Deborah Dunbar

An intestinal epithelium-specific cytochrome P450 (P450) reductase-knockout mouse model: direct evidence for a role of intestinal p450s in first-pass clearance of oral nifedipine.

To determine the in vivo function of intestinal cytochrome P450 (P450) enzymes, we have generated an intestinal epithelium (IE)-specific P450 reductase gene (Cpr) knockout mouse model (designated IE-Cpr-null). In the IE-Cpr-null mouse, CPR expression was abolished in IE cells; however, CPR expression was not altered in other tissues examined. The loss of CPR expression in the small intestine (SI) led to increased expression of several P450 proteins examined, including CYP1A1, CYP2B, CYP2C, and CYP3A. It is interesting to note that the expression of CYP1A1 was also increased in the liver, kidney, and lung of the IE-Cpr-null mice compared with wild-type (WT) littermates, a result strongly supporting the notion that SI metabolism of putative dietary CYP1A1 inducers can influence the systemic bioavailability of these inducers. The rates of SI microsomal metabolism of nifedipine (NFP) in the IE-Cpr-null mice were ∼10% of the rates in WT littermates, despite the compensatory expression of multiple P450 enzymes in the SI. Furthermore, the area under the concentration-time curve (AUC) values for blood NFP (dosed at 10 mg/kg) levels were 1.6-fold higher in IE-Cpr-null mice than in WT littermates when NFP was given orally; in contrast, the AUC values were comparable for the two strains when NFP was given intravenously. This result directly showed that P450-catalyzed NFP metabolism in the SI plays an important role in the first-pass clearance of oral NFP. Our findings indicate that the IE-Cpr-null mouse model can be used to study the in vivo function of intestinal P450 enzymes in the clearance of oral drugs and other xenobiotics.

Role of Small Intestinal Cytochromes P450 in the Bioavailability of Oral Nifedipine

To determine the effect of intestinal cytochrome P450 (P450) enzymes on the bioavailability of oral drugs, we have examined the metabolism of nifedipine, an antihypertensive drug and a model substrate of CYP3A4, in mouse models having deficient expression of the NADPH-cytochrome P450 reductase. Initial studies were performed on Cpr-low (CL) mice, which have substantial decreases in Cpr expression in all tissues examined, including the small intestine. In CL mice, area under the concentration-time curve (AUC) values for blood nifedipine after intraperitoneal and oral dosing were 1.8- and 4.0-fold, respectively, higher than in wild-type mice, despite increased expression of multiple P450 enzymes in both liver and intestine. The greater extent of the increase in the AUC value for oral than for intraperitoneal nifedipine suggested that intestinal P450s influence the bioavailability of oral nifedipine, a notion supported by results from further studies on LCN and CL-LCN mice. The LCN mice, which have liver-specific Cpr deletion, had 6.9-fold higher AUC values and 2.2-fold higher Cmax values for blood nifedipine than did wild-type mice after oral nifedipine, consistent with the critical role of hepatic P450s in systemic nifedipine clearance. However, in the CL-LCN mice, which have global decreases in Cpr expression in all tissues examined and Cpr deletion in the liver, AUC and Cmax values for oral nifedipine were, respectively, 2.2- and 1.8-fold higher than in LCN mice, confirming the fact that P450-catalyzed metabolism in the small intestine, the portal-of-entry organ for oral drugs, plays an important role in the first-pass clearance of oral nifedipine.

INTRACELLULAR LACTATE DEHYDROGENASE CONCENTRATION AS AN INDEX OF CYTOTOXICITY IN RAT HEPATOCYTE PRIMARY CULTURE

In searching for a reliable index for cytotoxicity testing in rat hepatocyte primary culture, lactate dehydrogenase (LDH) concentrations in lysates of attached hepatocytes and LDH released into the culture medium were compared under conditions of exposure to various dosages of sodium chloride, sodium salicylate, R-warfarin, acetaminophen, phenylbutazone, and furosemide (frusemide). The amount of intracellular LDH was assessed by inducing the cells to release the enzyme with 0.1% Tritron X-100. The induced LDH leakage was completed in 1 hr and the LDH activity was stable in storage at 10° for 2 weeks. We found that intracellular LDH is a direct indicator of the number of viable hepatocytes in contrast to the LDH released, because released LDH does not account for the significant number of cells detached from monolayer but which are not leaky, during the 6-hr test period. Based on IC50 values (50% inhibitory concentration), the relative cytotoxicities are R-warfarin > phenylbutazone > furosemide > acetaminophen > sodium salicylate > sodium chloride.

