Toxicon 91 (2014) 45e56
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors Fernando Rubio a, *, Lisa Kamp a, Justin Carpino a, Erin Faltin a, Keith Loftin b, mulo Ara oz c, ** c, R o Jordi Molgo a
Abraxis LLC, Warminster, PA, USA U.S. Geological Survey, Organic Geochemistry Research Laboratory, Kansas Water Science Center, Lawrence, KS 66049, USA Centre de recherche CNRS de Gif-sur-Yvette, Institut F ed eratif de Neurobiologie Alfred Fessard FR2118, Laboratoire de Neurobiologie et D eveloppement UPR 3294, Gif sur Yvette 91198, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2014 Received in revised form 22 August 2014 Accepted 27 August 2014 Available online 27 September 2014
Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated a-bungarotoxin (the tracer) and streptavidinhorseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Anatoxin-a Nicotinic acetylcholine receptor nAChRs Receptor binding assay Cyclic imines Pinnatoxin
1. Introduction Microalgae are natural components of marine and freshwater environments. Excessive growth of algae becomes a nuisance and in some instances a public health threat through consumption of contaminated fish/mollusks and drinking water, and from the use of water for
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected], oz). Rubio), [email protected] (R. Ara
[email protected]
http://dx.doi.org/10.1016/j.toxicon.2014.08.073 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
(F.
recreational activities. Thus, some members of cyanobacteria, dinoflagellates or diatoms may cause harm through the release of toxins. In continental waters, cyanobacteria harmful algal blooms (CyanoHABs) are being increasingly reported worldwide due to eutrophication and global climate change (Carmichael, 2001; Paerl and Huisman, 2008). Water managers have expressed serious concerns about public health and environmental quality as a result of CyanoHAB toxins in recreational and drinking waters. CyanoHABs occur in freshwater lakes, ponds, rivers, reservoirs, and brackish waters throughout the world. Organisms responsible include an estimated 40 species,
46
F. Rubio et al. / Toxicon 91 (2014) 45e56
primarily belonging to the genera Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nostoc, and Oscillatoria (Planktothrix) (Carpenter and Carmichael, 1995). Cyanobacterial toxins (cyanotoxins) include cytotoxins and biotoxins responsible for acute lethal, acute chronic, and sub-chronic poisoning of both wild and domestic animals as well as humans. Cyanotoxins include the neurotoxins anatoxin-a, anatoxin-a(s), and saxitoxin, and the hepatotoxins microcystins, nodularins and cylindrospermopsin (Carmichael, 1997). Marine biotoxins are naturally occurring chemicals produced by a type of microscopic algae known as phytoplankton. These toxins can cause widespread harm to or death of sea life and can also affect humans through multiple routes of exposure (oral, respiratory, skin), making them a growing cause of concern for public health (Landsberg et al., 2005). Marine toxins can accumulate in fish and shellfish and can lead to several types of poisoning: amnesic shellfish poisoning (ASP), diarrhetic shellfish poisoning (DSP), neurologic shellfish poisoning (NSP), and paralytic shellfish poisoning (PSP). More recently, another syndrome, azaspiracid poisoning (AZP) produced by ingestion of azaspiracid toxins (AZA), have been reported after consumption of shellfish (Twiner et al., 2008). The emerging cyclic imine toxins gymnodimines, spirolides, and pinnatoxins have been reported to act as potent antagonists of nicotinic acetylcholine receptors (Kharrat et al., oz et al., 2011; Hu et al., 1995; 2008; Bourne et al., 2010; Ara Uemura et al., 1995). Following a typical toxicity by mouse bioassay, shellfish farms in New Zealand and France have been forced to close due to harmful algal blooms dominated by Karenia selliformis, the producer of gymnodimine A, and by Alexandrium ostenfeldii, the producer of 13desmethyl spirolide C. Pinnatoxins have been isolated from shellfish of the genus Pinna, and were shown to be produced by Vulcanodium rugosum (Rhodes et al., 2011; zan and Chome rat, 2011; Hess et al., 2013). Ne The nicotinic acetylcholine receptors (nAChRs) belong to the family of ligand-gated ion channels. These receptors are widely expressed by various non-neural cell types including muscle, skin, pancreas, lungs and by neural cells in the central and peripheral nervous system. The transmembrane nAChRs are assembled from five subunits that are arranged around a central water-filled pore which opens following acetylcholine binding to its putative binding site. In vertebrate mammals, muscle nAChRs are of two types: embryonic (a12b1gd) or mature receptor type (a12b1d3). In contrast, neuronal nAChRs show more variability in terms of subunit composition: ab nAChR are made up from a combination of a2-a6 and b2-b4 subunits. Muscle nAChR mediate fast synaptic transmission at the neuromuscular junction playing a central role in regulating functions vital for life and escaping from predation such as muscle contraction, and neuronal nAChRs control autonomic and central nervous system function (Albuquerque et al., 2009). In addition, the basic structure of the ligand binding site of nAChRs has been retained with little variability throughout evolution, making it an excellent structural target for a toxin. There are many examples of compounds that target nAChRs that are used as both predatory weapons and defensive measures against predation (Daly, 2005).
