Solution Reactivity of Brevetoxins As Monitored by Electrospray

Measurement of reaction rate constants indicates the following order of reactivity under acidic conditions: Btx-1 > Btx-2 > Btx-9. Under basic conditi...
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Chem. Res. Toxicol. 1999, 12, 1268-1277

Solution Reactivity of Brevetoxins As Monitored by Electrospray Ionization Mass Spectrometry and Implications for Detoxification Yousheng Hua and Richard B. Cole* Department of Chemistry, University of New Orleans, New Orleans, Louisiana 70148 Received May 17, 1999

The reactivities of brevetoxin compounds in acid and base and under oxidizing conditions were studied using electrospray ionization mass spectrometry (ESI-MS) to monitor reaction products. Brevetoxins are determined to be unstable in acidic and basic solutions. Under acidic conditions, brevetoxins containing an aldehyde functional group in the terminal “tail” side chain are easily converted to acetal structures, while “head” side lactone ring opening proceeds more slowly. Measurement of reaction rate constants indicates the following order of reactivity under acidic conditions: Btx-1 > Btx-2 > Btx-9. Under basic conditions, hydroxide ion attack at the head portion leads to lactone ring opening. Base hydrolysis (0.01 N NaOH in 50:50 methanol/water) goes to completion in 120 min for Btx-2 and Btx-9, but Btx-1 did not react to completion. Both acid and base hydrolyses can lead to reversible lactone ring opening, but base hydrolysis proceeds faster than acid hydrolysis under comparable conditions. Acid treatment is not an effective method for detoxifying brevetoxins. Base treatment can open the lactone ring (type B brevetoxins proceed faster than type A brevetoxins), leading to a product that is reportedly nontoxic, but the reaction is reversible. Brevetoxins are shown to be readily oxidized by permanganate in an irreversible and relatively fast reaction, likely through addition to double bonds followed by bond cleavage, suggesting that it is a viable method for detoxification.

Introduction Brevetoxins are natural lipophilic polyether neurotoxins that are synthesized and maintained in a single-cell dinoflagellate, Gymnodinium breve (1). This algal marine organism can grow rapidly under appropriate conditions of water depth, nitrate concentration, and salinity (2, 3), imposing an impressive phenomenon termed a “red tide” bloom or outbreak. The toxicity afforded by the brevetoxin content in these blooms has caused massive fish kills, shellfish poisoning (4-6), human respiratory irritation, human intoxication, and adverse economic effects on the tourism and fishery industries (7-10). The physiological effects of brevetoxins are mainly characterized as neurotoxic symptoms expressed in fish, animals, and humans (11). Brevetoxins can induce central depression of respiratory and cardiac functions, spontaneous muscular contractions, twitching, and rhinorrhea (12). Since the first complete structural identification work reported in 1981 (13), nine brevetoxins produced by the dinoflagellate G. breve have been fully structurally elucidated (11, 14, 15). They can be divided into either brevetoxin A (containing a 10-cycle polyether ring backbone) or brevetoxin B types (containing an 11-cycle polyether ring backbone) (see Figure 1). Recently, brevetoxin metabolites isolated from greenshell mussels have been structurally identified. Derived from type B brevetoxins, these metabolites exhibit side chain modifications and/or D-ring opening (5, 16, 17). It is known that brevetoxins possess a strong binding tendency toward a specific receptor site inside the volt* To whom correspondence should be addressed.

Figure 1. General structures of the two main types of brevetoxins.

age-sensitive sodium channels (VSSC) of cellular membranes (15, 18, 19). The effects of this binding include the changing of the membrane potential toward more

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Solution Reactivity of Brevetoxins

negative values (depolarizing the membrane, while the normal potential inside the cell wall is between -90 and -40 mV), and the prolonging of the time during which the sodium channel remains activated (19, 20). The membrane potential change will increase the sodium ion flux and possibly increase the rate of neurotransmitter release in response to nerve stimulation. Over time, cells are no longer able to maintain proper potentials, causing a series of symptoms such as hypotension, temperature reversal, and arrhythmias (21). The brevetoxin receptor site has been numbered as site 5 in the VSSC, and this site is not a receptor for other natural toxins such as tetrodotoxin, saxitoxin, batrachotoxin, sea anemone toxin, and scorpion toxins. However, ciguatoxin is the only toxin known to date that also binds to site 5 of the VSSC (15). Recent work has elucidated more details about this site on the genetic level. Investigations show that the binding site is located on a 260 kDa R-subunit glycoprotein of the VSSC. The VSSC also contains two other subunits, i.e., a 36 kDa β1-subunit and a 33 kDa β2-subunit (22). The binding process has been described as a rather complex association and dissociation process that may require several hours to reach equilibrium (23). It is hypothesized that brevetoxin binding to site 5 stabilizes the open conformation of the VSSC, which in turn shifts the channel activation voltage and inhibits the inactivation function of the channel (15). More studies are necessary to fully characterize the structure and the basic neurological functions of the sodium channel, and brevetoxins will continue to serve as probes in the elucidation of these neurological processes. As a basic property affecting long-term toxicity, brevetoxin stability is of substantial interest. Brevetoxins have been described as air-sensitive compounds (24), and brevetoxins that are sold commercially are sometimes stored in vacuum vials. Certain literature reports have indicated that Btx-1 is unstable in alcohol (24) and Btx-3 is unstable in chloroform (25), but other reports state that Btx-2 and Btx-3 are stable for months in methanol, acetone, and chloroform (7, 15). Type B brevetoxins are reported to be more stable than type A brevetoxins (15). Btx-2 and Btx-3 were reported to be unstable at extreme pHs, e.g., pH >10 or 50 ppm) can detoxify brevetoxins. It was presumed that detoxification of brevetoxins was achieved through oxidative addition and cleavage at double bonds (15). We further examined this detoxification approach by con-

