The reaction was initiated with NADPH, progressed at 25o for 30 min, and was terminated 

 with 200 uL ice-cold 16 mM CaC12 to aggregate the microsomes. Samples were transferred to 

 test tubes containing dextran-coated charcoal (pellets from 200 uL of 1 percent activated 

 charcoal and 0.5 percent dextran in 10 mM Tris, pH 8.0), vortexed, shaken at 0-5oC for 15 

 min, and centrifuged at 5,000 G. Aliquots (200-uL) of the supernatant were counted in a 

 Packard Tri-Carb scintillation counter. 



Cytochrome P-450E homologue. Immunoblot ( "Western" blot) analyses were accomplished 

 with monoclonal antibody (MAb) 1-12-3 against P-450E, the major PAH-induced form isolated 

 from the marine fish scup (Klotz et «/., 1983). The characterization of this antibody and its 

 specificity in immunoblotting has been described (Park et ah, 1986; Kloepper-Sams et ah, 

 1987). Microsomal samples were incubated in a steaming water bath for 10 min, in 20 mM 

 Tris-HCl, pH 6.8, 1 percent SDS, 13.3 percent glycerol, and 1.7 percent B-mercaptoethanol 

 and bromophenol blue. Proteins were electrophoretically resolved and were transferred onto 

 0.2 urn nitrocellulose paper essentially as described by Towbin et al. (1979). The blots were 

 incubated in 5 percent dry milk in phosphate buffered saline (PBS) for 1 hour at 42°C, and 

 MAb 1-12-3 diluted in PBS for 2 hours at room temperature. They were washed 10 min each 

 in: PBS; 0.5 percent Tween, PBS; and PBS again, and then incubated for 1 hour in PBS with 

 5 ul-mL"l g 0at anti-mouse peroxidase-linked IgG. They were washed again as above, and 

 developed in PPB with HRP Color Developer (BioRad) containing 4-chloro-l-naphthol 

 added in cold methanol and 0.02 percent hydrogen peroxide for 20 to 30 min. Stain 

 deposition was measured by densitometric analysis with a soft laser densitometer (Helena 

 Labs., Inc.). 



All enzyme analyses were done at least in duplicate; some were done up to 10 

 replicates. Enzyme assays as well as spectral and immunoblot analyses of P-450 were 

 repeated for samples giving unusual results or results near the limits of detection. Some 

 samples were analyzed three or more times. Repeated analyses showed less than 15 percent 

 variation in the results for any given sample. 



Positive control samples. Selected individual P. stellatus captured at the BK and SP sites 

 were treated with a known inducer, B-naphthoflavone (BNF). The treatment with BNF 

 was done by R. Spies, and liver microsomes were prepared by WHOI personnel, at the 

 LLNL. Microsomes were prepared from fresh liver, according to methods employed at 

 WHOI. These microsomes were prepared to serve as a positive control, to validate the test 

 methods, and to provide a measure of the degree of response which might be possible in P. 

 stellatus. 



Fish Chemical Analyses 



Residues of chlorinated hydrocarbons in liver were determined at the LLNL using 

 methods similar to those of Ozretich and Schroeder (1986). Briefly, tissues were macerated 

 in pre-cleaned beakers, water was removed by addition of pre-combusted (600°C) anhydrous 

 Na2SC>4, acetonitrile (UV grade) and an internal standard of 4,4'-dibromoocto- 

 fluorobiphenyl was added, and the mixture was homogenized by a high-speed tissue 

 macerator (Polytron) to produce the first extract. The first extract was decanted after 

 settling and the mixture was extracted twice more, with the final extract clarified by 

 centrifugation. The extracts were combined, and made up to 100 mL and cooled overnight at 

 4°C. A 50 mL subsample was removed to gravimetrically determine extracted lipids. A 

 second subsample was removed for isolation, identification, and quantification of aromatic 

 compounds of interest (PCB, DDT, and pesticides). Interfering saturated compounds (e.g., 

 alkanes), remaining lipids, and fatty acids were removed by passing the extract through 

 disposable reverse-phase chromatography columns (Baker) with C|g and NH2 solid-phase 

 absorbents. The concentrated extract was analyzed by GC (Hewlett-Packard, 5880) using a 

 6 ^Ni ECD and a 0.25 mm inside diameter, 30-m fused-silica capillary column internally 

 coated with cross-linked methyl silicone. Chlorinated hydrocarbons of interest were 

 analyzed based on retention times and response factors of authentic external standards 



27 



