Molecular regulation of the induction of cytochrome P-450E in the estuarine fish Fundulus heteroclitus
Kloepper-Sams, Pamela J.
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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.
Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy at the Massachusetts Institute of Technology and the Woods Hole Oceanographic Institution December 1988
Suggested CitationThesis: Kloepper-Sams, Pamela J., "Molecular regulation of the induction of cytochrome P-450E in the estuarine fish Fundulus heteroclitus", 1988-12, DOI:10.1575/1912/5366, https://hdl.handle.net/1912/5366
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