title: In vivo and in vitro inhibition of CYP1A-dependent activity in Fundulus heteroclitus by the polynuclear aromatic hydrocarbon fluoranthene.
abstract: Certainpolynuclear aromatic hydrocarbons (PAHs) such as benzo[a]pyrene (BaP) induce CYP1A-dependent enzyme activities. Because PAHs are ubiquitous environmental contaminants, and some are aryl hydrocarbon agonists, CYP1A has been used as a biomarker for PAH exposure. However, PAHs exist in the environment in complex mixtures that may confound biomarker results. In in vitro studies, the PAH fluoranthene (FL) failed to increase or enhance CYP1A1-dependent ethoxyresorufin-O-deethylase (EROD) activity in cells, but rather inhibited activities induced by AhR agonists such as 2,3,7,8-tetrachlorodibenzo-p-dioxin and benzo(k)fluoranthene. In order to determine the in vivo effects of FL on CYP1A and DNA adduct levels, Fundulus heteroclitus were given single ip injections of either BaP (5 mg/kg), BaP + FL (5 mg/kg each), BaP + FL (5 and 50 mg/kg, respectively), FL (5 mg/kg), FL (50 mg/kg), or corn oil control. BaP-treated fish had liver microsome EROD activities significantly higher than controls, whereas FL-treated fish were not different from controls. EROD activities in BaP + FL cotreatments were significantly lower compared to fish treated with BaP alone. When FL was incubated with BaP-induced microsomes, the IC50 for inhibition of EROD activity was 1.4 x 10(-5) M FL. Kinetic studies indicated a significant noncompetitive component to the FL inhibition. When fish were treated with [(14)C]FL, the concentration of radiolabel associated with microsomal preparations was four orders of magnitude lower than the IC50. Therefore, the presence of FL or a FL metabolite was insufficient to account for the inhibition by a kinetic mechanism. In contrast to cell studies, CYP1A immunoreactive protein was significantly decreased in vivo by FL cotreatment, indicating that FL may inhibit EROD activity by down-regulating the CYP1A protein. A covalent interaction of [(14)C]FL with CYP1A was not detected. Total (32)P-postlabeled DNA adducts were not significantly changed by cotreatment of FL and BaP; however, cotreatment with 50 mg/kg FL decreased one adduct and increased another significantly. Because FL and perhaps other inhibitory PAHs, co-occur in the environment with CYP1A inducers, CYP1A-dependent bioassays may cause an underestimation of PAH exposures.
authors.affiliation: Department of Pharmacology and Environmental Toxicology Research Program, University of Mississippi, University, Mississippi 38677-1848, USA. kwillett@olemiss.edu
authors.forename: K L
authors.initials: KL
authors.lastname: Willett
authors.forename: D
authors.initials: D
authors.lastname: Wassenberg
authors.forename: L
authors.initials: L
authors.lastname: Lienesch
authors.forename: W
authors.initials: W
authors.lastname: Reichert
authors.forename: R T
authors.initials: RT
authors.lastname: Di Giulio
mesh_terms: D000818:Animals; D001564:Benzo(a)pyrene / Q000494:pharmacology; D001667:Binding, Competitive / Q000187:drug effects; D019363:Cytochrome P-450 CYP1A1 / Q000378:metabolism*; D018736:DNA Adducts / Q000032:analysis / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005260:Female; D005449:Fluorenes / Q000494:pharmacology*; D023781:Fundulidae / Q000378:metabolism*; D008099:Liver / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D008297:Male; D008862:Microsomes, Liver / Q000187:drug effects / Q000201:enzymology; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D011084:Polycyclic Aromatic Hydrocarbons / Q000494:pharmacology*
chemical_list: D018736:DNA Adducts; D004791:Enzyme Inhibitors; D005449:Fluorenes; D000072317:Polychlorinated Dibenzodioxins; D011084:Polycyclic Aromatic Hydrocarbons; D001564:Benzo(a)pyrene; C007738:fluoranthene; D019363:Cytochrome P-450 CYP1A1; C022921:benzo(k)fluoranthene