title: Induction of cytochrome P450 family 1 mRNAs and activities in a cell line from the frog Xenopus laevis.
abstract: Cytochrome P450 family 1 (CYP1) includes four subfamilies of enzymes: CYP1A, CYP1B, CYP1C, and CYP1D. In many vertebrates, CYP1A, 1B, and 1C expression is induced by agonists of the aryl hydrocarbon receptor, including toxic contaminants such as chlorinated dioxins, coplanar chlorinated biphenyls, and polynuclear aromatic hydrocarbons. Assessed at the level of mRNA, protein, or enzyme activity, CYP1s (especially CYP1As) represent potent and popular biomarkers of contaminant exposure in aquatic vertebrates. Alkylated resorufins are synthetic substrates used to detect, quantify, and describe catalytic activities of cytochrome P450s. The ability to oxidize specific resorufin-based substrates can distinguish the catalytic activities of individual CYP1s. Xenopus laevis, the African clawed frog, is the most widely employed amphibian model in aquatic toxicology, yet the number, inducibility, and activities of CYP1s have not been systematically characterized in this species. Here we report the cloning of cDNAs encoding two new CYP1 family members, X. laevis CYP1B and CYP1C, along with an integrated assessment of the induction of alkyloxyuresorufin-O-dealkylase (AROD) activities and mRNA expression of four known X. laevis CYP1s: CYP1A6, CYP1A7, CYP1B, and CYP1C. Using XLK-WG, an X. laevis kidney epithelial cell line, we determined that EROD (ethoxyresorufin substrate) and MROD (methoxyresorufin) were both induced 3000- to 5000-fold following 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD) exposure up to 250 nM, while BROD (benzyloxyresorufin) and PROD (pentyloxyresorufin) activity was not detectable regardless of TCDD treatment. TCDD induced CYP1A6 and CYP1A7 mRNAs by 2-3 orders of magnitude, while CYP1B and CYP1C were unchanged. The more potent AHR agonist, FICZ (6-formylindolo[3,2-b]carbazole), induced CYP1B up to 10-fold at concentrations between 0.1 and 250 nM, while CYP1C induction was less than 3-fold. CYP1B mRNA showed the highest constitutive mRNA expression, 5- to 75-fold greater than the other CYP1 transcripts. Taken together, these results suggest that CYP1A6 and CYP1A7 perform the bulk of EROD and MROD activities we observed in these cells. The ability of each X. laevis CYP1 to catalyze oxidation of individual resorufin substrates remains to be determined. Correlating CYP1 mRNA and induced AROD activity is a significant step toward clarifying the biochemical meaning of these biomarkers and the roles of CYP1 enzymes in X. laevis. The cell culture approach represents an important complement to the long standing use of frog embryos and tadpoles in toxicological studies, providing a well suited model system for determining the molecular mechanisms underlying the regulation of these important biomarkers of contaminant exposure.
authors.affiliation: Biology Department, Kenyon College, Gambier, OH 43022, USA.
authors.forename: Daniel V
authors.initials: DV
authors.lastname: Iwamoto
authors.forename: Chad M
authors.initials: CM
authors.lastname: Kurylo
authors.forename: Kelly M
authors.initials: KM
authors.lastname: Schorling
authors.forename: Wade H
authors.initials: WH
authors.lastname: Powell
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D015415:Biomarkers; D002460:Cell Line; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects* / Q000502:physiology*; D010802:Phylogeny; D000072317:Polychlorinated Dibenzodioxins / Q000633:toxicity*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D014982:Xenopus laevis
chemical_list: D015415:Biomarkers; D000072317:Polychlorinated Dibenzodioxins; D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System