms of K. brevis also intermittently occur on the U.S. Atlantic coast. Notably, between September 2007 and January 2008, a K. brevis bloom occurred along approximately 200 km of Florida Atlantic coastal waters, representing the longest duration and most extensive red tide recorded for the Atlantic coast of Florida (Wolny et al., 2008). Multi-species fish kills, water discoloration, and human respiratory irritation were reported during this red tide event (Douglas Adams, personal observation), and measured K. brevis cell concentrations peaked at 6,030,000 cells/l (FWC-FWRI, Harmful Algal Bloom database). Such a measured concentration is much higher than natural background concentrations (<103 K. brevis cells/l) and at levels known to have environmental impacts (e.g., >5 × 103 cells/l may cause filter-feeding shellfish to become toxic for human consumption; 0.5–10 × 106 cells/l typically cause fish mortality) (Fire et al., 2007; Pierce and Henry, 2008; Quick and Henderson, 1974; Steidinger et al., 1998b; Tester et al., 1998). One particular species at risk of PbTx intoxication are lemon sharks (Negaprion brevirostris). According to our recent findings, the nearshore waters at Cape Canaveral, which is located in the middle of the 2007–2008 K. brevis bloom, serves as a critical nursery ground for juvenile lemon sharks (Reyier et al., 2008). Juveniles repeatedly utilize clearly defined nursery habitats and thus can offer a good indication of regional exposures to marine neurotoxins. Although substantial concentrations of PbTxs have been reported in some marine animals (Fire et al., 2007; Flewelling et al., 2005; Landsberg et al., 2009; Naar et al., 2007), including sharks (Flewelling et al., 2010), this has not been studied in detail for the lemon shark. While PbTxs and their metabolites are of concern with regard to the health of Florida’s coastal ecosystems, limited information exists on PbTx levels in the tissues of top predatory species, and whether such exposures cause sub-clinical neurological impacts is unknown. In the present study, we conducted a case–control study whereby tissue PbTx concentrations (expressed as ng PbTx-3 eq/g) levels were compared between lemon sharks exposed to K. brevis blooms with non-exposed lemon sharks from the same area. We also investigated whether PbTx exposure could be associated with changes in key neurochemical parameters (MAO, ChE, mAChR, NMDAR) in lemon shark brain tissues. Since mercury (Hg) is a well-established neurotoxicant that was previously documented in the same animals studied here (Nam et al., unpublished results), a retrospective calculation was performed to determine if brain Hg (total and methyl Hg) levels could be related to the aforementioned neurochemical biomarkers as previously documented in other predatory wildlife (Basu et al., 2007).

2. Materials and methods 2.1. Study area The study area encompassed nearshore Atlantic Ocean waters directly off Cape Canaveral in east-central Florida. Overview information regarding the habitat characteristics, water quality parameters and general oceanography of the area, as well as specific details regarding lemon sharks and other shark species in the