Acute toxicity of fluorinated ether anesthetics: Role of 2,2,2-trifluoroethanol and other metabolites☆☆☆

The fluorinated ethers 2,2,2-trifluoroethyl vinyl ether (TFVE), 2,2,2-trifluoroethyl ethyl ether (TFEE), and 2,2,2-trifluoroethyl allyl ether (TFAE) are lethal to rats pretreated with a variety of cytochrome P-450-inducing agents at doses not toxic to uninduced rats. The hepatic microsomal cytochrome P-450-catalyzed metabolic pathways have been elucidated: TFVE yields 2,2,2-trifluoroethanol (TFE) and glycolaldehyde; TFEE yields TFE and acetaldehyde; and TFAE yields TFE and acrolein. Time courses of metabolite concentrations in blood after administration of toxic doses of metabolites or anesthetics to variously induced rats were compared. For TFVE, phenobarbital (PB) or pregnenolone-16 alpha-carbonitrile (PCN) induction increased acute lethality 3.1- and 2.4-fold respectively, and blood TFE concentrations reached lethal levels. beta-Naphthoflavone (BNF) induction did not produce lethality and TFE concentrations did not reach lethal levels. Glycolaldehyde concentrations did not approximate lethal levels in any case. For TFEE, PB, BNF, or PCN induction increased lethality 3.4-, 2.6-, and 2.0-fold respectively, and TFE concentrations exceeded lethal levels in all cases. Acetaldehyde concentrations did not approximate lethal levels in any case. For TFAE, BNF, or PCN induction increased lethality 3.2- and 4.6-fold respectively, and TFE concentrations approached lethal levels. PB induction did not produce lethality nor did TFE concentrations reach lethal levels. Blood acrolein concentrations could not be determined. Thus the lethal effects of the three anesthetics arise from specific cytochrome P-450 isozyme-catalyzed metabolism to TFE. The relative rates of formation of TFE from the three anesthetics, catalyzed by microsomes from untreated and variously induced rats, supported this conclusion.

Rat liver metabolism and toxicity of 2,2,2-trifluoroethanol

2,2,2-Trifluoroethanol (TFE) is a metabolite of anesthetic agents and chlorofluorocarbon alternatives. Its toxicity in rats is a consequence of its metabolism to 2,2,2-trifluoroacetaldehyde (TFAld) and then to trifluoroacetic acid (TFAA). The enzymes involved in the toxic metabolic pathway have been investigated in this study. For the reaction of TFE to TFAld, the major hepatic metabolism associated with toxicity (as assessed by pyrazole-inhibitability) was NADPH dependent and occurred in the microsomes, whereas for TFAld conversion to TFAA, NADPH-dependent microsomal metabolism was significant, but mitochondrial and cytosolic metabolism in the presence of NADPH were also major contributors. NADPH-dependent hepatic microsomal metabolism of TFE to TFAld and TFAld to TFAA was inhibited by carbon monoxide, 2-allyl-2-isopropylacetamide, SKF-525A, metyrapone, imidazole, and pyrazole, and both reactions were oxygen dependent. The metabolism of TFE to TFAld was inhibited by diethyldithiocarbamate, a specific inhibitor of cytochrome P450E1, and by a monoclonal antibody to P4502E1, whereas the metabolism of TFAld was inhibited by neither. Ethanol pretreatment of rats enhanced the Vmax for hepatic microsomal metabolism of TFE to TFAld from 5.3 to 9.7 nmol/mg protein/min, while for TFAld to TFAA the Vmax was increased from 4.3 to 6.5 and the Km was unaffected for both reactions. Phenobarbital pretreatment of the rats did not affect any of these kinetic parameters. Coadministration of ethanol and a lethal dose of TFE very markedly decreased the lethality. Both the lethality (LD50 0.21 to 0.44 g/kg) and the metabolic kinetic parameters [(Vmax/Km)H(Vmax/Km)D = 4.2] were affected markedly when deuterated TFE replaced TFE. In contrast, deuteration of TFAld did not affect its lethality or rates of metabolism, but did affect its Km. Taken together these results indicate that P4502E1 catalyzed toxicity-associated hepatic metabolism of TFE to TFAld, while TFAld metabolism was catalyzed by a P450 which was not P4502E1. The hepatic metabolism of TFAld was not associated with its toxicity, which has been determined previously to be associated with its intestinal metabolism.

