Properties and regional expression of a CYP3A-like protein in channel catfish intestine.
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.
James
Margaret O
MO
Department of Medicinal Chemistry, University of Florida, Gainesville, FL 32610-0485, USA. mojames@ufl.edu
Lou
Zhen
Z
Rowland-Faux
Laura
L
Celander
Malin C
MC
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*
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