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)

Brevetoxins (BTX) are a group of marine neurotoxins produced by the harmful alga Karenia brevis. Numerous studies have shown that BTX are rapidly accumulated and metabolized in shellfish and mammals. However, there are only limited data on BTX metabolism in fish, despite growing evidence that fish serve as vectors for BTX transfer in marine food webs. In this study, we aimed to investigate the in vitro biotransformation of BTX-2, the major constituent of BTX profiles in K. brevis, in several species of northern Gulf of Mexico fish. Metabolism assays were performed using hepatic microsomes prepared in-house as well as commercially available human microsomes for comparison, focusing on phase I reactions mediated by cytochrome P450 monooxygenase (CYP) enzymes. Samples were analyzed by UHPLC-HRMS(/MS) to monitor BTX-2 depletion and characterize BTX metabolites based on MS/MS fragmentation pathways. Our results showed that both fish and human liver microsomes rapidly depleted BTX-2, resulting in a 72–99% reduction within 1 h of incubation. We observed the simultaneous production of 22 metabolites functionalized by reductions, oxidations, and other phase I reactions. We were able to identify the previously described congeners BTX-3 and BTX-B5, and tentatively identified BTX-9, 41,43-dihydro-BTX-2, several A-ring hydrolysis products, as well as several novel metabolites. Our results confirmed that fish are capable of similar BTX biotransformation reactions as reported for shellfish and mammals, but comparison of metabolite formation across the tested species suggested considerable interspecific variation in BTX-2 metabolism potentially leading to divergent BTX profiles. We additionally observed non-enzymatic formation of BTX-2 and BTX-3 glutathione conjugates. Collectively, these findings have important implications for determining the ecotoxicological fate of BTX in marine food webs.