title: Properties and regional expression of a CYP3A-like protein in channel catfish intestine.
abstract: Biotransformation in the intestine may influence the bioavailability and toxicity of ingested xenobiotics. The objective of this study was to examine the expression and catalytic properties of a constitutive cytochrome P450 (CYP) 3A-like protein along the intestine of channel catfish, Ictalurus punctatus. Fish were maintained on commercial chow or nutritionally complete semi-purified diets. Polyclonal antibodies generated against rainbow trout CYP3A proteins reacted strongly with catfish washed intestinal microsomes on Western blots showing a major protein band with MW of 59 kDa. In catfish maintained on a standard chow diet, the expression of this protein was higher in the proximal segment (0.101 +/- 0.031 units/mg protein, mean +/- S.D., n = 4) than in the distal part (0.032 +/- 0.023 units/mg protein). Microsomal testosterone 6beta-hydroxylation activity was monitored as the catalytic indicator of CYP3A, and was higher in proximal than distal catfish intestine (263 +/- 80.3 and 88.6 +/- 15.6 pmol/min/mg protein for proximal and distal, respectively, mean +/- S.D., n = 4). CYP3A protein levels and testosterone 6beta-hydroxylation activities were lower in microsomes from the proximal segment of intestine from catfish maintained on a semi-purified diet, compared with commercial chow, but again the proximal intestine had higher CYP3A and 6beta-hydroxylase activities than distal intestine. Testosterone 6beta-hydroxylase activities in all samples correlated with the CYP3A protein levels, r2 = 0.8. Testosterone 6beta-hydroxylation was inhibited by specific CYP3A inhibitors, ketoconazole (IC50 = 0.02 microM) and erythromycin (IC50 = 41 microM), as well as general CYP inhibitors, metyrapone (IC50 = 2.8 microM) and SKF-525A (IC50 = 25 microM). There was evidence for the involvement of CYP3A in the mono-oxygenation of benzo(a)pyrene and of (-)-benzo(a)pyrene-7,8-dihydrodiol in intestinal microsomes from catfish maintained on the semi-purified diet. Mono-oxygenation of both substrates was increased in a concentration-dependent manner by in vitro addition of alpha-naphthoflavone. Benzo(a)pyrene hydroxylase activities were higher in proximal than in distal intestine; 3.72 +/- 0.77 pmol/min/mg protein, mean +/- S.D., n = 5 and 1.45 +/- 0.42 in these respective segments. The results of this study strongly suggest that CYP3A is important in the first pass metabolism of dietary xenobiotics in untreated fish.
authors.affiliation: Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, USA. mojames@ufl.edu
authors.forename: Margaret O
authors.initials: MO
authors.lastname: James
authors.forename: Zhen
authors.initials: Z
authors.lastname: Lou
authors.forename: Laura
authors.initials: L
authors.lastname: Rowland-Faux
authors.forename: Malin C
authors.initials: MC
authors.lastname: Celander
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D001189:Aryl Hydrocarbon Hydroxylases / Q000378:metabolism*; D001564:Benzo(a)pyrene / Q000378:metabolism; D015153:Blotting, Western; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D004032:Diet; D004101:Dihydroxydihydrobenzopyrenes / Q000378:metabolism; D004917:Erythromycin / Q000494:pharmacology; D007059:Ictaluridae / Q000378:metabolism*; D007413:Intestinal Mucosa / Q000378:metabolism*; D008797:Metyrapone / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*; D011335:Proadifen / Q000494:pharmacology; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015262:Xenobiotics / Q000378:metabolism*
chemical_list: D065607:Cytochrome P-450 Enzyme Inhibitors; D004101:Dihydroxydihydrobenzopyrenes; D015262:Xenobiotics; C017228:benzo(a)pyrene 7,8-dihydrodiol; D001564:Benzo(a)pyrene; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D011335:Proadifen; D006899:Mixed Function Oxygenases; D013250:Steroid Hydroxylases; D001189:Aryl Hydrocarbon Hydroxylases; D051544:Cytochrome P-450 CYP3A; C022657:steroid hormone 6-beta-hydroxylase; D010089:Oxidoreductases, N-Demethylating; D008797:Metyrapone