Fig. 1. Anatoxins and cylic imine toxins chemical structures.
F. Rubio et al. / Toxicon 91 (2014) 45e56
Anatoxin-a and its homologue homoanatoxin-a are bicyclic secondary amines that are potent agonists of the muscular a12b1d3 and neuronal a4b2 and a7 nAChRs (Spivak et al., 1980; Thomas et al., 1993; Amar et al., 1993). Anatoxin-a kills mice within 2e5 min after intraperitoneal injection; death is preceded by twitching, muscle spasms, paralysis, and respiratory arrest (i.p. mouse LD50: 200 mg kg1) (Devlin et al., 1977). Repetitive stimulation of the muscle-type a12b1d3 nAChR by anatoxin-a stabilizes the desensitized state of the cholinergic receptor that ultimately leads to the blockade of neuromuscular transmission of the depolarizing type (Spivak et al., 1980; Thomas et al., 1993). Cyanobacterial anatoxins are produced by species distributed worldwide from the genera Anabaena, Aphanizomenon, Cylindrospermum, Oscillatoria, Phormidium, Planktothrix, and Raphidiopsis (Wonnacott and Gallagher, 2006). Long term monitoring of water bodies has shown that the composition of the cyanobacteria population and toxin production changes with time and environmental factors (Baker et al., 2002; Havens et al., 2003; Briand et al., 2009). No predictions can be made to determine whether a toxic species will dominate a given cyanobacterial bloom or when it will produce toxins. Toxin occurrence therefore needs to be assessed on a case by case basis. The spirolides, gymnodimines, pinnatoxins, pteriatoxins, prorocentrolides, and spiroprorocentrimines are included in the cyclic imine group of marine phycotoxins due to the presence of an imine ring in their macrocyclic chemical structure (Fig. 1) depicts chemical structures of anatoxins and cyclic imine toxins. These toxins display a “fast acting toxicity” by mouse bioassay (MBA) (Kharrat oz et al., 2011; Hellyer et al., 2008; Bourne et al., 2010; Ara et al., 2011). Cyclic imine toxins are globally distributed; gymnodimines have been detected in New Zealand (Seki et al., 1995), Tunisia (Kharrat et al., 2008), and the United States (Van Wagoner et al., 2011). Spirolides have been reported in Canada (Cembella et al., 2000), Norway (Aasen et al., 2005), France (Amzil et al., 2007), Spain (Gonzalez et al., 2006), Italy (Ciminiello et al., 2006), Chile (Alvarez et al., 2010), the United States, Denmark, and Scotland ret and Brimble, 2010). Pinnatoxins have been found in (Gue Japan and China (Uemura et al., 1995), Australia and New Zealand (Selwood et al., 2010), Canada (McCarron et al., 2012), and Norway (Rundberget et al., 2011), and France (Hess et al., 2013). The evidence that spirolides and gymodimines target muscular and neuronal nAChR subtypes with high affinity (Bourne et al., 2010; Kharrat et al., 2008) has prompted the development of alternative analytical techniques using a receptor-based approach for the detection of spirolides and gymnodimines by fluores~ o et al., cence polarization and chemiluminescence (Vilarin oz et el., 2009; Rodríguez et al., 2011; Otero et al., 2011; Ara 2012). Anatoxin-a regulations and guidance levels are being established around the world. New Zealand has implemented a maximum acceptable level in drinking water of 6 mg/L, and in the United States, several states have recreational water guidance levels between 1 and 90 mg/L (Chorus, 2012; USEPA, 2014). Cyclic imines are not yet internationally regulated but as they display “fast-acting”
47
toxicity by MBA, their presence has resulted in the closures of shellfish aquaculture activities in Foveaux Strait, New Zealand (Seki et al., 1995) and in Arcachon, France (Amzil et al., 2007), leading to substantial economic losses. Recent developments on liquid chromatography mass spectrometry (LC/MS) technology allow fast and automated detection of an array of freshwater and marine toxins with high accuracy, selectivity, sensitivity and reproducibility. However, LC/MS methods will only detect known toxins for which toxin standards are available. Besides, given the sophistication of mass spectrometry-based methods, their application for routine detection of cyanobacterial anatoxins and cyclic imines could be hampered by natural components of freshwater reservoirs or marine samples such as