area are provided in recent nearshore assessment studies (Adams and Paperno, 2007; Reyier et al., 2008). 2.2. Animals Thirty juvenile lemon sharks (N. brevirostris) were sampled between 2005 and 2008. Of these 30 sharks, one male shark collected from a multi-species fish kill that occurred during a red tide event (December 3, 2007) had presumably died as a result of brevetoxin exposure. Another eleven sharks (7 males and 4 females) were obtained during the same HAB event on December 7, 2007. Accordingly, these twelve sharks enabled us to compare measurements in sharks collected during an ongoing bloom (HAB-exposed) with control (non-exposed or background) sharks. Stage of maturity was determined by examination of internal and external reproductive organs (characterized as the status of clasper calcification and umbilical scars) and by comparing shark size with size estimates at birth or maturity from previous studies (Reyier et al., 2008). For all sharks, the gender (18 males and 12 females), total body weight (3388 ± 1216 g TW), and total length (830.4 ± 80.5 mm TL) were recorded. Tissue samples (liver, kidney, brain, dorsal white muscle, gill, and intestine) were carefully extracted to avoid external contamination, placed in sterile polyethylene sample containers and stored at −20 ◦ C for metals determination or −80 ◦ C for neurochemical measurements. 2.3. Sample preparation After thawing, 0.3–5 g of wet tissues (liver, kidney, brain cerebral cortex, dorsal white muscle, gill, and intestine) were homogenized for 30 s in ice-cold phosphate buffer (50 mM NaH2 PO4 , 5 mM KCl, 120 mM NaCl, pH 7.4). Cellular membranes were then isolated for neurochemical analyses from these samples in homogenization and centrifugation steps described elsewhere (Basu et al., 2007). Subsamples (2 g) were extracted for brevetoxin analysis by homogenizing in the presence of organic 80% aqueous methanol (4 ml/g tissue). Homogenates were centrifuged (10 min at 3000 × g), the supernatants were retained, and the pellets were extracted a second time in the same manner. The supernatants were pooled and then partitioned with 100% hexane (1:1, v:v). The methanol fraction was retained and stored at −20 ◦ C until analyzed. 2.4. Brevetoxin analyses Brevetoxin concentrations in extracts were measured by competitive ELISA (Naar et al., 2002) with modifications described in elsewhere (Flewelling, 2008). Standard curves were generated using PbTx-3. Results are expressed in nanograms per gram as PbTx-3 equivalents and reflect the overall concentration of brevetoxins and brevetoxin-like compounds in the sample. As performed in this study, the limit of detection by ELISA was 5 ng PbTx-3 eq/g. 2.5. Neurochemical enzyme activity Neurochemical enzyme activities of ChE and MAO were measured according to published procedures (Basu et al., 2009a,b). Briefly, tissues were homogenized in cold phosphate buffer (50 mM NaH2 PO4 , 5 mM KCl, 120 mM NaCl, pH 7.4) including 0.5% (v/v) Triton X-100, and the supernatant was removed following a 10 min centrifugation at 15,000 × g (4 ◦ C). The concentration of protein in the supernatant preparation was determined with the Bradford assay using bovine serum albumin as the standard. For ChE activity, 0.5 ␮g of supernatant protein was mixed with 100 ␮M 10-acetyl-3,7-dihydroxyphenoxazine, 200 mU horseradish peroxidase, 20 mU choline oxidase, and 100 ␮M acetylcholine. For

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Table 1 Concentrations (ng PbTx-3 eq/g of wet tissue) of brevetoxins and their metabolites in several tissues of lemon sharks in relation to HAB exposure. Control (n = 18)

HAB-exposed (n = 11)

Mean ± SD Median Range Positive no.

6

Gill

ND-7 2

1307 ± 614 1194 429–2833 11

Liver

Mean ± SD Median Range Positive no.

14 ± 7 12 ND-36 14

Kidney

Mean ± SD Median Range Positive no.

c

Mortality (n = 1)a

p-Valueb

930

<0.001

764 ± 819 487 277–3112 11

1223

<0.001

9±2 9 ND-11 6

278 ± 150 206 124–541 11

803

<0.001

ND

12 ± 2 12 ND-15 4

15

<0.001

Muscle

Mean ± SD Median Range Positive no.

15

134

<0.001

Intestine

Mean ± SD Median Range Positive no. Mean ± SD Median Range Positive no.

ND

44

<0.001

Brain

a b c

0

ND-15 1

0

143 ± 80 110 39–304 11 35 ± 24 34 15–101 11

A shark that died due to brevetoxin exposure. Statistical difference was calculated between control and HAB-exposed sharks. Based on the brevetoxin-positive samples; ND: not detected (below level of detection: <5 ng PbTx-3 eq/g of wet tissue).

