REGULATION OF CYTOCHROME P 4501 A 1 EXPRESSION IN RAT SMALL INTESTINE

The predominant inducible cytochrome P450 (CYP) in rat small intestine is CYP1A1, which, when induced to elevated levels by xenobiotics or dietary constituents, has the potential to metabolize and consequently reduce the systemic uptake of low concentrations of orally ingested, bioactivatable polycyclic aromatic hydrocarbons and heterocyclic aromatic amines. We investigated the regulation of small intestinal CYP1A1 in an effort to develop its anticancer potential. The time courses of hepatic and intestinal CYP1A1 induction by b-naphthoflavone (BNF) were compared quantitatively at the protein and mRNA levels by immunoblot and competitive RNA-polymerase chain reaction analyses. CYP1A1 mRNA levels in both organs increased sharply and were maximal at ;6 hr and returned to near basal levels by 12 hr after BNF treatment. In contrast, hepatic CYP1A2 mRNA levels increased much more gradually. Small intestinal CYP1A2 mRNA concentrations were insufficient to support translation of detectable protein. Maximal levels of intestinal and hepatic CYP1A1 protein occurred between 12 and 24 hr, and 24 and 48 hr, respectively, after BNF. Intestinal CYP1A1 protein was detectable earlier and for a shorter duration than hepatic CYP1A1. CYP1A1 induction was first detected in crypt cells 3 hr before the appearance of activity in villous cells, and maximal levels of activity were reached in crypt cells 12 to 18 hr before maximal and 1.5-fold (per mg protein) higher responses in villous cells—induction thus occurs in both villous and crypt cells. Previously detected decreases in CYP1A1 inducibility from duodenum to ileum correlated with decreases in immunoblot determined-Ah receptor levels. Intestinal CYP1A1 induction does not involve the glucocorticoid receptor in contrast to hepatic induction. These studies have revealed several novel features of small intestinal CYP1A1 regulation. Gene products of the P450 1 gene superfamily are reasonably well represented in rat small intestinal epithelial cells—the enterocytes. Of the 33 P450 forms in families 1 to 3 identified to date in the rat (1), at least six forms have been demonstrated to be expressed in the small intestine, including CYP1A1, CYP2B1, CYP2C6, CYP2C11, CYP2D, and CYP3A1 (2–4). Enterocytes provide the first metabolic machinery for orally ingested xenobiotics and are the site for xenobiotic first-pass metabolism in the small intestine. Since some of the small intestinal P450s— such as CYP1A1, CYP2B1, and CYP3A1—are inducible (2), in some instances by constituents of a normal diet (5), these P450s can regulate, to variable extents depending on the level of induction of the small intestinal P450s, the metabolic fate of many ingested xenobiotics. In addition, the short life of enterocytes, 2 days in the rat (6), confers a correspondingly short life on small intestinal P450-activated adducts to DNA and other enterocyte macromolecules. A potential benefit is to diminish the carcinogenic consequences of ingested bioactivatable procarcinogens. In the rat small intestine, the most prominent inducible form of P450 is CYP1A1 (4). The inducibility of CYP1A1-related activity in rat small intestine was first reported over 20 years ago (7, 8). This form probably does not occur constitutively, but is inducible by constituents of commonly used rat chow and is, consequently, usually detected in small intestinal preparations in the absence of administration of known inducing agents to the rats (3, 9–11). Inducing agents, such as BNF, however, readily induce small intestinal CYP1A1 by up to 17-fold (2, 4) in rats on a normal chow diet. The induction of CYP1A1 mRNA in rat enterocytes by BNF has also recently been reported (12). Many PAH procarcinogens are metabolized by CYP1A1 and in this process are bioactivated to putative carcinogens (13). The potential of small intestinal cells containing CYP1A1 to reduce the systemic uptake of orally ingested PAHs by bioactivating them is under investigation in this laboratory. PAHs entering the small intestine can be: 1) bioactivated and covalently bound to enterocyte macromolecules and removed by sloughing off the enterocytes within 2 days; 2) metabolized and excreted back into the lumen and eliminated in the feces; and/or3) metabolized and the stable metabolites absorbed systemically. All three possibilities could diminish the carcinogenicity of orally ingested carcinogens, in the latter case because the small intestinal PAH metabolites are unlikely to undergo further metabolic bioactivation in what would otherwise be target organs. In an effort to incorporate the potential of the inducible small intestinal CYP1A1 to metabolize PAHs into cancer chemoprevention strategies, we have investigated aspects of its regulation, and these are reported herein. Materials and Methods BNF, DEX, peroxidase-conjugated rabbit anti-goat IgG, and peroxidaseconjugated goat anti-rabbit IgG were from Sigma Chemical Co. (St. Louis, MO). The BCA protein assay kit was obtained from Pierce Chemical Co. (Rockford, IL). The ECL kit was purchased from Amersham (Arlington Heights, IL). The TRI reagent was from Molecular Research Center, Inc. This research was funded by Grant ES06256 from the National Institutes of Health, Public Health Service. 1 Abbreviations used are: P450, cytochrome P450; CYP, cytochrome P450; BNF, b-naphthoflavone; PAH, polycyclic aromatic hydrocarbon; DEX, dexamethasone; IgG, immunoglobulin G; BCA, bicinchoninic acid; ECL, enhanced chemiluminescence; PCR, polymerase chain reaction; bp, base pair; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; AhR, arylhydrocarbon receptor; TBST, 20 mM Tris HCl (pH 7.4), containing 0.5 M NaCl and 0.05%