salts, organic and/or inorganic compounds. Although LC/MS will replace MBA as the official method for monitoring internationally regulated marine toxins in Europe (EC, 2011), there is a strong need for additional rapid, simple, and cost-effective methods such as enzymelinked immunosorbent assay (ELISA) and receptor-binding assay for the quantitative analysis of freshwater and marine toxins. Mechanism-based in vitro assays such as the receptor binding assay (RBA) described in this paper, provide tools that fulfill these requirements. It is known that immobilized Torpedo electrocyte membranes would behave as a suitable substrate for detection of nAChRs agonists and antagonists at environmentally relevant concentrations in surface waters (anaoz et al., 2010), and shellfish extracts (cyclic toxins) (Ara oz et al., 2012). Competitive, colorimetric, miimines) (Ara crotiter plate assay validation was conducted against known anatoxin analogues and cyclic imine toxins with known nAChR activity. Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) was used to validate anatoxin assay results. Solid-phase extraction methods were developed to increase assay sensitivity. Assay validation was confirmed by laboratory fortified samples, certified reference materials and environmental samples when available. 2. Materials and methods 2.1. Chemicals and biochemicals Chemicals were of reagent grade and were purchased from Sigma Chemical Company, St. Louis MO, USA, except as indicated. Anatoxin-a and homoanatoxin-a were obtained from NRC, Canada; 13-Desmethyl spirolide C and 12methyl gymnodimine from Marbio, Inc. Pinnatoxin-A and pinnatoxin-G were supplied by U. California, Department of Chemistry. Receptor Binding Assay (cat # 520050) and anatoxin-a solid-phase extraction (SPE) columns (cat. # 520051) were supplied by Abraxis LLC. Polystyrene microtiter plates (12 8) were obtained from Costar USA. 2.2. Purification of Torpedo electrocyte membranes Torpedo electrocyte membranes rich in nAChRs were purified from the electric organ of Torpedo marmorata as described previously (Hill et al., 1991) and diluted as oz et al. (2008). described by Ara
48
F. Rubio et al. / Toxicon 91 (2014) 45e56
Table 1 Summary of receptor binding assay and LC/MS/MS results for anatoxins for select environmental surface waters. Waterbody name
State Known potential anatoxin Total chlorophyll Receptor binding LC/MS/MS producers present?a (mg/L)a assay (mg/L as anatoxin-a) Anatoxin-a (mg/L)a Homoanatoxin-a (mg/L)
Fox River at Montgomery Patriots Park Lake Storey Lake Binder Lake East Okoboji Lake Rock Creek Lake Upper Gar Lake Bilby Ranch Reservoir Albert Lea Lake Loon Lake Okamanpeedan Lake
IL
NDb
ND
0.5
0.28
99% Microcystis sp. Yes, 6% Yes, 2e3% Yes, 28% Yes, 39% Yes, at least 32% Yes, 99% Yes, 0.2%
ND ND 4400 130 190,000 160 34 500 620 190
Contents lists available at ScienceDirect
Toxicon journal homepage: www.elsevier.com/locate/toxicon
Colorimetric microtiter plate receptor-binding assay for the detection of freshwater and marine neurotoxins targeting the nicotinic acetylcholine receptors Fernando Rubio a, *, Lisa Kamp a, Justin Carpino a, Erin Faltin a, Keith Loftin b, mulo Ara oz c, ** c, R o Jordi Molgo a
Abraxis LLC, Warminster, PA, USA U.S. Geological Survey, Organic Geochemistry Research Laboratory, Kansas Water Science Center, Lawrence, KS 66049, USA Centre de recherche CNRS de Gif-sur-Yvette, Institut F ed eratif de Neurobiologie Alfred Fessard FR2118, Laboratoire de Neurobiologie et D eveloppement UPR 3294, Gif sur Yvette 91198, France b c
a r t i c l e i n f o
a b s t r a c t
Article history: Received 21 June 2014 Received in revised form 22 August 2014 Accepted 27 August 2014 Available online 27 September 2014
