ToxSci Advance Access originally published online on October 12, 2005
Toxicological Sciences 2006 89(1):57-65; doi:10.1093/toxsci/kfj013
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Published by Oxford University Press 2005.
Characterization of In Vitro Oxidative and Conjugative Metabolic Pathways for Brevetoxin (PbTx-2)
* Marine Biotoxins Program, Center for Coastal Environmental Health and Biomedical Research, NOAA/National Ocean Service, 219 Fort Johnson Road, Charleston, South Carolina 29412; and Faculty of Science at Sohag, South Valley University, Egypt
1 To whom correspondence should be addressed at Coastal Research Branch, Center for Coastal Environmental Health and Biomolecular Research, NOAA-National Ocean Service, 219 Fort Johnson Road, Charleston SC 29412. Fax: 843-762-8700. E-mail: john.ramsdell{at}noaa.gov.
Received August 22, 2005; accepted September 30, 2005
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ABSTRACT |
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 TOP ABSTRACT IntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brevetoxins are potent marine toxins produced by the dinoflagellate Karenia brevis, the causative organism of Florida red tides. An in vitro metabolism of PbTx-2 was performed using purified cDNA-expressed rat liver cytochrome P-450 (CYP) enzymes and freshly isolated rat hepatocytes. The metabolic activities of six CYP enzymes, CYP1A2, CYP2A2, CYP2C11, CYP2D1, CYP2E1, and CYP3A1, were examined by incubation with PbTx-2 for up to 4 h in the presence of a NADPH-generating system. Further identification of the metabolites produced by CYP1A2 and CYP3A1 was preformed using high performance liquid chromatography-mass spectrometry (LC/MS). Both CYP1A2 and CYP3A1 metabolized PbTx-2 to PbTx-3 (MH+: m/z 897), PbTx-9 (MH+: m/z 899), and a newly recorded diol brevetoxin-2 metabolite (MH+: m/z 929). CYP3A1 also produced a considerably higher amount of BTX-B5 (MH+: m/z 911). Subsequent incubation of PbTx-2 with rat hepatocytes produced additional phase 1 metabolites of MH+: m/z 911, 913, 915, 917, and 931, indicating a CYP-catalyzed epoxidation at H-ring (C27,C28-double bond) and a subsequent A-ring hydrolysis of PbTx-2 metabolic products. A conjugation metabolism was identified by the production of a glutathione-brevetoxin conjugate (MH+: m/z 1222) and a cysteine-brevetoxin conjugate (MH+: m/z 1018). Structures of the new metabolites are postulated, and a likely CYP-catalyzed metabolism pathway of PbTx-2 metabolism are discussed.
Key Words: Karenia brevis; red tide bloom; brevetoxin metabolism; in vitro; cytochrome P450; CYP; conjugation.
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Introduction |
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TOPABSTRACTIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Brevetoxins are a family of naturally occurring trans-fused cyclic polyether compounds produced by the marine dinoflagellate Karenia brevis (Davis, 1948
). Brevetoxins are toxic to marine animals and ultimately to humans. Brevetoxins are also associated with fish and bird mortality, and toxin levels rapidly accumulate and persist in herbivorous fish exposed to K. brevis (Quick and Henderson, 1974; Woofter et al., 2005). Brevetoxins are notably toxic to marine mammals and affect dolphins via consumption of herbivorous fish and manatees via consumption of K. brevis cells associated with seagrass (Flewelling et al., 2005). In humans brevetoxins are the cause of neurotoxic shellfish poisoning (NSP) syndrome (McFarren et al., 1965), and they also produce respiratory and eye irritation via airborne exposure (Woodcock, 1948; Pierce, 1986).