ducting brevetoxin oxidation reactions and monitoring the products using ESI-MS. Potassium permanganate (KMnO4) was chosen as the oxidizing agent because of its oxidation strength, availability, and affordability. The results of oxidation experiments with Btx-9 employing KMnO4 (see Experimental Procedures for conditions) are shown in Figure 9. Figure 9a displays the ESI mass spectrum obtained from the reaction of 190 µL of 2.1 × 10-5 M Btx-9 in methanol, 200 µL of 0.4% TFA in methanol, and 400 µL of 10-5 M KMnO4 in water. The molar ratio of KMnO4 to Btx-9 is about 1:1. The peak at m/z 922 ([Btx-9 + Na]+, Figure 9a) represents unreacted Btx-9. The peak at m/z 956 (assigned as Btx-9D) is proposed to be the product of double bond oxidation to the 1,2-diol, while the peak at m/z 939 is assigned as [Btx-9 + Btx-9D + 2Na]2+. The peak at m/z 988 is proposed to be another oxidation product (Btx-9LD) formed by double bond oxidation and lactone ring opening with methanol addition (i.e., m/z 956 + 32). Figure 9b is obtained from the reaction of 200 µL of 2.1 × 10-5 M Btx-9 in methanol, 200 µL of 0.4% TFA in water, and 400 µL of 1.0 × 10-3 M KMnO4 in water. Here, the molar ratio of KMnO4 to Btx-9 is increased to about 100:1 (a 100-fold increase in oxidant relative to that from Figure 9a). Under these conditions, virtually all Btx-9 becomes oxidized and decomposes to smaller molecules, as only barely detectable peaks (e.g., at m/z 988, Figure 9b) remain in the region of the molecular ion. Brevetoxin Sample Storage. From the reaction modes identified above, brevetoxins show instability in acidic methanol solutions and in basic aqueous methanol solutions. Brevetoxin (Btx-1, -2, and -9) reactivity was also examined in an acidic aqueous methanol solution (0.025 M TFA in 1:1 methanol/water), and the results (not shown) indicate a much slower decrease in the parent brevetoxin ion abundances (more than 5 times slower for Btx-2), compared to acidic methanol solutions devoid of water. This result is in accordance with the proposed mechanism of aldehyde conversion to the dimethyl acetal. When reaction rates for acids and bases

Solution Reactivity of Brevetoxins

dissolved in similar concentrations in the same solvent systems were compared, for all brevetoxins, the base reactions (k ranging from 0.078 to 0.11 min-1, 298 K) were shown to proceed much faster than acid reactions. In neutral methanol or aqueous methanol solutions, our experience has been that brevetoxin-1, brevetoxin2, and brevetoxin-3 solutions are stable for months when refrigerated (∼269 K). When the lactone and aldehyde reaction schemes are considered, a neutral solution using aprotic solvents such as acetonitrile or acetone would be predicted to be even less reactive than protic solvents such as water or methanol for storage of brevetoxin standard solutions. Furthermore, brevetoxins are not likely to be air-sensitive. Brevetoxin functional group inspection also indicates that none of the compounds depicted in Figure 1 will be easily oxidized under normal atmospheric conditions. Brevetoxin Detoxification Considerations. The toxicity of brevetoxin compounds is highly related to their molecular conformation. As was postulated by Rein et al. (34), the common pharmacophore (bioactive portion) of brevetoxins is likely a roughly “cigar-shaped” molecular segment (about 30 Å long) bound to its receptor primarily by hydrophobic and nonpolar solvating forces, and possibly aided by hydrogen bonds near the site of the lactone carbonyl. Literature studies (15, 16, 26, 34) indicate that the most important brevetoxin structural segment is the backbone conformation. If the middle range backbone structure is broken (16, 34), or the head-side lactone ring is opened (26), the binding ability and toxicity will be substantially reduced. On the other hand, the chemical integrity of the side chain on the terminal ring of either the type A or type B backbone is not essential for binding activity and toxicity (5, 15, 17, 25). Using these literature guidelines, we can predict the efficiency of brevetoxin detoxification using acids, bases, and oxidants. As discussed above, under acidic conditions (e.g., 0.025 M TFA in methanol), brevetoxins containing a “tail-side” aldehyde group will convert to dimethyl acetal derivatives at relatively fast rates. But because this reaction only changes the structure of the terminal side chain, the toxicity is not expected to be altered significantly (15). The other principal reaction site for all brevetoxins is the head-side lactone ring, which will undergo a relatively slow and reversible methyl esterification process. This head-side lactone ring opening is purported to change brevetoxins to nontoxic derivatives (26), but with a slow reaction rate resulting in a low yield in methanol or aqueous methanol solutions, suggesting that acid treatment is not an efficient way to detoxify brevetoxins. On the other hand, exposure to acid will not greatly affect biological studies involving brevetoxins (e.g., investigations of Na+ channel binding and toxicity). If brevetoxins are treated with 0.01 M NaOH in 50% MeOH/H2O solutions, the head-side lactone ring will be open virtually quantitatively within 60 min for brevetoxin-2 and brevetoxin-9. But the lactone ring of brevetoxin-1 has been observed to open in a much smaller proportion (Figure 8), possibly due to the decreased reactivity of the five-membered ring as compared with that of the six-membered ring (37). At elevated base concentrations, the lactone ring will surely open more quickly. As mentioned in the literature (4), a 10 min exposure to 0.1 N NaOH will completely remove ichthyotoxicity of brevetoxin-2 and brevetoxin-3 (both type B brevetoxins). But for brevetoxin-1 (a type A brevetoxin),