Anatoxin-a and homoanatoxin-a, produced by cyanobacteria, are agonists of nicotinic acetylcholine receptors (nAChRs). Pinnatoxins, spirolides, and gymnodimines, produced by dinoflagellates, are antagonists of nAChRs. In this study we describe the development and validation of a competitive colorimetric, high throughput functional assay based on the mechanism of action of freshwater and marine toxins against nAChRs. Torpedo electrocyte membranes (rich in muscle-type nAChR) were immobilized and stabilized on the surface of 96-well microtiter plates. Biotinylated a-bungarotoxin (the tracer) and streptavidinhorseradish peroxidase (the detector) enabled the detection and quantitation of anatoxin-a in surface waters and cyclic imine toxins in shellfish extracts that were obtained from different locations across the US. The method compares favorably to LC/MS/MS and provides accurate results for anatoxin-a and cyclic imine toxins monitoring. Study of common constituents at the concentrations normally found in drinking and environmental waters, as well as the tolerance to pH, salt, solvents, organic and inorganic compounds did not significantly affect toxin detection. The assay allowed the simultaneous analysis of up to 25 samples within 3.5 h and it is well suited for on-site or laboratory monitoring of low levels of toxins in drinking, surface, and ground water as well as in shellfish extracts. © 2014 Elsevier Ltd. All rights reserved.
Keywords: Anatoxin-a Nicotinic acetylcholine receptor nAChRs Receptor binding assay Cyclic imines Pinnatoxin
1. Introduction Microalgae are natural components of marine and freshwater environments. Excessive growth of algae becomes a nuisance and in some instances a public health threat through consumption of contaminated fish/mollusks and drinking water, and from the use of water for
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected], oz). Rubio), [email protected] (R. Ara
[email protected]
http://dx.doi.org/10.1016/j.toxicon.2014.08.073 0041-0101/© 2014 Elsevier Ltd. All rights reserved.
(F.
recreational activities. Thus, some members of cyanobacteria, dinoflagellates or diatoms may cause harm through the release of toxins. In continental waters, cyanobacteria harmful algal blooms (CyanoHABs) are being increasingly reported worldwide due to eutrophication and global climate change (Carmichael, 2001; Paerl and Huisman, 2008). Water managers have expressed serious concerns about public health and environmental quality as a result of CyanoHAB toxins in recreational and drinking waters. CyanoHABs occur in freshwater lakes, ponds, rivers, reservoirs, and brackish waters throughout the world. Organisms responsible include an estimated 40 species,
46
F. Rubio et al. / Toxicon 91 (2014) 45e56
primarily belonging to the genera Anabaena, Aphanizomenon, Cylindrospermopsis, Lyngbya, Microcystis, Nostoc, and Oscillatoria (Planktothrix) (Carpenter and Carmichael, 1995). Cyanobacterial toxins (cyanotoxins) include cytotoxins and biotoxins responsible for acute lethal, acute chronic, and sub-chronic poisoning of both wild and domestic animals as well as humans. Cyanotoxins include the neurotoxins anatoxin-a, anatoxin-a(s), and saxitoxin, and the hepatotoxins microcystins, nodularins and cylindrospermopsin (Carmichael, 1997). Marine biotoxins are naturally occurring chemicals produced by a type of microscopic algae known as phytoplankton. These toxins can cause widespread harm to or death of sea life and can also affect humans through multiple routes of exposure (oral, respiratory, skin), making them a growing cause of concern for public health (Landsberg et al., 2005). Marine toxins can accumulate in fish and shellfish and can lead to several types of poisoning: amnesic shellfish poisoning (ASP), diarrhetic shellfish poisoning (DSP), neurologic shellfish poisoning (NSP), and paralytic shellfish poisoning (PSP). More recently, another syndrome, azaspiracid poisoning (AZP) produced by ingestion of azaspiracid toxins (AZA), have been reported after consumption of shellfish (Twiner et al., 2008). The emerging cyclic imine toxins gymnodimines, spirolides, and pinnatoxins have been reported to act as potent antagonists of nicotinic acetylcholine receptors (Kharrat et al., oz et al., 2011; Hu et al., 1995; 2008; Bourne et al., 2010; Ara Uemura et al., 1995). Following a typical toxicity by mouse bioassay, shellfish farms in New Zealand and France have been forced to close due to harmful algal blooms dominated by Karenia selliformis, the producer of gymnodimine A, and by Alexandrium ostenfeldii, the producer of 13desmethyl spirolide C. Pinnatoxins have been isolated from shellfish of the genus Pinna, and were shown to be produced by Vulcanodium rugosum (Rhodes et al., 2011; zan