Role of hepatic microsomal cytochrome P-450 in the toxicity of fluorinated ether anesthetics☆

Abstract To evaluate the role of cytochrome P-450 in anesthetic toxicity, we investigated the effects of hepatic microsomal cytochrome P-450 inducers [phenobarbital (PB), 3-methylcholanthrene (3-MC) and pregnenolone-16 α-carbonitrile (PCN)] and inhibitors [SKF 525-A, metyrapone, and 2allyl2isopropylacetamide (ALA)] on the potentiation of lethal effects to rats of i.p. administered 2,2,2-trifluoroethyl vinyl ether (TFVE), ethyl 2,2,2-trifluoroethyl ether (TFEE), allyl 2,2,2-trifluoroethyl ether (TFAE) and 2,3-epoxypropyl 2,2,2-trifluoroethyl ether (EPTFE). The time courses of tail-vein blood anesthetic concentrations and quantities of exhaled anesthetics together with the in vitro metabolism of the anesthetics and their binding to microsomal cytochromes P-450 were also determined. The results indicate that (1) the majority of the administered anesthetics make a single pass through the liver prior to exhalation and apparently are metabolized to toxic products, (2) the epoxide (EPTFE) exerts its lethal effects independently of cytochrome P-450 catalyzed metabolism and does not lie on the major path of TFAE metabolism, (3) all the anesthetics yield 2,2,2-trinuoroethanol (TFE) on metabolism in vitro but lethality does not always correlate with the rates of TFE formation, (4) PB induced cytochromes P-450 potentiate lethal effects of TFVE and TFEE but not of TFAE, and inhibitors differentiate mechanisms of TFVE and TFEE lethality, (5) PCN induced cytochromes P-450 potentiate the toxicity of TFVE, TFAE, and TFEE in a similar manner, and (6) 3-MC induction potentiates TFEE and TFAE lethality apparently independently of cytochrome P-450 catalyzed metabolism.

Trifluorinated ether anesthetic lethality in rats: The role of bacterial infection☆

The lethal effects of the fluorinated ether anesthetics fluroxene (2,2,2-trifluoroethyl vinyl ether) and its ethyl (TFEE) and allyl analogues in male Wistar rats have previously been demonstrated to be potentiated by specific hepatic microsomal cytochromes P-450, and mediated by the common metabolite 2,2,2-trifluoroethanol (TFE). We report here that administration of lethal combinations of anesthetic and cytochrome P-450-inducing agents or of lethal doses of TFE (0.21 g/kg and higher) to rats caused decreased white blood cell counts, necrosis of sternum bone marrow cells and lymphocytes in the thymic cortex, and resulted in Escherichia coli contamination of the blood, lungs, liver, and kidneys of treated rats. Control animals in identical environments were free of bacterial contamination. Pretreatment of rats with the antibiotic tetracycline-HCl in the drinking water (0.6 g/liter) from 24 hr before anesthetic or TFE administration significantly diminished the mortality. With TFEE and beta-naphthoflavone induction, mortality was reduced from 85 to 30% by the antibiotic. However, the antibody plaque assay following immunization with sheep erythrocytes indicated that the primary humoral immune response to a thymus-dependent antigen was not impaired in treated rats. These results considered together indicate that metabolic formation of TFE from the anesthetic agents produced a decreased host resistance with subsequent increased susceptibility to bacterial infection. If not administered the antibiotic, the animals succumbed to the infection.

[5] Azidowarfarin as photoaffinity probe of cytochromes P450

Publisher Summary This chapter discusses the azidowarfarin as photoaffinity probe of cytochromes P450. Photoaffinity labeling has the potential to be one of the best approaches for labeling cytochrome P450 substrate binding site amino acids, thus leading to their identification. The technique involves use of a substrate modified with a photoactivatable substituent, which specifically binds to the substrate binding site of an enzyme, is photoactivated in situ , and binds covalently to the substrate binding site. Aryl azides which on photoactivation yield nitrenes capable of inserting into carbon-hydrogen, oxygen-hydrogen, and nitrogen–hydrogen bonds, are potentially useful for broadly labeling the substrate binding site residues of cytochromes P450, which have more than a single amino acid residue interacting with substrates. The mechanism of action of cytochrome P450 lends itself to a substrate-based photoaffinity probe of substrate binding sites, as substrates bind to the oxidized enzymes.