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Figure 10. ESI mass spectra of a Btx-9 solution that has undergone (a) base treatment; and (b) acid treatment following initial base treatment. Base treatment condition of 5.2 × 10-5 M Btx-9 in 0.01 M NaOH with 1:1 methanol/water incubated at room temperature (25 °C) for 60 min. Subsequent acid treatment conditions consisted of adding 2 portions of the basetreated solution and 1 portion of 10% TFA for 60 min, and then 7 portions of methanol.

it is more difficult to quantitatively open the fivemembered ring lactone, so detoxification of type A brevetoxins using base treatment is more difficult. Figure 10, however, provides evidence that lactone ring opening on brevetoxins is a reversible process. In Figure 10a, Btx-9 incubated in 0.01 M NaOH (in 1:1 methanol/ water) has undergone complete base hydrolysis (no remaining [Btx-9 + Na]+, at m/z 922) to give Btx-9B as the predominant decomposition product ([Btx-9B + Na]+, m/z 962). Subsequently, trifluoroacetic acid and methanol were added to the basic solution and solution pH was adjusted from 12 to 1.5. The ESI mass spectrum of the resultant acidic solution is shown in Figure 10b with the apparent reappearance of Btx-9 ([Btx-9 + Na]+, m/z 922), representing Btx-9B conversion back to Btx-9. Another peak at m/z 975 (Figure 10b) is assigned as a salt cluster of the form (CF3COONa)nNa+, where n ) 7; other peaks in this series (n ) 1-8) were also observed (not shown) (39). These experiments demonstrate that the base hydrolysis product, Btx-9B, can largely revert back to the toxic brevetoxin-9 form upon re-acidification. From this reversible reaction, we can classify base treatment as a conditional detoxification method, i.e., not a permanent detoxification method. On the other hand, it is known that double bond oxidation reactions are irreversible reactions. Because there are two double bonds in the type A and type B brevetoxin backbones, irreversible double bond cleavage by oxidants such as KMnO4 and aqueous chlorine is quite certain to effectively decompose the backbone, and thereby irreversibly detoxify brevetoxin molecules (7, 12). Because strong oxidants will normally oxidize many organic compounds, especially those containing double bonds,

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conditions for detoxification using oxidants will largely depend on the matrix composition. High oxidant concentrations will usually be required for solution matrixes containing a high organic content. Oxidation methods can thus be more reliable than base treatment for brevetoxin detoxification. KMnO4 oxidation goes very fast, and the reaction can further be used to decontaminate apparatuses exposed to brevetoxins by rinsing with a dilute (e.g., 10-3 M) KMnO4 solution.

Conclusion Brevetoxins are determined to be reactive in acidic and basic solutions. In acidic solutions, those containing an aldehyde functional group on the terminal side chain are easily converted to dimethyl acetal structures, while acid reaction to form the methyl ester at the head-side lactone ring proceeds slowly. The reactivity of brevetoxins to acid attack decreases in the following order: Btx-1 > Btx-2 > Btx-9. Under basic conditions, head-side lactone ring opening initiated by hydroxide ion attack proceeds to completion in 120 min for Btx-2 and Btx-9, but that for Btx-1 never reached completion. Base hydrolysis proceeds faster than acid hydrolysis for comparable acidic or basic conditions. However, these acid and base hydrolyses can be reversible reactions, and thus, they may not be reliable for detoxification purposes. Brevetoxins are easily oxidized by KMnO4, through double bond addition and then cleavage. The brevetoxin oxidation treatment is an irreversible process and proceeds relatively fast, so it can be a good means of brevetoxin detoxification.

Acknowledgment. Financial support for this research was provided by the Louisiana Board of Regents Support Fund through Grant LEQSF(1998-01)-RD-B-17.

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