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M J Fasco

Publications and source records attributed to M J Fasco.

At least 37 records · Page 2Linked to original sources
PubMed1995

Human cytochromes P4501A1 and P4501A2: R-warfarin metabolism as a probe.

Two forms of the cytochrome P450 enzyme superfamily, P4501A1 and P4501A2, that are heterogeneously distributed in populations and are induced in response to environmental factors are important because of their capacity to bioactivate procarcinogens. Phenotyping P4501A1 and P4501A2 in individuals will thus provide assessments of those individuals' susceptibility to procarcinogens. The anticoagulant drug warfarin is metabolized by human P4501A1 and P4501A2, and we have characterized this metabolism for the R-warfarin enantiomer as a potential in vivo probe. cDNA-expressed human P4501A1 and P4501A2 are regioselective for R-warfarin 6- and 8-hydroxylation with very similar KM values: 1.4 mM (6-hydroxylation), 1.2 mM (8-hydroxylation), 1.6 mM (6-hydroxylation), and 1.4 mM (8-hydroxylation), respectively, indicating possible binding competition for R-warfarin between the two forms. However, when comparing 6- and 8-hydroxylation, P4501A1 showed weak regioselectivity for 8-hydroxylation, whereas P4501A2 exhibited strong regioselectivity for 6-hydroxylation, with 6-hydroxylation/8-hydroxylation ratios of 0.6 and 5.0, respectively. These findings were confirmed by using microsomes from 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated HepG2 and MCF-7 cells expressing only P4501A1 (ratios of 0.7), and from human hepatic microsomal preparations containing only P4501A2 (average ratios of 4.0). P4501A2 levels in the liver preparations, as assessed by densitometry of immunoblots, correlated with R-warfarin 6-hydroxylation rates (r2 = 0.83) and caffeine 3-demethylation rates (r2 = 0.67), but not with R-warfarin 8-hydroxylation rates. P450s 2A6, 2B6, 2C9, 2D6, 2E1, and 3A4 did not yield either 6- or 8-hydroxy-warfarin from R-warfarin. We conclude that R-warfarin 6-hydroxylation rates are markers for human hepatic P4501A2, whereas ratios of 6-hydroxylation/8-hydroxylation could be used in vitro as a marker for P4501A1.

Base Sequence↗
PubMed1993

Rat small intestinal cytochromes P450 probed by warfarin metabolism.

Small intestinal cytochromes P450 (P450s) provide potential first-pass metabolism of ingested xenobiotics. To investigate this system, this study addresses the procedure for elution of enterocytes from male rat small intestine, histological evaluation of the elution procedure, and assessment of the functional microsomal P450s in small intestine of untreated and induced rats, using warfarin metabolism as a probe. Histologically it was demonstrated that villous enterocytes are initially detached in sheets and are subsequently eluted without clear resolution into villous tip, midvillous, and lower villous cells, contrary to previous reports. Crypt cells are eluted after cells from the villus. The following functional P450s were identified, using stereo- and regioselectivity of warfarin metabolism, in small intestinal microsomes: P4502B1 in untreated rats, P4501A1 in beta-naphthoflavone-induced rats, P4502B1 in phenobarbital-induced rats, and P4503A1/2 in pregnenolone-16 alpha-carbonitrile-induced rats. In contrast to hepatic microsomes from untreated or induced rats, P4502C11 and -2C6 were not present or inducible by these inducing agents in rat intestine. Western immunoblots, warfarin assays, and P450 assays all indicated that beta-naphthoflavone induced P4501A1 in small intestinal villous and crypt cells, but in contrast to the liver neither apo-P4501A2 nor functional P4501A2 was induced.

Animals↗
PubMed1991

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.

Animals↗
PubMed1988

Loss of metastatic and primary tumor factor X activator capabilities by Lewis lung carcinoma cells cultured in vitamin K-dependent protein deficient serum.