MAO activity, 5 ␮g of protein was mixed with 100 ␮M 10-acetyl3,7-dihydroxyphenoxazine, 200 mU horseradish peroxidase, and 100 mM tyramine. Both assays were incubated for 30 min. The reaction end-product, resorufin, was monitored following a two-step enzymatic reaction catalyzed by choline oxidase and horseradish peroxidase at room temperature. Fluorescence (ex = 540 nm, em = 590 nm) of resorufin was measured every 5 min, between 30 and 60 min (HTS7000Plus, PerkinElmer). Enzyme activities were expressed as nmol of resorufin formed per min per unit (␮g or mg) protein and each sample was assayed in triplicate. 2.6. Neurochemical receptor binding For neurochemical receptor binding assays, cellular membranes were prepared from whole brain tissues using protocols described

elsewhere (Basu et al., 2009a,b). Binding to the mACh receptor (phosphate buffer: 50 mM NaH2 PO4 , 5 mM KCl, 120 mM NaCl, pH 7.4) and NMDA receptor (Tris: 50 mM Tris, 100 ␮M glycine, 100 ␮M l-glutamic acid, pH 7.4) were performed in the buffers indicated. Approximately 30 ␮g of cellular membrane preparation was diluted in 100 ␮l buffer and added to 1.0 ␮m GF/B glass filter (Millipore, Boston, MA, USA) microplate wells. For mACh receptor binding, samples were incubated with 1 nM [3 H]-QNB (3quinuclidinyl benzilate; 50.5 Ci/mmol; NEN/Perkin Elmer, Boston, MA, USA) for 60 min. For NMDA receptor binding, samples were incubated with 5 nM [3 H]-MK-801 (dizocilpine; 27.5 Ci/mmol; NEN/Perkin Elmer) for 120 min. Incubations were conducted at room temperature and reactions were terminated by vacuum filtration. All filters were rinsed three times with buffer and then allowed to soak for 72–96 h in 25 ␮l of OptiPhase Supermix Cocktail

Fig. 1. Relationships between tissue brevetoxin levels and body weight in HAB-exposed lemon sharks.

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(Perkin Elmer). Radioactivity retained by the filter was quantified by liquid scintillation counting in a microplate detector (Wallac Microbeta, Perkin Elmer) having a counting efficiency of approximately 50%. Specific binding to both receptors was defined as the difference in radioligand bound in the presence and absence of 100 ␮M unlabelled atropine and MK-801 for mACh and NMDA receptors, respectively. Binding was reported as fmol of radioisotope bound per mg of membrane protein (fmol/mg). All samples were assayed in quadruplicate for total and non-specific binding. 2.7. Statistical analyses Normality and homoscedasticity of data were tested by using the Kolmogorov-Smirnov test and the Levene’s test (Fmax -test), respectively. Mann–Whitney U-tests were conducted to discern differences between control and HAB-exposed groups with respect to PbTxs concentrations in tissues. The relationships among PbTxs, metals (Hg, organic Hg, Se), and neurochemical data in brains were determined using Spearman rank correlation tests. A p value less than 0.05 was considered statistically significant in all analyses. 3. Results Levels of tissue PbTx differed between control (non-HAB exposed) lemon sharks and sharks exposed to a K. brevis bloom. In the eighteen non-exposed sharks, PbTxs were detected in the gill (2 cases), liver (14 cases), kidney (6 cases), and intestine (1 case) tissues of some individuals but the measured values were close to the analytical limit of detection (5–10 ng PbTx-3 eq/g; Table 1). PbTxs were not detected in the muscle and brain tissues of control, non-exposed sharks (Table 1). In HAB-exposed sharks, all tissues examined contained PbTxs (range from 15 to 3112 ng PbTx-3 eq/g), except for the muscle tissue in which only four of 11 individual sharks had detectable PbTx levels. Comparison of PbTx concentra-