and Chome rat, 2011; Hess et al., 2013). Ne The nicotinic acetylcholine receptors (nAChRs) belong to the family of ligand-gated ion channels. These receptors are widely expressed by various non-neural cell types including muscle, skin, pancreas, lungs and by neural cells in the central and peripheral nervous system. The transmembrane nAChRs are assembled from five subunits that are arranged around a central water-filled pore which opens following acetylcholine binding to its putative binding site. In vertebrate mammals, muscle nAChRs are of two types: embryonic (a12b1gd) or mature receptor type (a12b1d3). In contrast, neuronal nAChRs show more variability in terms of subunit composition: ab nAChR are made up from a combination of a2-a6 and b2-b4 subunits. Muscle nAChR mediate fast synaptic transmission at the neuromuscular junction playing a central role in regulating functions vital for life and escaping from predation such as muscle contraction, and neuronal nAChRs control autonomic and central nervous system function (Albuquerque et al., 2009). In addition, the basic structure of the ligand binding site of nAChRs has been retained with little variability throughout evolution, making it an excellent structural target for a toxin. There are many examples of compounds that target nAChRs that are used as both predatory weapons and defensive measures against predation (Daly, 2005).
Fig. 1. Anatoxins and cylic imine toxins chemical structures.
F. Rubio et al. / Toxicon 91 (2014) 45e56
Anatoxin-a and its homologue homoanatoxin-a are bicyclic secondary amines that are potent agonists of the muscular a12b1d3 and neuronal a4b2 and a7 nAChRs (Spivak et al., 1980; Thomas et al., 1993; Amar et al., 1993). Anatoxin-a kills mice within 2e5 min after intraperitoneal injection; death is preceded by twitching, muscle spasms, paralysis, and respiratory arrest (i.p. mouse LD50: 200 mg kg1) (Devlin et al., 1977). Repetitive stimulation of the muscle-type a12b1d3 nAChR by anatoxin-a stabilizes the desensitized state of the cholinergic receptor that ultimately leads to the blockade of neuromuscular transmission of the depolarizing type (Spivak et al., 1980; Thomas et al., 1993). Cyanobacterial anatoxins are produced by species distributed worldwide from the genera Anabaena, Aphanizomenon, Cylindrospermum, Oscillatoria, Phormidium, Planktothrix, and Raphidiopsis (Wonnacott and Gallagher, 2006). Long term monitoring of water bodies has shown that the composition of the cyanobacteria population and toxin production changes with time and environmental factors (Baker et al., 2002; Havens et al., 2003; Briand et al., 2009). No predictions can be made to determine whether a toxic species will dominate a given cyanobacterial bloom or when it will produce toxins. Toxin occurrence therefore needs to be assessed on a case by case basis. The spirolides, gymnodimines, pinnatoxins, pteriatoxins, prorocentrolides, and spiroprorocentrimines are included in the cyclic imine group of marine phycotoxins due to the presence of an imine ring in their macrocyclic chemical structure (Fig. 1) depicts chemical structures of anatoxins and cyclic imine toxins. These toxins display a “fast acting toxicity” by mouse bioassay (MBA) (Kharrat oz et al., 2011; Hellyer et al., 2008; Bourne et al., 2010; Ara et al., 2011). Cyclic imine toxins are globally distributed; gymnodimines have been detected in New Zealand (Seki et al., 1995), Tunisia (Kharrat et al., 2008), and the United States (Van Wagoner et al., 2011). Spirolides have been reported in Canada (Cembella et al., 2000), Norway (Aasen et al., 2005), France (Amzil et al., 2007), Spain (Gonzalez et al., 2006), Italy (Ciminiello et al., 2006), Chile (Alvarez et al., 2010), the United States, Denmark, and Scotland ret and Brimble, 2010). Pinnatoxins have been found in (Gue Japan and China (Uemura et al., 1995), Australia and New Zealand (Selwood et al., 2010), Canada (McCarron et al., 2012), and Norway (Rundberget et al., 2011), and France (Hess et al., 2013). The evidence that spirolides and gymodimines target muscular and neuronal nAChR subtypes with high affinity (Bourne et al., 2010; Kharrat et al., 2008) has prompted the development of alternative analytical techniques using a receptor-based approach for the detection of spirolides and gymnodimines by fluores~ o et al., cence polarization and chemiluminescence (Vilarin oz et el., 2009; Rodríguez et al., 2011; Otero et al., 2011; Ara 2012). Anatoxin-a regulations and guidance levels are being established around the world. New Zealand has implemented a maximum acceptable level in drinking water of 6 mg/L, and in the United States, several states have recreational water guidance levels between 1 and 90 mg/L (Chorus, 2012; USEPA, 2014). Cyclic imines are not yet internationally regulated but as they display “fast-acting”