A highly metastatic line of Lewis lung tumor cells established in fetal bovine serum (10%) was subcultured into normal rodent (mouse or rat) serum or rodent serum made deficient in functional vitamin K-dependent proteins (barium sulfate adsorption or warfarin treatment of animals). Following injection of cells cultured in normal rodent serum into C57BL/6 mice, Factor X activator activity in the primary tumors increased at a near linear rate per gram tumor and attained 5- to 8-fold higher levels than did cells grown in either of the deficient sera. Secondary lung foci were also visible in all mice of the normal-rodent serum groups within 10 days after injection, and by 21 days extensive tumor growth in the lungs had developed. No secondary lung foci were apparent in any mice of the deficient serum groups throughout 21 days of tumor burden. Cells cultured in nonrodent serum (fetal bovine serum) were less proficient than cells grown in normal mouse serum in developing primary tumor Factor X activator activity and producing secondary tumors. Exposure of cells cultured in barium sulfate-treated mouse serum to normal mouse serum for 3 h and 3 weeks prior to injection partially restored primary tumor Factor X activator and metastatic competence. These data strongly suggest that in Lewis lung tumor cells at least one species selective, plasma/serum vitamin K-dependent protein plays a major role in the regulation of metastatic events and demonstrate that there is a positive correlation between primary tumor Factor X activation activity and metastasis.

Animals↗
PubMed1988

Normal and warfarin-resistant rat hepatocyte metabolism of vitamin K 2,3-epoxide: evidence for multiple pathways of hydroxyvitamin K formation.

Vitamin K and 3- (and/or 2)-hydroxy-2,3-dihydro-2-methyl-3-phytyl-1,4-naphthoquinone (hydroxyvitamin K) have been identified as metabolites of vitamin K 2,3-epoxide incubated with hepatocytes isolated from normal and warfarin-resistant rats. Dithiothreitol added to the extracellular medium differentially enhanced the formation of both metabolites: hydroxyvitamin K formation, almost undetectable in the absence of dithiothreitol, was particularly affected. Addition of the vitamin K 2,3-epoxide reductase inhibitors warfarin (5 to 100 microM) and brodifacoum (1 to 5 microM) to normal rat hepatocyte cultures produced a slight increase in hydroxyvitamin K formation and a marked inhibition of vitamin K formation. Brodifacoum was a weak inhibitor of hydroxyvitamin K formation at higher concentrations. Hepatocytes from warfarin-resistant rats catalyzed hydroxyvitamin K formation 1.5 to 2 times faster and vitamin K formation 1.5 to 2 times slower than did normal rat hepatocytes. The addition of warfarin to these cultures had no effect on epoxide metabolism to hydroxyvitamin K and only partially diminished metabolism to vitamin K. In contrast, brodifacoum (1 microM) addition produced 50% inhibition of hydroxyvitamin K formation and almost complete inhibition of vitamin K formation. These data suggest that in resistant, but not in normal rat hepatocytes, the vitamin K 2,3-epoxide reductase makes a significant contribution to hydroxyvitamin K formation. A second sulfhydryl-dependent pathway, present in both strains, is also involved in the formation of this metabolite. They also suggest that in resistant rats, warfarin inhibition of the vitamin K 2,3-epoxide reductase, and presumably the sulfhydryl-dependent vitamin K reductase, is incomplete and independent of concentration.

Animals↗
PubMed1988

Comparative effects of cefoxitin and cefotetan on vitamin K metabolism.

The effects of cefoxitin and cefotetan on vitamin K metabolism and clotting parameters in five healthy subjects were investigated. No changes in prothrombin time or in the formation of abnormal prothrombin were seen either during or following the cefoxitin or cefotetan phase. However, when phytonadione (10 mg) (vitamin K1) was administered at the completion of each course of antibiotics, formation of vitamin K 2,3-epoxide was observed only during the cefotetan phase. It is probable, therefore, that cefotetan, a cephamycin antibiotic containing the N-methylthiotetrazole side chain, inhibits hepatic vitamin K 2,3-epoxide reductase. While hypoprothrombinemia and formation of abnormal prothrombin were not seen in healthy subjects, the effect of cefotetan on the coagulation status of vitamin K-depleted patients may be adverse.

Adult↗
PubMed1987

Evidence for a warfarin-sensitive serum factor that participates in factor X activation by Lewis lung tumor cells.