tions between tissues of control and HAB-exposed sharks showed significant differences in each of the six tissues studied. Levels of PbTxs in HAB-exposed sharks were comparable to levels detected in a shark that presumably died from PbTx exposure. When PbTx levels were compared between tissues on an intra-individual basis, significant positive correlations were found among several tissues (r = 0.812, p < 0.01 for liver vs. kidney; r = 0.982, p < 0.05 for kidney vs. muscle; r = 0.783, p < 0.01 for gill vs. intestine; r = 0.858, p < 0.01 for liver vs. gill) in HAB-exposed sharks. However, there were no correlations between PbTx levels in the brains with any other tissue type. When body size was considered, hepatic (but not other tissues including brain) PbTx levels were negatively correlated with body weight (and to a lesser degree, total body length; data not shown) (Fig. 1A and B). There were no gender-related effects on PbTx levels in any tissue type. Several associations between brain PbTx levels and changes in neurochemical biomarkers were found, and these were not influenced by gender. For the two neurochemical enzymes studied (AChE and MAO), HAB-exposed sharks had mean enzyme activities that were 84.3% (AChE; p = 0.034) and 69.7% (MAO; p = 0.012), respectively, of values measured in control (non-HAB exposed) sharks (Fig. 2A and B). Likewise, a negative correlation was found between brain PbTx levels and AChE (r = −0.438, p = 0.020) and MAO (r = −0.472, p = 0.011) activity. For the two receptors, significant differences in specific binding of radioligands were found between the two test groups (Fig. 2C and D). For the mACh receptor, HABexposed sharks had mean receptor levels that were 160.3% greater than control sharks (p < 0.001) (Fig. 2C), and a significant positive correlation was measured between brain PbTx levels and mACh receptor levels (r = 0.693, p < 0.001). For the NMDA receptor, HABexposed sharks had mean receptor levels that were 74.2% lower than control sharks (p < 0.001) (Fig. 2D), and a significant negative correlation was measured between brain PbTx levels and NMDA receptor levels (r = −0.513, p = 0.006).

Fig. 2. Comparisons of neurochemical enzyme activities (A,B) and receptor densities (C,D) in brain tissues of control (non-HAB exposed) and HAB-exposed lemon sharks (* p < 0.05; *** p < 0.001).

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Fig. 3. Brevetoxin concentrations (ng PbTx-3 eq/g) in tissues (L: liver; K: kidney; B: brain) of juvenile lemon sharks (control: open; HAB-exposed: shaded; mortality: black) in relation to past studies showing health effects. Studies are separated according to LRT (Laboratory Rodents Tests), ICT (In vitro Cellular Tests), and ET (Ecological Tests) as indicated: (1) (Walsh et al., 2008, 2005) [ICT]; (2) (Flewelling et al., 2005) [ET]; (3) (Ishida et al., 2004; Murata et al., 1998; Selwood et al., 2008) [ET]; (4) (Baden and Mende, 1982; Rein et al., 1994; Templeton et al., 1989b) [LRT, ICT, ET]; (5) (Sayer et al., 2005) [ICT]; (6) (Flewelling et al., 2005) [ET]; (7) (Washburn et al., 1994; Washburn et al., 1996) [ICT]; (8) (Baden, 1983, 1989; Rein et al., 1994; Risk et al., 1979a,b) [ICT, ET]; (9) (Radwan and Ramsdell, 2008; Sayer et al., 2005) [ICT]; (10) (Colman and Ramsdell, 2003) [ICT, ET]; (11) (Apland et al., 1993; Atchison et al., 1986; Dravid et al., 2004; Dravid and Murray, 2003; Gawley et al., 1995; Huang et al., 1984; LePage et al., 2003; Tsai and Chen, 1991; Wu et al., 1985) [LRT, ICT]; (12) (George et al., 2009; Jeglitsch et al., 1998) [LRT, ICT]; (13) (Berman and Murray, 1999; Dravid et al., 2005; LePage et al., 2003) [LRT, ICT]; (14) (Canete and Diogene, 2008; Manger et al., 1995) [ICT]; (15) (Risk et al., 1982) [ICT]; (16) (Bakke and Horsberg, 2007; Berman and Murray, 1999; Choich et al., 2004) [LRT, ET]; (17) (Edwards et al., 1992; Gawley et al., 1995; Trainer and Baden, 1999) [LRT, ICT]; (18) (Cohen et al., 2007) [ET].