K. brevis produces several brevetoxins that are grouped according to their backbone structures into A- or B- types (Lin et al., 1981; Shimizu et al., 1986). Principal A-types are PbTx-1 and –7, and principal B-types are PbTx-2, –3, and –9 (Poli et al., 1986). PbTx-1 and PbTx-2 are presumed to be the parent molecules containing the precursor backbone of other natural derivatives or metabolites of each type. PbTx-2 is the major natural constituent of a K. brevis toxin extract (approx. 74% of toxin extract); however, it is chiefly metabolized to PbTx-3 and other brevetoxin metabolites in contaminated shellfish (Dickey et al., 1999; Plakas et al., 2002; Wang et al., 2004). Similar metabolites have been described as major contributors to the toxicity in New Zealand shellfish (Nozawa et al., 2003).
Brevetoxin metabolites were also reported in tissues of different animal models used for brevetoxin exposure studies, such as fish (Kennedy et al., 1992; Washburn et al., 1994, 1996; Flewelling et al., 2005), rodents (Poli et al., 1990; Radwan et al., 2005), and Florida red tide exposed dolphin (unpublished observations). Metabolism is an essential step for brevetoxin detoxification; however, certain structural features among brevetoxins, including their natural derivatives or metabolic products, have been found to assign unique physiological activities that modify both potency and the molecular mechanism of action (Jeglitsch et al., 1998; Purkerson et al., 1999; Baden et al., 2005).
In the course of understanding the mechanism of brevetoxin metabolism in mammals using laboratory rats, we recently defined a rapid in vivo metabolism of PbTx-2 (Fig. 1). Within a period of 24 h post-exposure, PbTx-2 was mostly eliminated in urine as a water-soluble cysteine conjugate (MH+: m/z 1018) (Radwan et al., 2005). Initial CYP-mediated reduction and oxidation are likely mechanisms because PbTx-3 (MH+: m/z 897) and a carboxylic brevetoxin BTX-B5 (MH+: m/z 911, named by Ishida et al., 2004) were also detected. Nevertheless, additional transitory metabolites were likely produced, but not readily detected in vivo.
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A potential CYP catalysis and thiol conjugation in brevetoxin metabolism was established about 10 years ago by Washburn and co-workers. They reported that brevetoxin exposure induced CYPs (the oxidation/reduction-mediating enzyme) and also activated glutathione S-transferases (GST) (the conjugation-mediating enzymes) in fish (Washburn et al., 1994
; 1996). Washburn and co-workers reported that PbTx-3 induces CYP1A activity in redfish (Scaienops ocellatus); however, PbTx-2 induces the GST in the striped bass (Morone saxatilis). Those findings suggested an H-ring epoxidation of PbTx-2 (C27,C28-double bond, see Fig. 1), which may produce a metabolite similar to the naturally occurring brevetoxin, PbTx-6. Epoxide formation induces GST and microsomal epoxide hydrolase (Washburn et al., 1996). In this study, in vitro metabolism experiments were performed by incubation of PbTx-2 with purified cDNA-expressed rat CYP enzymes and rat hepatocytes. The goal of this study was to characterize the in vitro metabolism of PbTx-2 and to identify the role of CYP-catalyzed metabolic pathway(s) that may produce "short-lived" intermediate metabolites of potential toxicologic significance.
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MATERIALS AND METHODS |
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TOPABSTRACTIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
cDNA-expressed rat cytochrome P-450 enzymes (CYPs), including CYP1A2, CYP2A1, CYP2D1(+ reductase), CYP2E1, CYP2C11, and CYP3A1 (+ reductase + b5) were purchased from BD Gentest (BD Supersomes Enzymes, BD Bioscience, Woburn, MA). All CYPs used in this study have the same mobility as in their similar rat liver microsomes, which were confirmed by the vendor with a Western immunoblotting technique (www.gentest.com). A microsome preparation using wild-type virus (BD Gentest Catalog No. 456201) was used as a control for native activity. A nicotinamide adenine dinucleotide phosphate hydrogenase (NADPH)–generating system consisting of two reagents, Solution A (NADP+ and glucose-6-phosphate [Glc-6-PO4]) and Solution B (glucose-6-phosphate dehydrogenase [G6PDH]) was purchased separately (BD Bioscience, Woburn, MA). PbTx-2 (MH+: m/z 895, see Fig. 1) purified from Karenia brevis cultures was provided by Dr. Steve Morton (National Ocean Service/NOAA, Charleston, SC). Standards of brevetoxin A-ring hydrolysis products for PbTx-2 (MH+: m/z 913, MH–: m/z 911), PbTx-3 (MH+: m/z 915, MH–: m/z 913), PbTx-9 (MH+: m/z 917, MH–: m/z 915) and BTX-B5 (MH+: m/z 929, MH–: m/z 927) were provided by Dr. Zhihong Wang (National Ocean Service/NOAA, Charleston, SC). All other analytical grade chemicals used in this study were purchased from either Sigma (St. Louis, MO) or Fisher Scientific (Sewanee, GA) unless otherwise stated.