Mouse Lewis lung (LL) carcinoma cells possess a factor X activator (procoagulant) that is inhibited in vivo by warfarin treatment or diet-induced vitamin K deficiency. This inhibition suggests that vitamin-K-dependent proteins are involved in LL cell activation of factor X. A LL primary tumor clone (LL13) was isolated which contained a warfarin-sensitive vitamin-K cycle of metabolism and expressed factor X procoagulant activity. LL13 cells exposed to media containing warfarin or deficient in vitamin K grew as well as cells in normal media, and activated factor X to similar extents. In contrast, administration of warfarin to mice bearing LL13 cells inhibited factor X procoagulant activity as well as the vitamin K cycle of metabolism in the primary tumors. In relation to LL13 cells grown in media containing fetal bovine serum, those incubated for 20 hr in media containing mouse serum or the sera from LL13-bearing mice exhibited 9- to 10-times higher levels of factor X procoagulant activity. However, LL13 cells exposed to media containing the sera of warfarin-treated LL-13-bearing mice or to barium-sulfate-adsorbed normal mouse serum activated factor X much less efficiently. Collectively, these data suggest that inhibition of vitamin K function in LL cells does not affect the extent of factor X activation and thus the intrinsic factor X procoagulant is not a vitamin-K-dependent protein. They further suggest that both a warfarin-sensitive (vitamin-K-dependent) protein present in normal mouse serum and a LL13 cell component participate in factor X procoagulant activity.

Animals↗
PubMed1987

Identification of vitamin K1 chromenol--a novel metabolite of vitamin K1 formed in vitro by a component in blood.

A newly recognized metabolite of vitamin K1, vitamin K1 chromenol, is produced when the vitamin is added to the plasma or serum of a number of species. The metabolite was identified by comparison of its uv and mass spectra and high-performance liquid chromatographic retention times with those of the synthetic vitamin K1 chromenol. In aqueous solution vitamin K chromenol decomposed to a variety of products and reacted with nucleophilic substances. Optimal conditions for its formation and evidence that chromenol formation may be an enzyme catalyzed reaction are presented.

Animals↗
PubMed1986

Vitamin-K-dependent proteins in microsomes of primary Lewis lung tumors.

Microsomes isolated from Lewis lung (LL) primary tumors raised in C57BL/6 mice have been shown to (i) contain a 4-hydroxycoumarin (warfarin)-sensitive cycle of vitamin K metabolism which is at least qualitatively similar to that of liver, and (ii) catalyze the incorporation of NaH14 CO3 into endogenous protein in a vitamin-K hydroquinone-dependent reaction to produce gamma-carboxyglutamate. As in liver microsomes, LL microsomal reduction of vitamin K 2,3-epoxide to vitamin K was greatly enhanced by exogenous dithiols such as dithiothreitol, but under identical conditions the former was 10-fold faster. The R(+) and S(-) warfarin enantiomers were highly and equally effective inhibitors of both the liver and tumor vitamin K 2,3-epoxide reductases-the average I50 against the tumor enzyme was 0.25 microM. Partially purified reductases isolated by centrifugation of sodium-cholate-treated liver and LL tumor microsomes over a discontinuous sucrose gradient were also inhibited by the sulfhydryl reagent N-ethylmaleimide following their reduction by dithiothreitol. Like the activity of the epoxide reductase, that of the gamma-carboxylase was much lower in tumor than in liver microsomes and was only detectable in microsomes isolated from tumor-bearing mice previously administered S(-) warfarin. In view of the reported inhibition of LL tumor metastasis by warfarin and diet-induced vitamin-K deficiency, vitamin-K-dependent proteins may play a role in the spread and/or subsequent growth of LL cells.

Animals↗
PubMed1985

Identification of a warfarin-sensitive protein component in a 200S rat liver microsomal fraction catalyzing vitamin K and vitamin K 2,3-epoxide reduction.

