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.

In Vitro Glucuronidation of Caribbean Ciguatoxins in Fish: First Report of Conjugative Ciguatoxin Metabolites.

Ciguatoxins (CTX) are potent marine neurotoxins, which can bioaccumulate in seafood, causing a severe and prevalent human illness known as ciguatera poisoning (CP). Despite the worldwide impact of ciguatera, effective disease management is hindered by a lack of knowledge regarding the movement and biotransformation of CTX congeners in marine food webs, particularly in the Caribbean and Western Atlantic. In this study we investigated the hepatic biotransformation of C-CTX across several fish and mammalian species through a series of in vitro metabolism assays focused on phase I (CYP P450; functionalization) and phase II (UGT; conjugation) reactions. Using liquid chromatography high-resolution mass spectrometry to explore potential C-CTX metabolites, we observed two glucuronide products of C-CTX-1/-2 and provided additional evidence from high-resolution tandem mass spectrometry to support their identification. Chemical reduction experiments confirmed that the metabolites were comprised of four distinct glucuronide products with the sugar attached at two separate sites on C-CTX-1/-2 and excluded the C-56 hydroxyl group as the conjugation site. Glucuronidation is a novel biotransformation pathway not yet reported for CTX or other related polyether phycotoxins, yet its occurrence across all fish species tested suggests that it could be a prevalent and important detoxification mechanism in marine organisms. The absence of glucuronidation observed in this study for both rat and human microsomes suggests that alternate biotransformation pathways may be dominant in higher vertebrates.

Effect of Salinity on DMSP Production in Gambierdiscus belizeanus (Dinophyceae).

Dimethylsulfoniopropionate (DMSP) is produced by many species of marine phytoplankton and has been reported to provide a variety of beneficial functions including osmoregulation. Dinoflagellates are recognized as major DMSP producers; however, accumulation has been shown to be highly variable in this group. We explored the effect of hyposaline transfer in Gambierdiscus belizeanus between ecologically relevant salinities (36 and 31) on DMSP accumulation, Chl a, cell growth, and cell volume, over 12 d. Our results showed that G. belizeanus maintained an intracellular DMSP content of 16.3 pmol cell-1 and concentration of 139 mM in both salinities. Although this intracellular concentration was near the median reported for other dinoflagellates, the cellular content achieved by G. belizeanus was the highest reported of any dinoflagellate thus far, owing mainly to its large size. DMSP levels were not significantly affected by salinity treatment but did change over time during the experiment. Salinity, however, did have a significant effect on the ratio of DMSP:Chl a, suggesting that salinity transfer of G. belizeanus induced a physiological response other than DMSP adjustment. A survey of DMSP content in a variety of Gambierdiscus species and strains revealed relatively high DMSP concentrations (1.0-16.4 pmol cell-1 ) as well as high intrageneric and intraspecific variation. We conclude that, although DMSP may not be involved in long-term (3-12 d) osmoregulation in this species, G. belizeanus and other Gambierdiscus species may be important contributors to DMSP production in tropical benthic microalgal communities due to their large size and high cellular content.

Changes in reproductive biomarkers in an endangered fish species (bonytail chub, Gila elegans) exposed to low levels of organic wastewater compounds in a controlled experiment.

In arid regions of the southwestern United States, municipal wastewater treatment plants commonly discharge treated effluent directly into streams that would otherwise be dry most of the year. A better understanding is needed of how effluent-dependent waters (EDWs) differ from more natural aquatic ecosystems and the ecological effect of low levels of environmentally persistent organic wastewater compounds (OWCs) with distance from the pollutant source. In a controlled experiment, we found 26 compounds common to municipal effluent in treatment raceways all at concentrations <1.0 microg/L. Male bonytail chub (Gila elegans) in tanks containing municipal effluent had significantly lower levels of 11-ketotestosterone (p=0.021) yet higher levels of 17beta-estradiol (p=0.002) and vitellogenin (p=0.036) compared to control male fish. Female bonytail chub in treatment tanks had significantly lower concentrations of 17beta-estradiol than control females (p=0.001). The normally inverse relationship between primary male and female sex hormones, expected in un-impaired fish, was greatly decreased in treatment (r=0.00) versus control (r=-0.66) female fish. We found a similar, but not as significant, trend between treatment (r=-0.45) and control (r=-0.82) male fish. Measures of fish condition showed no significant differences between male or female fish housed in effluent or clean water. Inter-sex condition did not occur and testicular and ovarian cells appeared normal for the respective developmental stage and we observed no morphological alteration in fish. The population-level impacts of these findings are uncertain. Studies examining the long-term, generational and behavioral effects to aquatic organisms chronically exposed to low levels of OWC mixtures are needed.