Since these lemon shark brain tissues were previously analyzed for Hg (total Hg and organic Hg) and selenium (Se) (Nam et al., unpublished results), a retrospective calculation was performed to determine if any of the neurochemical biomarkers associated with the aforementioned elements. There were no Hg-associated relationships, but brain Se concentrations significantly correlated with AChE activity (r = 0.53, p < 0.01) and mACh receptors (r = −0.44, p < 0.05). When these aforementioned measures of brain Hg and Se were compared between HAB-exposed sharks and controls, there were no significant differences. 4. Discussion The key finding of this study was that significant changes in neurochemical enzymes (AChE, MAO) and receptors (mAChR, NMDAR) were related to levels of PbTx in brains of lemon sharks that were naturally exposed to an extensive K. brevis bloom (Figs. 2 and 3). Though several laboratory-based studies have documented PbTx neurotoxicity, to our knowledge this is the first to characterize exposure-related neurochemical effects in any free-ranging marine biota. There were clear differences in PbTx concentrations between the control and HAB-exposed sharks. In the control sharks, only a few samples had detectable PbTx levels in gill, intestine, and kidney, whereas PbTx was detected in about 80% of the livers (Table 1).

Others have demonstrated that PbTxs can persist in the tissues of marine organisms for weeks or months after a K. brevis bloom (Naar et al., 2007; Steidinger et al., 1998a,b), and PbTxs are often detected in teleost fish and elasmobranchs collected during non-bloom periods (Flewelling, 2008; Flewelling et al., 2010; Naar et al., 2007). The presence of PbTx in tissues during non-bloom periods may represent residual PbTx from past exposure. However, before the HAB event in 2007, there were no documented K. brevis blooms on the Atlantic coast of Florida since late 2002. It is possible that PbTx in tissues during non-bloom periods may result from the persistence of PbTxs in prey items, from exposure of the animal or its prey to a bloom in a different location, or from exposure to an undetected K. brevis bloom (Flewelling, 2008; Flewelling et al., 2010). Regardless, PbTx concentrations in our control lemon sharks were extremely low (and often not detected) compared to the HABexposed sharks and no PbTx was detected in the brains of any of the control sharks. Our findings clearly indicate that in sharks collected from a K. brevis bloom, PbTx uptake into several tissues is significant. For example, appreciable levels in the liver (764 ng/g), intestine (143 ng/g), and gill (1307 ng/g) likely reflect a combination of adsorption (e.g., across gill membrane) and active uptake (e.g., through digestive tract) (Abbott et al., 1975; Catterall and Risk, 1981; Tester et al., 2000). When the current results are compared against previous studies focused on health effects, it is clear that