A partially purified, 200S submicrosomal fraction exhibiting thiol-dependent vitamin K1 (vitamin K) and epoxide reductase activities has been isolated by partial solubilization of rat hepatic microsomes with sodium cholate and separation by centrifugation at 105 000 g into a discontinuous sucrose gradient. At pH 7.4, the rates of vitamin K and vitamin K 2,3-epoxide reduction per milligram of 200S fraction protein were equivalent and were 2.5-3.0 times faster than in microsomes. Reduction of vitamin K 2,3-epoxide occurred in a tightly coupled, two-step reaction initially to vitamin K and subsequently to vitamin K hydroquinone (vitamin KH2). Incorporation of glycerol or sucrose and of sodium cholate into reaction mixtures equivalently affected the rates of both vitamin K and vitamin K 2,3-epoxide reduction, but in the case of epoxide metabolism, the ratios of vitamin KH2/vitamin K were much lower, suggesting that the second reaction has been partially uncoupled from the first. A 14 000-17 000-dalton warfarin-sensitive protein (WSP) that participates in vitamin K and vitamin K 2,3-epoxide reduction in the 200S fraction was identified by incorporation of N-[3H]ethylmaleimide ([3H]NEM) into the catalytically active reduced form of one or more attached disulfides. Reduction of WSP with dithiothreitol was required for reaction with [3H]NEM, and the substrates vitamin K and vitamin K 2,3-epoxide and the inhibitor warfarin all effectively blocked the reaction. 2-Mercaptoethanol could not substitute for dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1984

Metabolism of vitamin K and vitamin K 2,3-epoxide via interaction with a common disulfide.

The effects of thiols and sulfhydryl blocking reagents on the reduction of vitamin K to vitamin K hydroquinone and vitamin K 2,3-epoxide to vitamin K and vitamin K hydroquinone catalyzed by rat hepatic microsomes were investigated to determine the mechanism(s) for these reactions. Both vitamin K and vitamin K 2,3-epoxide reductions were catalyzed more effectively with dithiols than with monothiols as the reductant. The sulfhydryl reagent N-ethylmaleimide (NEM) inhibited vitamin K and vitamin K 2,3-epoxide reduction much more effectively when microsomes were initially treated with dithiothreitol (prereduced). In prereduced microsomes iodoacetamide was approximately half as effective an inhibitor of vitamin K and vitamin K 2,3-epoxide reduction as NEM, but in microsomes not prereduced it was more effective. Iodoacetic acid was ineffective as an inhibitor. Vitamin K or vitamin K 2,3-epoxide added to prereduced microsomes blocked subsequent inhibition by NEM of vitamin K and vitamin K 2,3-epoxide metabolism, respectively. Vitamin K added to prereduced microsomes also blocked inhibition by NEM of vitamin K 2,3-epoxide metabolism, and vitamin K 2,3-epoxide addition blocked inhibition by NEM of vitamin K metabolism. Vitamin K did not diminish the rate of vitamin K 2,3-epoxide metabolism, however, nor did vitamin K 2,3-epoxide diminish the rate of vitamin K metabolism. These data establish that exogenous thiol compounds promote the reduction of at least one protein disulfide which participates in the metabolism of vitamin K and vitamin K 2,3-epoxide. Presumably, the resultant sulfhydryl groups are reoxidized to the disulfide form during the metabolism of either vitamin which protects them from reaction with NEM.

Animals↗
PubMed1983

Warfarin inhibition of vitamin K 2,3-epoxide reductase in rat liver microsomes.

Warfarin is a potent inhibitor of vitamin K 2,3-epoxide reduction to vitamin K in vitro and in vivo. Dithiothreitol, an in vitro reductant for the vitamin K 2,3-epoxide reductase, antagonizes inhibition of the reductase by warfarin via mechanisms that have not been determined [Zimmermann, A., & Matschiner, J. T. (1974) Biochem. Pharmacol. 23, 1033-1040]. Experiments with rat hepatic microsomes were undertaken to characterize the interactions that exist between vitamin K 2,3-epoxide, warfarin, and dithiothreitol. Increasing concentrations of dithiothreitol decreased inhibition of the reductase by warfarin. When dithiothreitol was present prior to exposure of the reductase to warfarin, there was less inhibition than when the same concentration of dithiothreitol was present after its exposure to warfarin. Moreover, maximum inhibition of the reductase by warfarin occurred at a much slower rate when dithiothreitol was present initially. Inhibition of the reductase by warfarin was greater when the substrate concentration was 100 microM vitamin K 2,3-epoxide than when it was 10 microM epoxide. On the basis of these data, we conclude that (i) dithiothreitol reduces either directly or indirectly a critical disulfide within the reductase that it reoxidized during reduction of the epoxide substrate, (ii) warfarin and vitamin K 2,3-epoxide are not competitive with respect to one another, and (iii) warfarin binding, which produces inhibition, occurs solely to the disulfide form of the reductase. Once it is bound, warfarin inhibits further reduction of the critical disulfide by dithiothreitol. Dithiothreitol therefore antagonizes warfarin by maintaining the reductase in the reduced state.