Rat hepatocytes isolation.
Primary rat hepatocytes were isolated from 2-month-old (approx. 250 g body weight) male Sprague-Dawley rats (Charles River Laboratories Inc., Wilmington, MA) by a two-step in situ perfusion procedure (McQueen, 1993) with some modifications. Briefly, liver was perfused with Hanks' balanced salt solution (HBSS) containing 0.5 mM EGTA (ethyleneglycol-bis (P-aminoethyl ether)-N,N,N',N'-tetraacetic acid) and 10 mM HEPES (4–2-hydroxyethyl-1-piperazineethanesulfonic acid), pH 7.4, for 5 min, then perfused with a liver digest medium containing collagenase type IV (0.7 U/ml) (Gibco, Invitrogen Corporation) for 10 min. The liver was removed and dissociated by massaging and filtering through nylon gauze (250 µm). Hepatocytes were isolated by slow-speed centrifugation (
50 x g for 3 min) and resuspended in incubation medium (William's Medium E supplemented with 10% [v/v] FBS, L-glutamine [2 mM], HEPES [10 mM], and gentamicin [50 µg/ml]). Cell viability was assessed by trypan blue (0.2%) uptake. Hepatocyte preparations attaining initial cell viability 
90% were used. Hepatocytes were counted in a hemocytometer in the presence of 0.04% trypan blue in protein-free medium. All protocols involving animals were carried out in compliance with animal care guidelines identified by laws and regulations.
Incubation of PbTx-2 with CYPs.
The standard incubation mixture (final volume 0.5 ml) in a pre-warmed 0.1 M potassium phosphate buffer (pH 7.4) contained 50 pmol CYP and 50 µM PbTx-2 (final mixture contained 1% methanol). After pre-incubation at 37°C for 3 min, reactions were initiated by addition of an NADPH-generating system consisting of 1.3 mM NADP+, 3.3 mM Glc-6-PO4, 0.4 U/ml G6PDH, and 3.3 mM magnesium chloride. Further incubations were carried out for 0 min, 30 min, 60 min, and 240 min at 37°C with constant shaking. The reaction was terminated by the addition of 1 ml of acetonitrile/methanol mixture (1:1) to each incubate. After centrifugation at 900 x g for 10 min, pellets of each reaction were washed, using another 1 ml of acetonitrile/methanol mixture, and the products formed in the pooled supernatant were evaporated to dryness under a stream of nitrogen and stored at –20°C until use in LC/MS (/MS) analysis.
The incubations were carried out in duplicates. Controls were assayed in the same manner using a CYP-inactive microsome preparation as a control for native activity. For those controls established as time 0 min, an NADPH-generating system was added after termination of the reactions.
Incubation of PbTx-2 with rat hepatocytes.
