Basal and inducible CYP1 mRNA quantitation and protein localization throughout the mouse gastrointestinal tract.
ABSTRACT The CYP1A1, CYP1A2, and CYP1B1 enzymes are inducible by benzo[a]pyrene (BaP) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD); metabolism of BaP by these enzymes leads to electrophilic intermediates and genotoxicity. Throughout the gastrointestinal (GI) tract, we systematically compared basal and inducible levels of the CYP1 mRNAs by Q-PCR, and localized the CYP1 proteins by immunohistochemistry. Cyp1(+/+) wild-type were compared with the Cyp1a1(-/-), Cyp1a2(-/-), and Cyp1b1(-/-) single-knockout and Cyp1a1/1b1(-/-) and Cyp1a2/1b1(-/-) double-knockout mice. Oral BaP was compared with intraperitoneal TCDD. In general, maximal CYP1A1 mRNA levels were 3-10 times greater than CYP1B1, which were 3-10 times greater than CYP1A2 mRNA levels. Highest inducible concentrations of CYP1A1 and CYP1A2 occurred in proximal small intestine, whereas the highest basal and inducible levels of CYP1B1 mRNA occurred in esophagus, forestomach, and glandular stomach. Ablation of either Cyp1a2 or Cyp1b1 gene resulted in a compensatory increase in CYP1A1 mRNA - but only in small intestine. Also in small intestine, although BaP- and TCDD-mediated CYP1A1 inductions were roughly equivalent, oral BaP-mediated CYP1A2 mRNA induction was approximately 40-fold greater than TCDD-mediated CYP1A2 induction. CYP1B1 induction by TCDD in Cyp1(+/+) and Cyp1a2(-/-) mice was 4-5 times higher than that by BaP; however, in Cyp1a1(-/-) animals CYP1B1 induction by TCDD or BaP was approximately equivalent. CYP1A1 and CYP1A2 proteins were generally localized nearer to the lumen than CYP1B1 proteins, in both squamous and glandular epithelial cells. These GI tract data suggest that the inducible CYP1A1 enzyme, both in concentration and in location, might act as a "shield" in detoxifying oral BaP and, hence, protecting the animal.
Article: Targeted knockout of Cyp1a1 gene does not alter hepatic constitutive expression of other genes in the mouse [Ah] battery.[show abstract] [hide abstract]
ABSTRACT: Using the Cre-lox system, we have generated a cytochrome P450 1A1 Cyp1a1(-/-) knockout mouse by deletion of the translated portions of the Cyp1a1 gene. These mice are viable and demonstrate no obvious phenotype, compared with wild-type littermates. As a first step toward characterizing genes that might be expected to compensate for loss of CYP1A1, constitutive expression of [Ah] gene battery members was examined. In a cultured hepatoma CYP1A1 metabolism-deficient mutant line that does not express Cyp1a2, we have previously shown that constitutive transcriptional up-regulation of other [Ah] gene battery members occurs; these results are consistent with the elevation of a putative endogenous ligand (EL) for the Ah receptor that is a substrate for CYP1A1. The [Ah] battery includes Cyp1a2, NAD(P)H:quinone oxidoreductase (Nqo1), and three other Phase II genes. Examining mRNA, protein, and enzyme activity, we demonstrate that the absence of CYP1A1 has no effect on the hepatic constitutive expression of Cyp1a2 or Nqo1. We postulate that CYP1A1 and CYP1A2 might have overlapping substrate specificity for metabolism of the EL, such that basal CYP1A2 in the liver can compensate for the loss of CYP1A1.Biochemical and Biophysical Research Communications 02/2000; 267(1):184-9. · 2.48 Impact Factor
Article: The effects of dietary brussels sprouts and Schizandra chinensis on the xenobiotic-metabolizing enzymes of the rat small intestine.[show abstract] [hide abstract]
ABSTRACT: After an initial equilibration period of 7 days on a semi-synthetic basal diet, male Sprague-Dawley rats were fed for 2 wk on either the basal diet (controls), the basal diet containing 5% Schizandra chinensis or 25% Brussels sprouts, or on rat chow. One group of chow-fed rats was pretreated with 20 mg 3-methylcholanthrene (3-MC)/kg body weight, 24 hr before they were killed. Microsomal and cytosolic fractions were prepared from small intestine mucosa. Microsomes were assayed for cytochrome P-450, aryl hydrocarbon hydroxylase (AHH), ethoxycoumarin O-deethylase (ECD) and epoxide hydrolase (EH) activities, and for metabolism of benzo[a]pyrene (BaP), Cytosols were assayed for glutathione S-transferase (GST) activity. The largest increase in intestinal mixed-function oxidase activity over levels in the controls was seen in the 3-MC-treated group. However, EH and GST activities in these animals were not significantly increased. Increases in cytochrome P-450 levels and significant increases in AHH, ECD, EH and GST activities occurred in the rats fed Brussels sprouts. Rats in the S. chinensis group showed inhibition of AHH activity relative to controls, but increased activity of ECD, EH and GST. In the rats fed chow there were significant increases in the activities of all the enzymes assayed except GST. The percentage conversion of BaP to metabolites reflected the results of the AHH assay and the groups were ranked in the following order: 3-MC greater than Brussels sprouts greater than rat chow greater than basal diet greater than S. chinensis. The profile of BaP metabolites showed a larger proportion of the BaP diols and 3-hydroxybenzo[a]pyrene, and a smaller proportion of BaP-4,5-epoxide and the BaP quinones, for the Brussels sprouts- and S. chinensis-fed groups. The significance of these results is discussed in regard to the role of the small intestine as a mediator of toxicity induced by ingested chemicals.Food and Chemical Toxicology 02/1985; 23(1):57-65. · 3.00 Impact Factor
Article: Development and validation of real-time quantitative reverse transcriptase-polymerase chain reaction for monitoring gene expression in cardiac myocytes in vitro.[show abstract] [hide abstract]
ABSTRACT: In this article we present validation of a real-time RT-PCR method to quantitate mRNA expression levels of atrial natriuretic peptide and c-fos in an in vitro model of cardiac hypertrophy. This method requires minimal sample and no postreaction manipulation. In real-time RT-PCR a dual-labeled fluorescent probe is degraded concomitant with PCR amplification. Input target mRNA levels are correlated with the time (measured in PCR cycles) at which the reporter fluorescent emission increases beyond a threshold level. The use of an oligo(dt) magnetic bead protocol to harvest poly(A) mRNA from cultured cells in 96-well plates minimized DNA contamination. We show that the GAPDH gene chosen for normalization of the RNA load is truly invariant throughout the biological treatments examined. We discuss two methods of calculating fold increase: a standard curve method and the DeltaDelta Ct method. Real-time quantitative RT-PCR was used to determine the time course of c-fos induction and the effect of varying doses of four known hypertrophy agents on atrial naturitic factor messenger RNA expression in cultured cardiac muscle cells. Our results agree with published data obtained from Northern blot analysis.Analytical Biochemistry 06/1999; 270(1):41-9. · 3.00 Impact Factor