Michael J. Fasco

Small intestinal cytochromes P450

Small intestinal cytochromes P450 (P450) provide the principal, initial source of biotransformation of ingested xenobiotics. The consequences of such biotransformation are detoxification by facilitating excretion, or toxification by bioactivation. P450s occur at highest concentrations in the duodenum, near the pylorus, and at decreasing concentrations distally--being lowest in the ileum. Highest concentrations occur from midvillus to villous tip, with little or none occurring in the crypts of Lieberkuehn. Microsomal P4503A, 2C8-10, and 2D6 forms have been identified in human small intestine, and P450s 2B1, possibly 2B2, 2A1, and 3A1/2 were located in endoplasmic reticulum of rodent small intestine, while P4502B4 has been purified to electrophoretic homogeneity from rabbit intestine. Some evidence indicates a differential distribution of P450 forms along the length of the small intestine and even along the villus. Rat intestinal P450s are inducible by xenobiotics--with phenobarbital (PB) inducing P4502B1, 3-methylcholanthrene (3-MC) inducing P4501A1, and dexamethasone inducing two forms of P4503A. Induction is most effectively achieved by oral administration of the agents, and is rapid--aryl hydrocarbon hydroxylase (AHH) was increased within 1 h of administration of, for example, 3-MC. AHH, 7-ethoxycoumarin O-deethylase (ECOD), and 7-ethoxyresorufin O-deethylase (EROD) have been used most frequently as substrates to characterize intestinal P450s. Dietary factors affect intestinal P450s markedly--iron restriction rapidly decreased intestinal P450 to beneath detectable values; selenium deficiency acted similarly but was less effective; Brussels sprouts increased intestinal AHH activity 9.8-fold, ECOD activity 3.2-fold, and P450 1.9-fold; fried meat and dietary fat significantly increased intestinal EROD activity; a vitamin A-deficient diet increased, and a vitamin A-rich diet decreased intestinal P450 activities; and excess cholesterol in the diet increased intestinal P450 activity. The role of intestinal P450 in toxifying or detoxifying specific xenobiotics has been clearly demonstrated to only a limited extent. However, elevated intestinal P450 levels have been indirectly linked to gastrointestinal cancer. Intestinal metabolism of 2,2,2-trifluoroethanol produces intestinal lesions with consequent systemic bacterial infection.

[16] Production and application of antibodies to rat liver cytochrome P-450

Publisher Summary This chapter discusses the production and application of antibodies to rat liver cytochrome P-450. Hepatic cytochrome P-450 is a heme protein that functions as the terminal oxidase of the microsomal mixed-function oxidase system. The system detoxifies by catalyzing the oxidation of a wide variety of hydrophobic xenobiotics and of endogenous compounds, including fatty acids and steroids. The metabolism of the anticoagulant warfarin to multiple the products catalyzed by cytochrome P-450 has provided a regioselective criterion for the assessment of the multiplicity of cytochrome P-450 in tissues and of the homogeneity of purified cytochrome P-450. The use of both warfarin enantiomers enhances the utility of the method by indicating stereoselective differences between the forms of cytochrome P-450. Antibodies against highly purified forms of cytochrome P-450 have provided a further powerful probe of cytochromes P-450, when used together with warfarin metabolism, by immunoselectively inhibiting the formation of metabolites. The separation of 8-hydroxywarfarin from benzylic hydroxywarfarin is critically dependent on solvent pH. The homogeneity of purified cytochromes P-450 is assessed by performing the metabolism of R- and S-warfarin.

Expression of an estrogen receptor alpha variant protein in cell lines and tumors.

Human estrogen receptor alpha (ER) mRNA is a mixture of wild type and alternatively spliced variants. Many studies have examined the potential of ER mRNA profiles to serve as diagnostic/prognostic cancer biomarkers, but only a few have attempted to correlate ER mRNA profiles with protein expression. Representative ER mRNA pools were reproduced from the cDNAs of MCF-7 cells, a human breast tumor and human uterus and translated in a protease-free environment by reticulocyte lysates to determine relative translation efficiencies between the various ER mRNA transcripts and to facilitate identification of translated proteins. Cell line and tumor extracts were then examined for expression of the ER variant proteins identified in reticulocyte lysate translations. Each of the ER mRNA pools were translated by reticulocyte lysates into two ER proteins with molecular weights of approximately 60 and 52 kD. Western immunoblotting with various C- and N-terminal-directed, anti-ER antibodies and comparison with expressed ER protein standards established that the 52 kD protein (ERDelta7P) was translated from the predominant splice variant mRNA in each pool, which is missing exon 7. The 60 kD protein contained wild type ER sequence minus 61 C-terminal amino acids lost due to an intentional run off truncation. ERDelta7P expression was subsequently demonstrated in MCF-7 cells by Western immunoblotting with the site-directed antibodies. A protein corresponding to ERDelta7P was also detected in other ER positive breast tumor cell lines, and extracts of ER positive breast and uterine tumors. This widespread expression of ERDelta7P in vivo suggests that it may have some biological function. ERDelta7P may also affect immunohistochemical evaluation of ER positivity in tumors depending upon the level of its expression and the antibody used.

Cytochrome P450 mRNA induction: quantitation by RNA-polymerase chain reaction using capillary electrophoresis.

Publisher Summary This chapter discusses the cytochrome P450 mRNA induction. A highly sensitive, quantitative method for the determination of P450 mRNAs can provide a solid basis for many studies of P450 regulation. Quantitative reverse transcription and polymerase chain reaction (RNA-PCR) provides a valuable technique for achieving these goals. RNA-PCR has the potential as a quantitative measure of specific cellular mRNAs present in low copy numbers. All quantitative RNA-PCR methods are labor-intensive and require careful preparation and analysis of many samples per data point. Capillary electrophoresis employing liquid or solid polymers as molecular sieves is highly efficient in separating DNA fragments in the size range frequently used in quantitative RNA-PCR. However, the UV detection methods used are not adequately sensitive for RNA-PCR products derived from low copy numbers.

Development of the hepatic mixed-function oxidase system and its metabolism of warfarin in the perinatal rat.

The development of the rat hepatic microsomal mixed-function oxidase system was investigated by analyzing its components in fetal and young pups of postconceptional age 18-54 days. Cytochrome P-450 and cytochrome b5 concentrations and NADPH-cytochrome c reductase and R-warfarin hydroxylase activities were determined as a function of postconceptional age. The cytochromes and reductase were present and increased with age before birth, and major increases were detected in the first 2 days after birth. The cytochrome P-450 concentration increased again markedly at puberty, whereas reductase activity increased before puberty and reached the adult level at puberty. Metabolism of warfarin to specific metabolites indicated that at 37 days postconception the isozyme cytochrome P-450 UT-C began to predominate. Its domination of the hepatic cytochrome P-450 pool increased with age.