PbTx-2 was prepared in methanol and diluted in the incubation medium so that the final methanol concentration was 0.5% (v/v); it was then pre-incubated at 37°C for 10 min. Aliquots of hepatocyte suspension (250 µl; 1.0 x 106 cells/ml) were transferred into new incubation wells and pre-incubated at 37°C for 10 min. Metabolism was initiated by adding 250 µl of PbTx-2–enriched medium to a well that contained cells. Testosterone (10 µM) was prepared similarly and used as a positive control to assess the hepatocytes activity of CYPs (Specificity: 2A1, 2C11, 3A1/2; Sonderfan et al., 1987). Also, negative control reactions were preformed by incubating PbTx-2 without cells to monitor aqueous stability and/or nonspecific adsorption. The final reaction mixture contained either 10 µM PbTx-2 (for the metabolic stability study) or 50 µM PbTx-2 (for the metabolites identification study) and a cell count of approximate 0.5 x 106 cells/ml. All reactions were performed in duplicate and the reaction products were incubated at 37°C in a 5% CO2 humidified atmosphere. Metabolism reactions were terminated at 30, 60, 120, and 240 min. For further LC/MS (/MS) metabolic product identification, reactions were terminated at 180 min. Reactions were terminated using 3 volumes of 100% acetonitrile; supernatants containing metabolic products were cleaned by centrifugation at 1700 x g for 10 min and then evaporated to dryness. Residues were used for LC/MS (/MS) analysis.
LC/MS (/MS) analyses.
Residues from CYPs or hepatocyte incubations (collected as mentioned above) were dissolved in 50% methanol (contains 0.2% formic acid) for immediate LC/MS (/MS) analysis, as follows:
- PbTx-2 metabolic stability profile. Reaction products of each time point were analyzed using a Shimadzu VP HPLC system equipped with a DuraGel C18 guard cartridge (2.1 x 10 mm, 5µm) (Peeke Scientific, Redwood City, CA). The mobile phase consisted of water (A) and methanol (B) in a binary system, with 0.2% formic acid as an additive. The elution gradient was from 50–95% B for 2 min after a 0.75 min wash at flow rate 0.4 ml/min. Analytes were detected by an Applied Biosystems/MDS Sciex API 3000 mass spectrometer with an interface Turbo ion spray adjusted to 400°C and Q1/Q3 ions [M+H]+ : m/z 895/877 for PbTx-2 (Fig. 1). Metabolic half-life was calculated using all time points unless otherwise noted. The number of time points used was defined by fitting an exponential regression curve to the maximum number of points with linearity (R2) of 0.85.
- Metabolite identification. PbTx-2 metabolites were analyzed with a Shimadzu VP HPLC system equipped with a Hypersil Beta-Basic C18 column (1 x 50 mm, 3µm) (Thermo Electron Co., San Jose, CA). The mobile phase consisted of water (A) and methanol (B) in a binary system, with 0.2% formic acid as an additive. The elution gradient was 45–75% B for 30 min and the flow rate was 0.l ml/min. Analytes were analyzed with a Sciex API QSTAR quadrupole/time-of-flight (Qq-TOF) mass spectrometer. The mass spectrometer detected positive ions over the mass range m/z 300–1400 or 100–1400 amu for intense parent molecule ion scan at an orifice potential of 30 V. In some experiments, a negative ion scan was conducted (from 300–1400 amu) to determine acidic hydrogen. Standards of brevetoxin A-ring hydrolysis products for PbTx-2 (MH+: m/z 913, MH–: m/z 911), PbTx-3 (MH+: m/z 915, MH–: m/z 913), PbTx-9 (MH+: m/z 917, MH–: m/z 915) and BTX-B5 (MH+: m/z 929, MH–: m/z 927) were utilized to identify the presence of similar hydrolytic products of PbTx-2 and its metabolites.
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RESULTS |
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TOPABSTRACTIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In Vitro Metabolic Stability of PbTx-2
Figure 2 shows that all CYPs used in our experiments, except CYP2E1, yield substantial catalysis of PbTx-2 metabolism. Table 1 summarizes the metabolic activities for each of the individual CYPs. CYP3A1 and CYP2D1 caused the highest depletion of PbTx-2 at the 240 min incubation period. For example, CYP3A1 metabolized 88% of PbTx-2 within 240 min at a metabolic rate of 8 pmol/pmol CYP/min and a half-life (t1/2) 63.9 min (Table 1). A subsequent analysis (particularly of 30–120 min incubations) suggests that rat CYP catalysis may fall into two patterns of metabolic activity, one including both CYP1A2 and CYP2C11 and the other including CYP2A2, CYP2D1, and CYP3A1 (Fig. 2). From among these patterns, we selected CYP1A2 and CYP3A1 to pursue identification of metabolic products produced after 240 min of incubation with PbTx-2.