Animals↗
PubMed1983

Formation of hydroxyvitamin K by vitamin K epoxide reductase of warfarin-resistant rats.

A new metabolite of vitamin K, 2(3)-hydroxy-2,3-dihydro-2-methyl,3-phytyl-1,4-naphthoquinone (hydroxyvitamin K), has been identified as a product of vitamin K epoxide metabolism in hepatic microsomes from warfarin-resistant rats, but not in those derived from normal rats. The structure was determined by comparison of the high performance liquid chromatography retention times, UV, IR, CD, and mass spectra of the unknown with chemically synthesized standards. Alterations in the formation of hydroxyvitamin K occur in parallel with alterations in total vitamin K epoxide conversion with respect to reaction time, extent of reaction, detergent stimulation, and inhibition by warfarin. Thus, hydroxyvitamin K appears to be a product of the warfarin-resistant vitamin K epoxide reductase. It is neither a substrate nor an inhibitor of epoxide reduction. Hydroxyvitamin K is formed from both enantiomers of racemic vitamin K epoxide with little stereoselectivity for the configuration of either the oxirane ring or the phytyl side chain. The reaction is stereospecific; however, the biologically formed (+)-vitamin K epoxide yields exclusively (+)-3-hydroxyvitamin K. Observation of this product is discussed as a key to understanding the normal reaction mechanism of the enzyme.

Animals↗
PubMed1982

Evidence that warfarin anticoagulant action involves two distinct reductase activities.

The dithiothreitol-dependent vitamin K and vitamin K 2,3-epoxide hepatic microsomal reductase activities of warfarin-susceptible and warfarin-resistant rats were compared to gain insight into the role(s) of these activities in vitamin K metabolism and function. In microsomes from resistant rats, 3- to 4-fold more warfarin was required to produce 50% inhibition (I50) of vitamin K reduction to vitamin K hydroquinone than in microsomes from susceptible rats. For the reduction of vitamin K 2,3-epoxide to vitamin K a 6-fold higher warfarin concentration was required. In microsomes from resistant rats, the I50 warfarin concentration required to inhibit gamma-carboxylation of microsomal precursor protein was also 4-fold higher with vitamin K as substrate and was 6-fold higher with the epoxide as substrate than in microsomes from susceptible rats. Collectively, these data suggest that the vitamin K reductase contributes to the metabolism of vitamin K in intact rats and that warfarin inhibition of both the vitamin K and vitamin K 2,3-epoxide reductases is involved in its anticoagulant effect.

Animals↗
PubMed1982

R- and S-Warfarin inhibition of vitamin K and vitamin K 2,3-epoxide reductase activities in the rat.

Reduction of vitamin K 2,3-epoxide and vitamin K catalyzed by hepatic microsomal enzymes is required for normal, postribosomal, gamma-carboxyglutamate formation in the prothrombin complex Factors II, VII, IX, and X. The R- and S-warfarin enantiomers differentially inhibit (S-warfarin is 2 to 5 times more active) vitamin K function by mechanisms which have not been unambiguously determined. As a step toward determining the physiologically relevant site(s) of warfarin-antivitamin K activity we investigated in Wistar rats the effects of R- and S-warfarin on vitamin K 2,3-epoxide and vitamin K reductase activities and correlated them with effects on plasma concentrations of the Factors II, VII, and X. Based on the results of these studies we conclude that: 1) warfarin inhibition of the vitamin K 2,3-epoxide and vitamin K reductases is essentially irreversible; 2) S-warfarin stereoselectively inhibits both reductases in vivo but not in vitro; 3) the vitamin K reductase which utilizes dithiothreitol as cofactor in vitro is primarily responsible for vitamin K reduction to vitamin K hydroquinone under physiological conditions; 4) warfarin initially inhibits gamma-carboxyglutamate formation by inhibiting simultaneously the vitamin K 2,3-epoxide and vitamin K reductases; and 5) following enantiomer administration there is an apparent lack of correlation between the restoration of the reductase activities and the reinitiation of coagulation factor synthesis.

Animals↗
PubMed1981

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.

Aging↗