PNNL

Abstract

We characterized the toxicokinetics of trenbolone after water exposure to 500 ng/L in female rainbow trout (Oncorhynchus mykiss; fitted with an intra-arterial catheter and repetitively sampled) and fathead minnows (Pimephales promelas; serially terminated). Separate groups of trout were also administered 0.5 mg/Kg intraarterial (IA) doses of trenbolone. Other experiments measured toxicodynamic effects of trenbolone exposure including changes in circulating levels of estradiol (E2) and follicle stimulating hormone (FSH), E2-d3 pharmacokinetics after IA (trout) or intracoelomic (IC) injection (minnows) and plasma binding of E2. In both trout and minnows, trenbolone was rapidly absorbed from the water and reached peak plasma levels within 8 hrs of exposure. After peak levels were achieved, trenbolone concentrations in trout (66-95 ng/ml) were 2-6 times higher compared to minnows (15-29 ng/ml). This difference appeared to be due to greater plasma binding in trout, where the percentage of unbound trenbolone was 4.5% compared to 18.3% in minnows. In trout, trenbolone had a relatively large steady-state volume of distribution (Vss) of 2.18 ml/g and was rapidly eliminated after IA injection or during depuration after water exposure with a half-life of 14.5 -24 hrs. During water exposure to trenbolone, circulating levels of E2 rapidly decreased in both species to a concentration that was 25-40% of control values by 8-24 hrs of exposure and then remained relatively unchanged for the subsequent six days of exposure. In trout, changes in circulating levels of FSH were also significantly greater after trenbolone exposure relative to controls. In both species, the pharmacokinetics of injected E2-d3 was altered by trenbolone exposure with increased total body clearance and a corresponding decrease in elimination half-life observed. The unbound percentage of E2 in trout plasma was 0.25%, which was similar in pre- or post-vitellogenic female trout. Subsequent incubation with trenbolone caused the unbound percentage to significantly increase to 2.4 % in the pre-vitellogenic trout plasma. iTRAQ based toxicoproteomic studies in minnows exposed to 5, 50 and 500 ng/L trenbolone identified a total of 148 proteins in minnow plasma at > 95% confidence. There were 19 proteins considered down-regulated and 18 up-regulated. The number one ranked protein was fathead minnow vitellogenin (VTG; Q9W6I2_PIMPR), which displayed a dose dependent decrease in its abundance relative to controls. Other down-regulated proteins were fibrinogens, a-2-macroglobulin and transferrin. Up-regulated proteins included amine oxidase, apolipoproteins, parvalbumin, complement system proteins and several uncharacterized proteins. A sex hormone binding globulin (SHBG-a) was modestly increased in the 500 ng/L treatment. The results indicate trenbolone exposure is a highly dynamic process in fish with uptake and tissue equilibrium quickly established leading to both rapid and delayed effects on E2 turnover.