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ecologically relevant exposures to PbTx may be associated with several sub-clinical and clinical effects in lemon sharks and other marine organisms (Fig. 3). Though, such comparisons should be carefully interpreted owing to vast differences across studies with respect to exposure schemes (e.g., organisms exposed in laboratory, test tube, field) as well as inter-species differences in sensitivity to PbTXs. Brevetoxins are persistent and bioaccumulative neurotoxins in fish and marine mammals (Flewelling et al., 2005; Fire et al., 2007; Naar et al., 2007). In addition to route and duration of exposure, the extent to which organisms accumulate PbTxs depends on their solubility, stability, and toxicokinetics. When body size was considered, hepatic (and to a lesser degree, renal) PbTx levels were negatively correlated with body weight, but this was not found in other tissues including brain (Fig. 1A and B). It has been reported that PbTxs are distributed to the brain much slower than to other tissues (Benson et al., 1999; Cattet and Geraci, 1993; Tibbetts et al., 2006), and within the brain they may accumulate in lymphocytes and microglial cells (Bossart et al., 1998). Brevetoxins are known to cross the blood brain barrier (Baden, 1989; Templeton et al., 1989b) and reach significant levels within the central nervous system (Cattet and Geraci, 1993). Studies in laboratory animals have shown that ecologically relevant PbTx concentrations (in the nanomolar range) can cause neurochemical and synaptic alterations, neuronal injury and death, and neurobehavioral changes (Fig. 3). However, little observational evidence exists from wild populations that free-ranging animals accumulate PbTx in brain tissues and that levels are sufficient to induce sub-clinical or clinical neurotoxicity. Here, PbTx was detected in the brains of all HAB-exposed sharks, and the levels (range: 15–101 ng PbTx-3 eq/g, mean: 35 ng PbTx-3 eg/g) were comparable to a shark that presumably died following brevetoxin exposure (44 ng PbTx-3 eg/g) (Table 1). Though acute effects from K. brevis bloom are known, little is known about long-term exposure to sublethal concentrations (Fire et al., 2007; Leverone et al., 2006). To our knowledge, the results from the current study are the first to relate exposure of marine biota to PbTxs (including brain tissue levels) with sub-clinical alterations in brain neurochemistry. Here, one of the main findings was that significant changes in cholinergic function were associated with PbTx exposure in lemon sharks. Specifically, PbTx exposure was related to reduced AChE activity and increased mAChR levels (Fig. 2A and C). The fundamental role of AChE is to terminate neurotransmission by rapidly hydrolysing synaptic levels of acetylcholine (ACh) (Massoulie, 2002; Taylor and Radic, 1994), and thus has various biological roles on neurodevelopment (e.g., neuronal cell differentiation, adhesion, neuritogenesis, and neurodegeneration) and behavioral properties (Parnetti et al., 2002; Rasool et al., 1986). It has been reported that PbTxs can promote ACh release from guinea pig ileum (Poli et al., 1990; Risk et al., 1982) and rat phrenic nerve hemidiaphragms (Baden et al., 1984). Similar to the current study, brain AChE activity was significantly reduced in mice exposed to PbTx metabolites and analogues (Husain et al., 1996). Interestingly, the clinical symptoms of PbTx intoxication are similar to those following AChE inhibition (Martin and Chatterjee, 1969; Sasner et al., 1972). For example, clinical outcomes in mice exposed to synthesized PbTx metabolites included hyperactivity, tremors, and convulsions (Husain et al., 1996), which are commonly found in animals that have brain AChE activity reduced >40% (Hayes, 1982). Here, it is unclear if reduced AChE activity in HAB-exposed sharks is a primary response (direct inhibition of AChE by PbTx) or a secondary consequence of toxic action elsewhere. Nonetheless, prolonged suppression of AChE activity may have deleterious health consequences (e.g., delayed neurogenesis, behavioral impairment) in wild shark populations as previously demonstrated in laboratory studies on AChE knock-out mice (Duysen et al., 2002) and anticholinesterase agents

