Ann Skillman

Dose-response relationships and pharmacokinetics of vitellogenin in rainbow trout after intravascular administration of 17α-ethynylestradiol

Abstract A commonly used endpoint in bioassays testing the estrogenicity of chemicals is the induction of the egg yolk precursor vitellogenin (VTG) in male fish. However, relatively little is known about the kinetics of induction and elimination of VTG in fish exposed to xenoestrogens. In this study, we administered graded intra-arterial doses (0.001, 0.1, 1.0 and 10.0 mg/kg) of 17α-ethynylestradiol (EE 2 ) to male rainbow trout via a dorsal aortic cannula which allowed repetitive blood sampling from individual fish for up to 48 days after injection. The plasma concentrations of VTG was quantified using an enzyme-linked immunosorbent assay procedure and the simultaneous concentrations of EE 2 were determined by gas chromatography-mass spectrometry. The pattern of VTG induction was similar for all doses of EE 2 , with a 12-h lag-time before increase from basal levels (0.006–0.008 μg/ml), then increasing sharply to maximum levels within 7–9 days ( C max =0.05, 711, 1521 and 2547 μg/ml VTG for the 0.001, 0.1, 1.0 and 10.0 mg/kg doses, respectively). After induction by EE 2 , VTG declined mono-exponentially with an elimination half-life of 42–49 h. The half-life of VTG increased to 145 h in the 10 mg/kg treated fish. The pharmacokinetics of EE 2 were distinctly nonlinear with substantial increases in the elimination half-life with increasing dose. The plasma concentration–time profiles of EE 2 were influenced by enterohepatic recirculation that caused multiple or secondary peaks in the profiles. In a separate experiment, the pharmacokinetics of purified VTG was characterized after intra-arterial injection in trout. After direct injection of VTG, plasma levels declined tri-exponentially with an apparent steady-state volume of distribution of 837 ml/kg; total body clearance was 31.1 ml/h per kg, and the elimination half-life was 43.7 h.

Immunocompetence of juvenile chinook salmon against Listonella anguillarum following dietary exposure to polycyclic aromatic hydrocarbons.

Juvenile chinook salmon (Oncorhynchus tschawytscha) were fed a mixture of 14 polycyclic aromatic hydrocarbon (PAH) compounds that reflected the PAH composition of salmon stomach contents in an urban estuary of Puget Sound, Washington (USA). Following a 28-d dietary exposure, a standardized Listonella anguillarum challenge model was used to determine whether PAH exposure (16, 64, and 252 mg/kg wet wt feed) causes reduced disease resistance under the conditions examined in this study. To assess innate immunity, five replicate groups of fish per dose were acclimated for one week, exposed to a lethal concentration 60 of bacteria, and monitored for 14 d. In a parallel experiment, the effects of PAH exposure on the acquired immune response were examined by immersion vaccinating fish against L. anguillarum and allowing specific immunity to develop for three weeks prior to challenge. All mortalities were aseptically sampled to confirm L. anguillarum infections. No significant differences in fish length, weight, or coefficient of condition were observed. These controlled laboratory experiments suggest that dietary exposures to an environmentally relevant mixture of PAH compounds do not alter the immunocompetence or growth of juvenile chinook salmon.

Domoic acid toxicokinetics in Dungeness crabs: new insights into mechanisms that regulate bioaccumulation.

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.

Domoic acid excretion in dungeness crabs, razor clams and mussels

Domoic acid (DA) is a neurotoxic amino acid produced by several marine algal species of the Pseudo-nitzschia (PN) genus. We studied the elimination of DA from hemolymph after intravascular (IV) injection in razor clams (Siliqua patula), mussels (Mytilus edulis) and Dungeness crabs (Cancer magister). Crabs were also injected with two other organic acids, dichloroacetic acid (DCAA) and kainic acid (KA). For IV dosing, hemolymph was repetitively sampled and DA concentrations measured by HPLC-UV. Toxicokinetic analysis of DA in crabs suggested most of the injected dose remained within hemolymph compartment with little extravascular distribution. This observation is in sharp contrast to results obtained from clams and mussels which exhibited similarly large apparent volumes of distribution despite large differences in overall clearance. These findings suggest fundamentally different storage and elimination processes are occurring for DA between bivalves and crabs.