Kloepper-Sams Pamela J.

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Pamela J.

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  • Thesis
    Molecular regulation of the induction of cytochrome P-450E in the estuarine fish Fundulus heteroclitus
    (Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 1988-12) Kloepper-Sams, Pamela J.
    Induction of the aryl hydrocarbon hydroxylase (AHH) P450IA1 occurs in many organisms following exposure to polycyclic aromatic hydrocarbons (PAH). Regulation of induction of P450IA 1 (called P-450E) was examined in the estuarine teleost Fundulus heteroclitus. Antibodies were a primary tool in this work; their specificity and cross reactivity with other species were investigated by immunoblot and catalytic inhibition studies. Scup (Stenotomus chrysops) P-450E protein had been previously purified (Klotz et al., 83) and antibodies generated against it (Park et al., 86a). Monoclonal antibody (MAb) 1-12-3 reacted only with P-450E when tested in immunoblot analysis with five scup P-450 fractions. This and six other MAbs recognized purified P-450E, as well as a single comigrating band in microsomes from β-naphthoflavone- (BNF) treated scup. Polyclonal antibodies (PAb) reacted with P-450E but not with other scup P-450 fractions, and reacted strongly with the BNF-induced, comigrating band. PAb also faintly recognized other microsomal proteins, which were not changed in intensity by xenobiotic treatment. MAb 1-12-3 recognized P-450E induced by 3,4,5,3',4',5'hexachlorobiphenyl and Aroclor treatment and the P-450E orthologue in teleost species including rainbow and brook trout, winter flounder, and Fundulus. P-450E was induced in these fish by BNF and other xenobiotics. P-450E protein content in all fish analyzed correlated with ethoxyresorufin O-deethylase (EROD) activity. EROD was strongly inhibited by MAb 1-12-3 in scup and trout. PAb inhibited AHH and EROD more than 90%, inhibited ethoxycoumarin O-deethylase by about 60%, and did not inhibit aminopyrine N-demethylase, confirming the identity of P-450E as the major inducible EROD and AHH catalyst in these fish. Several MAbs and the PAb recognized purified rat P450IA1 and a BNF-induced, comigrating band in microsomes. MAb 1-12-3 and the PAb also recognized a second band, which comigrates with P450IA2, in microsomes from BNF-treated rats. These results establish the identity of P-450E in scup and other fish and the immunochemical relationship of P-450E with rat P450IA 1. The mode of PAH-type induction was investigated by examining hepatic P-450E content, catalytic activity, and mRNA levels in Fundulus after exposure to a single dose of BNF. In a 20 day experiment, EROD was elevated in BNFtreated animals from Day 4 through Day 20. Increases in immunodetectable P-450E showed the same trend, with low control values and at least a 19-fold increase in the BNF-treated fish. Teleost RNA was used in in vitro translation reactions in the presence of [3H]-leucine. Precipitation of Fundulus liver RNA translation products with anti-P-450E PAb gave no detectable signal from control fish, while the BNF-treated animals showed incorporation of [3H]-leucine in a single 56,000 Mr band. In a 48 hour experiment, EROD and P-450E levels were again coordinately increased in response to BNF treatment, and immunoprecipitation of translation products showed increased signal at all times 6 hours or more post-treatment. eDNA pfPt450-3', which encodes trout P450IA1 (Heilmann et al., 88), yielded unique bands on Southern blots with scup, trout and Fundulus DNA. A Northern blot of RNA from BNF-treated Fundulus showed increases in a single band with time when probed with the trout eDNA. P-450E mRNA increases preceded P-450E protein and EROD increases by about 25 hr, supporting the hypothesis that transcriptional activation is involved in induction of P-450E in fish. In another BNF study, Fundulus P-450E mRNA levels declined rapidly, returning to control levels by 5 days, while protein and activity levels remained elevated for at least 13 days. Thus, P-450E expression also appears to be under other forms of regulatory control. Microsomal protein, P-450E protein, and P-450E heme half-lives (t1/2) were examined in Fundulus during elevated P-450E expression. Decay in incorporated radiolabel ([3H]-leucine and [14C]-ALA) was followed over time. The immunoprecipitation method used for RNA translation products was modified for precipitation of P-450E protein from microsomes. A preliminary experiment indicated that Fundulus microsomes contained no free labeled amino acid at 2 or 23 hr after injection, and that specific radioactivity was higher at 2 than at 23 hr. In a longer experiment, [14C] counts were not detectable in total microsomes, but peak [3H] incorporation into microsomal protein was observed at 1.5 hr after injection, followed by a rapid decrease and stabilization at 30 hr. A calculation of the "rapid" and "slow" phases indicated that microsomal proteins had a "fast" t112 of 9.3 hr and a "slow" t112 of 190 hr. Both [14C] and [3H] were detectable in PAb-precipitated P-450E. Leucine incorporation peaked at 1.5 hr, with a second peak at 190 hr. Using only the early time points, P-450E protein was calculated to have a t1/2 of 32 hr. This was consistent with the 43 hr calculated from the time for P-450E to reach half the induced steady state. [14C] incorporation peaked at 8 hr, indicating a lag between leucine and ALA incorporation into the holoenzyme. The subsequent decline in [14C] was relatively slow, leading to a calculated heme t1/2 of 104 hr. Further studies of heme and apoprotein turnover will be needed to firmly establish the roles of these players in the regulation of P-450E expression. This study addressed, on a molecular level, how xenobiotics in the marine environment elicit a biochemical response - induction of P-450E - in marine teleosts.