(Abou-Donia et al., 2003; Costa et al., 1982; McDonald et al., 1988). Cholinergic effects were also realized at the level of the muscarinic acetylcholine receptor, which is the main cholinergic receptor in the central nervous system. Inhibition of AChE activity has been associated with a reduction in muscarininc cholinergic receptor levels, likely a compensatory response to limit over-stimulation of cholinergic signaling and maintain neural homeostasis (Costa et al., 1982). Concomitant changes in AChE and muscarinic cholinergic receptor levels exemplify the autoregulatory nature of cholinergic neurotransmission, particularly during periods of toxicant stress. Levels of mAChR were significantly higher in the HAB-exposed lemon sharks and there was negative correlation between AChE activity and mAChR levels (data not shown). Similar to the discussion on AChE, it is not clear whether changes in mAChR are a primary or secondary response to PbTX exposure, however there exists much cross-talk between mAChR and AChE levels. In rat brain, manipulation of mAChRs confers drastic changes on AChE transcription (Nitsch et al., 1998; Salmon et al., 2005). Stimulation of mAChRs may also result in decreased hydrolysis of ACh, possibly through reduced AChE expression (Kobayashi et al., 2007). The mAChR belongs to a highly conserved class of transmembrane receptors that involve intracellular signaling cascades coupled with a G-protein (Felder, 1995; Rosenblum et al., 2000; Wess, 2004), and thus alteration of endogenous ligand (i.e., ACh) binding to the mAChR can have profound impacts on diverse functions, including memory and locomotion (Wess, 2004). With respect to the dopaminergic system, we found brain MAO activity was reduced in PbTxs-exposed animals (Fig. 2B). Pharmaceutical studies have documented synaptic catecholamines (e.g., dopamine) are released via an exocytotic mechanism following Na+ channel activation in neuronal cells (Toner and Stamford, 1997). Others have shown that PbTxs (PbTx-2 or PbTx-3) potentiate catecholamine release following Na+ and Ca2+ influx, although the stimulatory modes of PbTxs vary according to the pharmacological properties of Na+ and Ca2+ channels in neuronal tissues (Poli et al., 1986; Risk et al., 1982). Here, the significant reduction in MAO activity in HAB-exposed sharks would be expected to result in a net increase of synaptic catecholamines such as dopamine, epinephrine, and norepinephrine, and possibly also serotonin. Additional studies are warranted to better understand the effects of PbTxs on dopaminergic, serotonergic, and noradrenergic neurotransmitter functions, including impacts on dopamine receptors and transporters, given that dopamine, serotonin, and norepinephrine play important roles in fish reproduction (Peter et al., 1986; Trudeau, 1997). Experiments performed on neuronal cells and rat brain synaptosomes have shown that PbTxs interact with binding site 5 on the ␣-subunit of voltage-gated sodium channels (VGSCs) (LePage et al., 2003; Poli et al., 1986). Activation of VGSCs by PbTxs promotes the release of excitatory amino acids (Dravid et al., 2005). PbTxs may also cause an increase of Na+ influx through VGSC depolarization (dependent on coincident activation of Src kinase), activation of NMDAR (tyrosin phosphorylation of NR2B by Src kinase and/or PKC-dependent pathway), NMDAR-mediated Ca2+ influx, and excitotoxicity by ERK activation switch which may involve excessive NMDAR activation (Cao et al., 2007; Churn et al., 1995; Dravid et al., 2004, 2005; Hardingham and Bading, 2002; Massensini et al., 2003; Yu, 2006). The NMDAR is the main excitatory neurotransmitter receptor in the vertebrate nervous system and plays an important role in synaptic plasticity and in the consolidation of learning and memory (Collingridge and Singer, 1990; Dravid et al., 2004; El-Nabawi et al., 2000; Meldrum, 2000). Thus, glutamateinduced excitotoxicity results from prolonged impairment of ion (e.g., Na+ , Ca2+ ) balance coupled with NMDAR hyperstimulation. Here we showed that bioaccumulation of PbTxs in shark brains

