title: Domoic acid toxicokinetics in Dungeness crabs: new insights into mechanisms that regulate bioaccumulation.
abstract: Domoic acid (DA) is an excitatory neurotoxic amino acid produced by several marine algal species and is the causative agent of amnesic shellfish poisoning. Profound differences in the toxicokinetics of DA have been identified in a wide variety of shellfish. We characterized the toxicokinetics of DA in Dungeness crabs (Metacarcinus magister) after oral and intravascular dosing (IV) using a variety of doses ranging from 0.1 to 20mg/kg. After a 1mg/kg oral dose, DA disappeared from the foregut within 2h and largely accumulated in the hepatopancreas, with hemolymph and other tissues having 100-1000 times lower concentrations. After IV dosing, hemolymph concentrations of DA were unexpectedly high and toxicokinetic analysis indicated the steady-state volume of distribution (Vss) was 123-197 ml/kg, which is well below the hemolymph volume of 350 ml/kg for crabs. This indicated only limited extravascular distribution of DA was occurring after IV injection, which is surprising considering the capacity of the hepatopancreas to sequester DA after oral dosing. Additional studies measured the partitioning of DA in hepatopancreas cellular and subcellular fractions. The subcellular distribution of DA was primarily associated with the S8 fraction and could be filtered through a 30,000 MW cut-off filter, indicating DA was not appreciably bound to macromolecules. Interestingly, very little (<0.4%) of the total hepatopancreas DA tissue content was associated with the cellular fraction isolated after dissociation and separation from tissue fragments. The in vivo and in vitro results led us to hypothesize that DA uptake and distribution is regulated by crustacean orthologs of ATP-binding cassette (ABC) type transporters. We tested this hypothesis by co-exposing crabs to DA and known inhibitors of ABC transporters (verapamil, cyclosporine A and MK-571) and through in vitro studies using isolated hepatopancreas tissue and mixed cell suspensions prepared from hepatopancreas tissue. The in vivo results were inconclusive in that the toxicokinetics of DA was not consistently altered by co-administration of the inhibitors. Two exceptions were MK-571, which significantly increased the total body clearance of DA and co-administration of verapamil, which significantly increased the hepatopancreas tissue content of DA 24h after IV injection. Isolated pieces of hepatopancreas tissue were able to readily absorb DA from incubation media, but mixed cell suspensions did not. The absorption of DA or lack thereof was largely unaffected by co-incubation with verapamil although cell suspensions appeared to accumulate small quantities of DA in the presence of verapamil. Collectively, the results of this study suggest DA accumulates in the extracellular spaces of the hepatopancreas, such as the tubular lumen. Under natural circumstances, crabs feeding on contaminated shellfish would be expected to readily absorb DA, which is then stored and slowly eliminated in urine. If the DA exposure level exceeds the storage capacity of the tissue (as occurred with the 20mg/kg dose), breakthrough occurs resulting in much higher systemic exposure and potential for DA toxicity.
authors.affiliation: Battelle Pacific NW Division-Marine Sciences Lab, 1529 West Sequim Bay Rd, Sequim, WA 98382, USA. ir_schultz@pnl.gov
authors.forename: Irvin R
authors.initials: IR
authors.lastname: Schultz
authors.forename: Ann
authors.initials: A
authors.lastname: Skillman
authors.forename: Siobhan
authors.initials: S
authors.lastname: Sloan-Evans
authors.forename: Dana
authors.initials: D
authors.lastname: Woodruff
mesh_terms: D000818:Animals; D003386:Brachyura / Q000737:chemistry / Q000187:drug effects* / Q000378:metabolism*; D006458:Hemolymph / Q000737:chemistry; D043143:Hepatopancreas / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D007608:Kainic Acid / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism / Q000633:toxicity; D008387:Marine Toxins / Q000032:analysis / Q000378:metabolism / Q000633:toxicity*; D010599:Pharmacokinetics
chemical_list: D008387:Marine Toxins; C012301:domoic acid; D007608:Kainic Acid
keywords: Disposition; Extracellular; Hepatopancreas; Transporters