Regulation of cytochrome P450 expression in a novel liver cell line from zebrafish (Brachydanio rerio).
The expression and induction of cytochrome P450 by 2,3,7,8-tetrachlordibenzo-p-dioxin (TCDD) and beta-naphthoflavone (BNF) in a new liver cell line from adult zebrafish (Brachydanio rerio) were studied. Subcellular fractions from control, BNF- or TCDD-treated cells did not show detectable bands in immunoblots probed with antibodies to the constitutive forms of trout P450 (LMC1, LMC2, LMC3, LMC4, and LMC5), suggesting that either zebrafish liver cells lack P450s closely related to those constitutively expressed in trout or that the concentrations of the orthologous P450s were too low to be detected. However, upon exposure to TCDD, the cells expressed a major immunoreactive 54-kDa protein and a minor 50-kDa protein recognized by antibodies to rainbow trout P4501A1. These immunoreactive proteins were observed in microsomal and mitochondrial fractions of TCDD-treated cells but were not detected in cell cultures treated with dimethyl sulfoxide (DMSO) (vehicle control) or BNF. The activities of ethoxyresorufin O-deethylase (EROD) and 7,12-dimethylbenzanthracene (DMBA) hydroxylase were markedly increased by TCDD but not by BNF in this cell line. EROD activity was more sensitive than DMBA hydroxylase activity of TCDD-treated liver cells to diagnostic inhibitors such as alpha-naphthoflavone and anti-trout P4501A1 IgG. The TCDD-treated cells converted DMBA to various metabolites, one of which is the putative proximate carcinogen, DMBA-3,4-diol. These results suggest that TCDD, but not BNF, induces one or possibly two forms of P450 immunochemically and functionally related to trout P4501A1, in cultured zebrafish liver cells.
Miranda
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CL
Department of Agricultural Chemistry, Oregon State University, Corvallis 97331-7307.
Collodi
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Zhao
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Barnes
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Buhler
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D015127:9,10-Dimethyl-1,2-benzanthracene / Q000378:metabolism; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism; D001571:Benzoflavones / Q000494:pharmacology; D015153:Blotting, Western; D002460:Cell Line; D019363:Cytochrome P-450 CYP1A1; D003577:Cytochrome P-450 Enzyme System / Q000276:immunology / Q000378:metabolism*; D004790:Enzyme Induction / Q000187:drug effects; D008099:Liver / Q000201:enzymology*; D010088:Oxidoreductases / Q000378:metabolism; D000072317:Polychlorinated Dibenzodioxins / Q000494:pharmacology; D015027:Zebrafish / Q000378:metabolism*; D019324:beta-Naphthoflavone
D001571:Benzoflavones; D000072317:Polychlorinated Dibenzodioxins; D015127:9,10-Dimethyl-1,2-benzanthracene; D019324:beta-Naphthoflavone; D003577:Cytochrome P-450 Enzyme System; D010088:Oxidoreductases; C037450:7,12-dimethylbenz(a)anthracene hydroxylase; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1