Human metabolites of brevetoxin PbTx-2: Identification and confirmation of structure

Abstract

Four metabolites were identified upon incubation of brevetoxin (PbTx-2) with human liver microsomes. Chemical transformation of PbTx-2 confirmed the structures of three known metabolites BTX-B5, PbTx-9 and 41, 43-dihydro-BTX-B5 and a previously unknown metabolite, 41, 43-dihydro-PbTx-2. These metabolites were also observed upon incubation of PbTx-2 with nine human recombinant cytochrome P450s (1A1, 1A2, 2C8, 2C9, 2C19, 2D6, 2E1, 3A4 and 3A5). Cytochrome P450 3A4 produced oxidized metabolites while other CYPs generated the reduced products. Keywordsbrevetoxins; PbTx-2; metabolism; cytochrome P450; human liver microsome Brevetoxins, produced by the "Florida red tide" dinoflagellate Karenia brevis, are a suite of neurotoxins which are responsible for massive fish kills and marine mammal mortalities (Landsberg et al., 2009). In humans, the brevetoxins are responsible for a syndrome known as Neurotoxic Shellfish Poisoning (NSP) which results from the consumption of tainted shellfish (Watkins et al., 2008) as well as respiratory distress in beachgoers upon exposure to aerosolized toxins (Fleming et al., 2005). Brevetoxin PbTx-2 (Fig. 1) is the primary constituent of this suite of toxins.Several studies of brevetoxin excretion and metabolism in mammals have demonstrated that the hepatobiliary system plays a key role in the detoxification and elimination of brevetoxins. Radwan found that in rats, PbTx-2 is rapidly detoxified and excreted in urine as cysteine conjugates and an unidentified oxidized metabolite. (Radwan et al., 2005) On the other hand, when fed to rats, [ 3 H]-PbTx-3, which is unable to form cysteine conjugates, is eliminated in both the urine and feces. (Poli, et al., 1990;Cattet and Geraci, 1993 Conflict of interestThe authors declare that there are no conflicts of interest.Supplementary information: Supplementary data associated with this article including experimental details may be found at XXXXXXX Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. NIH Public Access Author ManuscriptToxicon. Author manuscript; available in PMC 2011 September 15. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript brevetoxin-like activity which was later confirmed by LC-MS to be due to the presence of PbTx-3 and other unidentified metabolites. (Poli et al., 2000) Wang reported the conversion of PbTx-2 to PbTx-3 and the hydrolyzed A-ring lactone by rat liver microsomes. Later Radwan examined the metabolic activities of purified cDNA-expressed rat liver cytochrome P-450 (CYP) enzymes toward PbTx-2. All six CYPs studied (CYP1A2, CYP2A2, CYP2C11, CYP2D1...

Cellular and Transcriptional Responses of Crassostrea gigas Hemocytes Exposed in Vitro to Brevetoxin (PbTx-2)

“…To our knowledge, the metabolizing pathways of brevetoxins have not been described yet in these organisms. On the other hand, PbTx-2 metabolization associated to cytochrome P450 activity was confirmed in human, rat and fish CYPs [ 59 , 60 , 61 ]. Within metabolites described in these studies, some corresponded to metabolites identified in oyster tissues, e.g., PbTx-3 and PbTx-9 [ 62 , 63 ].…”

Contribution of Mass Spectrometry to the Advances in Risk Characterization of Marine Biotoxins: Towards the Characterization of Metabolites Implied in Human Intoxications

“…Guo et al [ 79 ] performed in vitro experiments incubating PbTx-2 with human liver microsomes. Three metabolites previously described, BTX-B5, PbTx-9, 41,43-dihydro-BTX-B5, and an additional unknown metabolite 41,43-dihydro-PbTx-2 were confirmed by LC–MS/MS ( Table 4 ).…”

Identification and cross-species comparison of in vitro phase I brevetoxin (BTX-2) metabolites in northern Gulf of Mexico fish and human liver microsomes by UHPLC-HRMS(/MS)

“…The B-type toxins are considered more prevalent than the A-type toxins, with BTX-2 representing the most prevalent algal congener in K. brevis in both bloom and culture settings ( Twiner et al, 2007 ; Pierce et al, 2008 ; Errera et al, 2010 ; Lekan and Tomas, 2010 ). Aside from algal congeners, much of the present knowledge on BTX derivatives comes from studies with humans and rodents ( Poli et al, 2000 ; Wang et al, 2005 ; Radwan and Ramsdell, 2006 ; Abraham et al, 2008 ; Guo et al, 2010 ), as well as with shellfish ( Morohashi et al, 1995 , 1999 ; Murata et al, 1998 ; Poli et al, 2000 ; Plakas et al, 2002 , 2004 ; Ishida et al, 2004b ; Wang et al, 2004 ). These studies have shown that BTX-2 is rapidly absorbed, distributed, and eliminated primarily as metabolites, indicating that biotransformation plays a major role in the post-exposure processing and excretion of BTX ( Poli et al, 1990 ; Radwan et al, 2005 ).…”

“…At least 70 BTX derivatives have been reported to date, although only about half of these have been structurally characterized ( ANSES, 2021 ; Hort et al, 2021 ). Phase I BTX-2 metabolites result mainly from cytochrome P450 monooxygenase (CYP)-dependent functionalization to several reduction and hydrolysis products (e.g., BTX-3, -9, the carboxylic acid derivative BTX-B5, as well as epoxide, diol, and A-ring hydrolysis products) ( Ishida et al, 2004a ; Radwan and Ramsdell, 2006 ; Guo et al, 2010 ). A variety of phase II metabolites have additionally been identified in mammals and shellfish, including cysteine and cysteine-sulfoxide conjugates arising from glutathione S-transferase (GST) pathways involving intermediate glutathione (GSH) metabolites ( Plakas et al, 2002 , 2004 ; Wang et al, 2004 ; Radwan and Ramsdell, 2006 ).…”