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Incubation of PbTx-2 with rat hepatocytes revealed a metabolic rate of 313 pmol min–1/106 cells, which occurred primarily in the first 60 min of toxin exposure (Fig. 3). This rate of metabolism accounted for an approximate depletion of about 92% of PbTx-2 within 240 min (Table 2).
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Metabolite Identification
Characteristic positive ion products of parent molecule PbTx-2 used in our experiments, including combinations of precursor/product ions (MH+: m/z 895/877/473/455), were next determined (see Fig. 1). Positive ions m/z 455, 473, 753, and/or 779 in B-type brevetoxin were typically observed in further mass fragmentation spectra. Metabolites were defined as they contained all or part of the above characteristic product ions. A metabolic pathway was established according to LC/MS ion products (examined in both positive and negative modes) and the common LCMS/MS fragments shared by a metabolite and its potential precursor molecule(s).
Figure 4 shows the LC/MS analysis of PbTx-2 metabolic products caused by the catalysis of CYP3A1 and CYP1A2. Both CYPs produced PbTx-3 (MH+: m/z 897), PbTx-9 (MH+: m/z 899), and a newly recorded MH+: m/z 929 metabolic product that has a similar positive ion mass, but a different LC/MS(/MS) characteristics than BTX-B5 hydrolytic standard (i.e. retention, fragments and negative ion). CYP3A1 also catalyzed the production of substantial amounts of the carboxylic brevetoxin BTX-B5 (MH+: m/z 911, MH–: m/z 909) with an estimated concentration equal to 64.7 % of the total metabolites produced in PbTx-2 equivalents. Further LC/MS (/MS) fragmentation of each metabolic product was performed. Representatives of positive product ion spectra are included in Figure 5.
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Several additional brevetoxin metabolites were detected in rat hepatocyte in vitro metabolism of PbTx-2 (Fig. 6). Those metabolites are a combination of CYP-catalyzed metabolism (activation phase) and subsequent thiol conjugation (conjugation phase). The activation phase metabolism was characterized by subsequent epoxidation of the H-ring conjugate bond, producing unique metabolic products such as m/z 911 (PbTx-6) and m/z 913. The metabolite m/z 913 has similar positive ion mass of PbTx-2 hydrolytic product standard, but different LC/MS(/MS) characteristics. Hydrolysis of the epoxide products m/z 911 and m/z 913 by the microsomal epoxide hydrolase, likely produces new metabolites, including m/z 929 and m/z 931; the former of which was previously identified using purified CYPs (Fig. 5 and Fig. 6 for MS/MS spectra). The PbTx-2 reduction metabolites PbTx-3 (m/z 897) and PbTx-9 (m/z 899) were hydrolyzed in the presence of rat hepatocytes, producing products of positive ions m/z 915 (MH–: m/z 913) and m/z 917 (MH–: m/z 915) that matched the retention characteristics of the respective A-ring hydrolytic standards. They may result from either a catalytic or a spontaneous hydrolysis reaction. The new metabolites are listed according to their approximate ion mass abundances at 180 min incubation as follows MH+: m/z 917
m/z 915 > m/z 929, 911(PbTx-6) > m/z 931> m/z 913.
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Conjugation phase metabolism was characterized by the detection of two (GSH) PbTx conjugates (MH+: m/z 1204 and m/z 1222). The former is analogous to PbTx-type B-GSH in shellfish (MH+: m/z 1204) (Wang et al., 2004
); whereas the mass of later is consistent with GSH conjugation of PbTx-2 after H-ring epoxidation. A PbTx-cysteine conjugate (MH+: m/z 1018) has been detected that is similar to a cysteine conjugate previously recovered from rat urine extracts (Radwan et al., 2005). The fragmentation patterns of both GSH and cysteine conjugates are shown in Figure 7.