47
toxicity by MBA, their presence has resulted in the closures of shellfish aquaculture activities in Foveaux Strait, New Zealand (Seki et al., 1995) and in Arcachon, France (Amzil et al., 2007), leading to substantial economic losses. Recent developments on liquid chromatography mass spectrometry (LC/MS) technology allow fast and automated detection of an array of freshwater and marine toxins with high accuracy, selectivity, sensitivity and reproducibility. However, LC/MS methods will only detect known toxins for which toxin standards are available. Besides, given the sophistication of mass spectrometry-based methods, their application for routine detection of cyanobacterial anatoxins and cyclic imines could be hampered by natural components of freshwater reservoirs or marine samples such as salts, organic and/or inorganic compounds. Although LC/MS will replace MBA as the official method for monitoring internationally regulated marine toxins in Europe (EC, 2011), there is a strong need for additional rapid, simple, and cost-effective methods such as enzymelinked immunosorbent assay (ELISA) and receptor-binding assay for the quantitative analysis of freshwater and marine toxins. Mechanism-based in vitro assays such as the receptor binding assay (RBA) described in this paper, provide tools that fulfill these requirements. It is known that immobilized Torpedo electrocyte membranes would behave as a suitable substrate for detection of nAChRs agonists and antagonists at environmentally relevant concentrations in surface waters (anaoz et al., 2010), and shellfish extracts (cyclic toxins) (Ara oz et al., 2012). Competitive, colorimetric, miimines) (Ara crotiter plate assay validation was conducted against known anatoxin analogues and cyclic imine toxins with known nAChR activity. Liquid chromatography triple quadrupole mass spectrometry (LC/MS/MS) was used to validate anatoxin assay results. Solid-phase extraction methods were developed to increase assay sensitivity. Assay validation was confirmed by laboratory fortified samples, certified reference materials and environmental samples when available. 2. Materials and methods 2.1. Chemicals and biochemicals Chemicals were of reagent grade and were purchased from Sigma Chemical Company, St. Louis MO, USA, except as indicated. Anatoxin-a and homoanatoxin-a were obtained from NRC, Canada; 13-Desmethyl spirolide C and 12methyl gymnodimine from Marbio, Inc. Pinnatoxin-A and pinnatoxin-G were supplied by U. California, Department of Chemistry. Receptor Binding Assay (cat # 520050) and anatoxin-a solid-phase extraction (SPE) columns (cat. # 520051) were supplied by Abraxis LLC. Polystyrene microtiter plates (12 8) were obtained from Costar USA. 2.2. Purification of Torpedo electrocyte membranes Torpedo electrocyte membranes rich in nAChRs were purified from the electric organ of Torpedo marmorata as described previously (Hill et al., 1991) and diluted as oz et al. (2008). described by Ara
48
F. Rubio et al. / Toxicon 91 (2014) 45e56
Table 1 Summary of receptor binding assay and LC/MS/MS results for anatoxins for select environmental surface waters. Waterbody name
State Known potential anatoxin Total chlorophyll Receptor binding LC/MS/MS producers present?a (mg/L)a assay (mg/L as anatoxin-a) Anatoxin-a (mg/L)a Homoanatoxin-a (mg/L)
Fox River at Montgomery Patriots Park Lake Storey Lake Binder Lake East Okoboji Lake Rock Creek Lake Upper Gar Lake Bilby Ranch Reservoir Albert Lea Lake Loon Lake Okamanpeedan Lake
IL
NDb
ND
0.5
0.28
99% Microcystis sp. Yes, 6% Yes, 2e3% Yes, 28% Yes, 39% Yes, at least 32% Yes, 99% Yes, 0.2%
ND ND 4400 130 190,000 160 34 500 620 190
