Bioaccumulation and Metabolic Effects of the Endocrine Disruptor Methoprene in the Lobster, Homarus americanus1

In vitro Incorporation of 3H D-glucosamine by expiant cultures

Postmolt juvenile lobsters were sacrificed 18 hr after ecdysis (molt stage A2) and the carapace was dissected on ice. After removal of muscle and connective tissue, the carapace was cut into 0.5 × 1 cm strips. About 4 strips were placed in each well of a 6-well plate and covered with 3 ml of expiant culture media: Dulbecco's modified Eagle's medium containing 2 mM L-glutamine, 5% glucose, 0.49 M sodium chloride, 10% fetal bovine serum, and ABAM (penicillin/streptomycin/amphotericin B). After addition of methoprene (final concentration = 25 ppb) to experimental cultures or acetone carrier vehicle to controls, the samples were pre-incubated at 18°C for 4 hours. After addition of ^sup 3^H D-glucosamine (50 µCi/ml; PerkinElmer Life Sciences; Specific activity = 30 Ci/mmol) to each well, the samples were incubated for an additional 16 hr. Afterward, epithelial tissue was removed from the cuticle with a plastic scraper. Samples of shell and epithelial tissue from control or methoprene treated wells were pooled, extracted and processed as described below. In this way, each individual postmolt animal served as its own control.

Tissue homogenization, differential centrifugation and extraction

Each tissue was homogenized with a Potter-Elvejhem homogenizer fitted with a Teflon plunger. Samples were homogenized ten strokes at 50% maximal rpm setting in Homogenization buffer: 20 mM Tris, pH 7.8, containing 0.4 M NaCl, 10 mM MgCl^sub 2^, 0.2 mM phenylmethanesulfonylchloride and protease inhibitor cocktail (Calbiochem). Shell samples were preextracted with 0.5 M EDTA, pH 7, containing PMSF and protease inhibitor cocktail for 12 hr at 18 °C. Homogenates were centrifuged (500 × g for 15 min) to remove cell debris and nuclei. The supernatant was centrifuged at 5,500 × g (15 min) to remove mitochondria and finally at 30,000 × g (45 min) to obtain a crude microsomal fraction ("16Kp") and cytosol (16Ks). The latter fraction was dialyzed against distilled water using 12 kDa cutoff dialysis membranes.

The 5,500 × g and 16Kp pellets were extracted with 8 M urea containing 0.2% dithiothreitol and PMSF. After centrifugation (10,000 × g for 15 min) the urea soluble supernatant was removed and dialyzed. After washing with 10 mM Tris, pH 7.4, the crude microsomes were collected by centrifugation (30,000 × g for 45 min). The microsomal pellet was then extracted with boiling 2% SDS in 10 mM Tris buffer, pH 7.4 for 5 min. The samples were filtered through Nytex screen and the residue was washed sequentially with water, ethanol and acetone. The final shell residue was air dried at room temperature to constant weight. Radioactivity in all fractions was measured and expressed as cpm/mg sample.

SDS-PAGE procedures

Samples of control and pesticide treated fractions were prepared for SDS-PAGE by boiling in 10 mM Tris, pH 7.0 containing 2% SDS, 15% glycerol, 0.001% brom phenolblue and 0.2% dithiothreitol for 3 min. Samples (15 µl) were applied to either precast 4-20% gradient gels (PageR Gold, Cambrex) or 10% acrylamide gels and separated according to Laemmli (1970). At the completion of the experiment, gels were either fixed and stained for total protein with Sypro Ruby or Coomassie Blue or electroblotted to PVDF membranes using a semi-dry technique. The membranes were then blocked with 5% Blotto (Bio-Rad) in Tris buffered saline (TBS) at 4°C overnight. Blots were probed with biotinylated Tomato Lectin (Pierce Chemical Co) followed by detection with Streptavidin-HRP conjugate (Dako). After final washing, the blots were treated with ECL Plus reagent (Amersham) and positive bands detected using Probe Plus X-ray film (Pierce). After exposure for 1-10 min, films were developed using an automated (Xomat) processor.