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DISCUSSION |
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TOPABSTRACTIntroductionMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Although purified CYPs have the advantage to investigate metabolism selective to individual CYP enzymes, the advantages of using intact hepatocytes include the existence of using interacting enzyme systems, physiological cofactor concentrations, and three to four times higher enzyme activities than those recognized in microsomal preparations (Wortelboer et al., 1990
; Donato, 1993). Consistent with our results, the PbTx-2 metabolic rate as well as CYP-catalyzed metabolic products varied with respect to the CYP preparation studied, whether purified or natively found in hepatocytes. Also, catalysis of BTX-B5 production by CYP3A1 clearly indicates a unique CYP selective metabolism. High amounts of m/z 929 generated by purified CYP3A1 and CYP1A2 were consistent with CYP-catalyzed H-ring epoxidation of PbTx-2 to an epoxide intermediate – (a likely PbTx-6 congener of a positive ion mass m/z 911), followed by an epoxide hydrolysis that likely was catalyzed by the microsomal epoxide hydrolase enzyme. Epoxidation in the presence of intact hepatocytes, however, results in the formation of detectable amounts of intermediate metabolic products such as m/z 911 (a metabolic product similar to the naturally occurring PbTx-6) and m/z 913 (27,28 epoxy-PbTx-3 (Fig. 5)). However, a subsequent epoxide breakage results in the formation of m/z 929 and m/z 931 metabolites. Hydrolysis of both PbTx-3 and PbTx-9 was identified by the detection of A ring hydrolytics m/z 915 and m/z 917, respectively. The metabolic pathway and the fragmentation patterns of new metabolites are postulated in Figure 8.
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In summary, PbTx-2 is metabolized by two separate pathways; initial CYP-dependent activation and a GST-catalyzed GSH conjugation that was followed by metabolic cleavage of the peptide to leave only the PbTx-cysteinyl residue. The present findings, in conjunction with our previous in vivo metabolism studies (Radwan et al., 2005
), show that PbTx-2 is metabolized predominantly by oxidation of the aldhyde group (C42) forming BTX-B5 and by reduction, but to lesser extent, to hydroxyl forming PbTx-3 (as confirmed by CYP incubations at 240 min). Additional reduction of the C41–C50 double bond yields PbTx-9. A CYP-catalyzed activation was more efficient in producing reactive brevetoxin epoxide precursors via oxidation at the C27–C28 double bond, which occurred to the greatest extent in hepatocytes/toxin incubations. The substantial amounts of m/z 929 produced by CYP3A1 and CYP1A2-catalysis suggest an important role of these enzymes in triggering an initial toxin epoxidation which could be neutralized by the formation of H-ring diol metabolites. Epoxide metabolites are strong electrophilic species that may not last long in biological compartments without binding to cellular micro- or macromolecules, although they can impose an ultimate genotoxic activity. |
ACKNOWLEDGMENTS |
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This work was carried out while F. R. held a National Research Council Associateship Award at the Marine Biotoxins Program, NOAA/NOS/CCEHBR (National Oceanic and Atmospheric Administration/National Ocean Service/Center for Coastal Environmental Health and Biomolecular Research). The authors thank Stephen Eaker and Robert Roberts for the production of purified PbTx-2, Jake Pritchett (IAS, Inc. Burlingame, CA) for MS analyses, Dr. Zhihong Wang for helpful insights, and our anonymous reviewers for valuable remarks. This work was funded by NOAA-NOS.
The National Ocean Service (NOS) does not approve, recommend, or endorse any proprietary product or material mentioned in this publication. No reference shall be made to NOS, or to this publication furnished by NOS, in any advertising or sales promotion which would indicate or imply that NOS approves, recommends, or endorses any proprietary product or proprietary material mentioned herein or which has as its purpose any intent to cause directly or indirectly the advertised product to be used or purchased because of NOS publication.
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