isosafrole and ellipticine

Induction of cytochrome P450 1A (CYP1A) can be used as a biomarker of exposure to planar halogenated aromatic hydrocarbons (PHAHs). Our objective was to characterize the induction of CYP1A activity and protein in three avian species following in vivo treatment with beta-naphthoflavone (BNF) and/or isosafrole. Alkoxyresorufin-O-dealkylase (alk-ROD) activities of hepatic microsomes from Herring Gulls (Larus argentatus) (HGs), Double-crested Cormorants (Phalacrocorax auritus) (DCCs) and chickens (Gallus domesticus) were measured using ethoxy-, methoxy-, pentoxy- and benzyloxy-resorufin, in the presence and absence of the inhibitors ellipticine or furafylline. Immunoreactivity of microsomal proteins with antibodies to several CYP1A proteins was investigated. CYP1A protein and alk-ROD activities of HGs and DCCs, but not chickens, were induced by isosafrole. Ellipticine was a potent and non-selective inhibitor of alk-ROD activity in all three species, while furafylline inhibition of alk-ROD activities varied among species and treatments. In all three species, BNF induced a protein immunoreactive with monoclonal antibody to CYP1A1 from the marine fish Stenotomus chrysops (scup), but a CYP1A2-like protein was not detected in avian microsomes probed with polyclonal antibodies to mouse CYP1A2. Variations in responses among avian species indicate that CYP1A proteins and substrate specificities should be characterized for each species used in PHAH biomonitoring programs.

Topics: Animals; Antibodies; beta-Naphthoflavone; Catalysis; Chickens; Cytochrome P-450 CYP1A1; Cytochrome P-450 CYP1A2; Cytochrome P-450 CYP1A2 Inhibitors; Ellipticines; Enzyme Induction; Enzyme Inhibitors; Hepatocytes; Immunoblotting; Immunochemistry; Safrole; Species Specificity

Liver microsomes from juvenile trout metabolized DMBA to unknown highly polar metabolites (X) and to DMBA-t-5,6-diol, DMBA-t-8,9-diol, 7-OHM-12-MBA, 7M-12-OHMBA, 2-OH-DMBA, 4-OH-DMBA, and trace amounts of DMBA-t-3,4-diol. Treatment of trout with beta-naphthoflavone (BNF) and isosafrole (ISF) increased the formation of these products except for the hydroxymethyl derivatives of DMBA. The production of DMBA-t-3,4-diol, 2-OH-DMBA, and 4-OH-DMBA was much greater in BNF-induced liver microsomes than that in ISF-induced liver microsomes. In contrast, the yield of DMBA-t-8,9-diol and 7-OHM-12-MBA was greater in ISF-induced microsomes than that in BNF-induced microsomes. Trout CYP1A1 (P450 LM4b) purified from BNF-treated trout catalyzed the formation of the same metabolites generated by BNF-induced microsomes in the presence of added human microsomal EH. The constitutive forms of P450 isolated from untreated trout such as P450s LMC3, LMC4, and LMC5, CYP2M1 (P450 LMC1), and CYP2K1 (P450 LMC2) did not produce any of the DMBA metabolites (except for DMBA-t-8,9-diol by CYP2K1) generated by the trout microsomes. Generation of DMBA-DNA and DMBA-protein adducts in vitro was enhanced by treatment of trout with BNF and by ISF to a lesser extent. Formation of adducts and DMBA diols by BNF-induced liver microsomes and by trout CYP1A1 was completely blocked by the CYP1A inhibitor ellipticine (100 microM). These results suggest that the BNF-inducible trout P450 (CYP1A), not the constitutive P450s, is the major catalyst for the biotransformation of DMBA to metabolites that bind to macromolecules.

Topics: 9,10-Dimethyl-1,2-benzanthracene; Animals; beta-Naphthoflavone; Biotransformation; Clofibrate; Cytochrome P-450 CYP1A1; Cytochrome P-450 Enzyme System; Dexamethasone; DNA Adducts; Drug Synergism; Ellipticines; Enzyme Induction; Enzyme Inhibitors; Isoenzymes; Microsomes, Liver; Oncorhynchus mykiss; Proteins; Safrole