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was associated with a decreased number of NMDAR in the brain, as determined by [3H]-MK801 binding (Figs. 2D and 3D). Reduced NMDAR is a common mechanism to minimize excitotoxic damage caused by physical (e.g., anoxia and ischemia) (Gascon et al., 2005; Klein et al., 1989) as well as chemical (e.g., mercury) stressors (Basu et al., 2007, 2009a). Accordingly, the observation of reduced NMDAR in response to PbTxs exposure may reflect a compensatory, neurochemical response to PbTx-induced excitotoxicity. The longterm ecological significance of prolonged alterations in NMDAR is not clear. Though, in rodents, experimental manipulation of NMDAR has been associated with functional deficits in motor and cognitive activity (Cheli et al., 2006; Himori et al., 1991; Mohn et al., 1999). In summary, PbTxs are of increasing health concern in marine coastal ecosystems. Here we found that exposure of lemon sharks to PbTxs, under an ecologically relevant scenario, may result in the disruption of key neurochemical enzymes and receptors. As disruptions to neurochemistry precede structural and functional damage to the nervous system, these results suggest that relevant exposures to PbTxs may be causing sub-clinical effects in lemon sharks and raise questions about the ecological and physiological impacts of PbTxs on marine biota. Acknowledgements Thank you to Eric Reyier (IHA-NASA) for assistance with collection of lemon sharks, and to Karen Atwood and April Granholm (FWC-FWRI) for assistance with brevetoxin analyses. Funding for the brevetoxin analyses was provided by the State of Florida and the National Oceanic and Atmospheric Administration Oceans and Human Health Initiative (grant no. NA05NOS4781246) and the neurochemical biomarkers were provided by the University of Michigan School of Public Health. References Abbott, B.C., Siger, A., Spiegelstein, M., 1975. Toxins from blooms of Gymnodinium breve. In: LoCicero, V.R. (Ed.), Proceedings of the First International Conference on Toxic Dinoflagellate Blooms. Massachusetts Science and Technology Foundation, Wakefield, pp. 355–366. Abou-Donia, M.B., Abdel-Rahman, A., Goldstein, L.B., Dechkovskaia, A.M., Shah, D.U., Bullman, S.L., Khan, W.A., 2003. Sensorimotor deficits and increased brain nicotinic acetylcholine receptors following exposure to chlorpyrifos and/or nicotine in rats. Arch. Toxicol. 77, 452–458. Abraham, W.M., Bourdelais, A.J., Ahmed, A., Serebriakov, I., Baden, D.G., 2005. Effects of inhaled brevetoxins in allergic airways: toxin-allergen interactions and pharmacologic intervention. Environ. Health Perspect. 113, 632–637. Adams, D.H., Paperno, R., 2007. Preliminary assessment of a nearshore nursery ground for the scalloped hammerhead off the Atlantic coast of Florida. In: American Fisheries Society Symposium, vol. 50, pp. 165–174. Apland, J.P., Adler, M., Sheridan, R.E., 1993. Brevetoxin depresses synaptic transmission in guinea pig hippocampal slices. Brain Res. Bull. 31, 201–207. Atchison, W.D., Luke, V.S., Narahashi, T., Vogel, S.M., 1986. Nerve membrane sodium channels as the target site of brevetoxins at neuromuscular junctions. Br. J. Pharmacol. 89, 731–738. Backer, L.C., Kirkpatrick, B., Fleming, L.E., Cheng, Y.S., Pierce, R., Bean, J.A., Clark, R., Johnson, D., Wanner, A., Tamer, R., Zhou, Y., Baden, D.G., 2005. Occupational exposure to aerosolized brevetoxins during Florida red tide events: effects on a healthy worker population. Environ. Health Perspect. 113, 644–649. Baden, D.G., 1983. Marine food-borne dinoflagellate toxins. Int. Rev. Cytol. 82, 99–150. Baden, D.G., 1989. Brevetoxins: unique polyether dinoflagellate toxins. FASEB J. 3, 1807–1817. Baden, D.G., Bikhazi, G., Decker, S.J., Foldes, F.F., Leung, I., 1984. Neuromuscular blocking action of two brevetoxins from the Florida red tide organism Ptychodiscus brevis. Toxicon 22, 75–84. Baden, D.G., Bourdelais, A.J., Jacocks, H., Michelliza, S., Naar, J., 2005. Natural and derivative brevetoxins: historical background, multiplicity, and effects. Environ. Health Perspect. 113, 621–625. Baden, D.G., Mende, T.J., 1982. Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis. Toxicon 20, 457–461. Baden, D.G., Mende, T.J., Bikhazi, G., Leung, I., 1982. Bronchoconstriction caused by Florida red tide toxins. Toxicon 20, 929–932.

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