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M J Fasco

Publications and source records attributed to M J Fasco.

At least 19 recordsLinked to original sources
PubMed1998

12-O-tetradecanoylphorbol-13-acetate upregulates the Ah receptor and differentially alters CYP1B1 and CYP1A1 expression in MCF-7 breast cancer cells.

Elevated expression of cytochrome P450 1B1 (CYP1B1) and estradiol 4-hydroxylation have been reported to be biomarkers of tumorigenesis in humans. The aromatic hydrocarbon receptor (AhR) regulates expression of human cytochrome P450 1A1 (CYP1A1) and CYP1B1, 17beta-estradiol (E2) 2- and 4-hydroxylases, respectively. There is also evidence that expression of estrogen receptor alpha (ERalpha) potentiates CYP1A1 inducibility in breast cancer cells. To characterize these relationships further, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), which downregulates ERalpha, and the high-affinity AhR ligand, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the expression of AhR, ERalpha, CYP1A1, and CYP1B1 in MCF-7 human breast cancer cells. Treatment with TPA, which suppressed ERalpha mRNA levels, caused a greater than fourfold elevation of AhR mRNA and protein levels, whereas treatment with TCDD caused a decrease in AhR protein but no change in ERalpha or AhR mRNA levels. In MCF-7 cells treated with TPA prior to treatment with TCDD, the AhR mRNA level was elevated, the ERalpha mRNA level remained suppressed, and the ratio of CYP1B1 to CYP1A1 mRNA was increased compared with treatment with TCDD alone. A corresponding increase in the ratio of the rates of 4- to 2-hydroxylation pathways of E2 metabolism was also observed in response to pretreatment with TPA prior to the addition of TCDD. These results demonstrate differential regulation of the human CYP1A1 and CYP1B1 genes and provide a cellular model to investigate further the mechanisms that may be involved in the elevated expression of CYP1B1 in tumorigenesis.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Estrogen receptor mRNA splice variants produced from the distal and proximal promoter transcripts.

Relative proportions of the estrogen receptor (ER) alternatively spliced mRNA variants from the proximal (A) and distal (B) promoter pre-mRNA transcripts were measured in normal human uterus, an endometrial tumor, and in T47D, MCF-7, and BT-20 breast tumor cell lines. A single tube RNA-PCR method was developed to determine the proportions of the individual transcripts and a nested, competitive RNA-PCR method to determine the proportions of the alternatively spliced variants. Except for the BT-20 cells, the patterns of splice variants produced from each transcript were very similar. In BT-20 cells no splice variants were detected for the minor (< or = 1%) A promoter transcript, although the B promoter transcript was alternatively spliced similarly to the other samples, with the exon 7 variant as the major mRNA form. These results indicate that the mRNA spliced variant patterns in most tissues and tumors will be essentially unaffected by any changes in the A and B promoter ER mRNA transcript ratios that may occur. At least one exception does exist, however, and only more comprehensive studies can determine whether the BT-20 cells are unique or part of a larger subgroup.

Alternative Splicing↗
PubMed1998

Differential expression of CYP1A1 and CYP1B1 in human breast epithelial cells and breast tumor cells.

Human cytochromes P450 1A1 (CYP1A1) and P450 1B1 (CYP1B1) catalyze the metabolic activation of a number of procarcinogens and the hydroxylation of 17beta-estradiol (E2) at the C-2 and C-4 positions, respectively. The aromatic hydrocarbon receptor (AhR) agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) has a marked effect on estrogen metabolism in MCF-7 breast-tumor cells by induction of these two enzymes. To investigate whether induction of CYP1A1 and CYP1B1 by AhR agonists and the associated increase in E2 metabolism are common to all breast epithelial cells and breast-tumor cells, we determined the effects of TCDD on E2 metabolism, and CYP1A1 and CYP1B1 mRNA levels in a series of non-tumor-derived breast epithelial (184A1 and MCF-10A) and breast-tumor (MCF-7, T-47D, ZR-75-1, BT-20, MDA-MB-157, MDA-MB-231 and MDA-MB-436) cell lines. In 184A1 cells, which did not express detectable estrogen receptor (ER) alpha mRNA, CYP1A1 mRNA and activity were induced by TCDD, and enhanced E2 metabolism in TCDD-treated cells was predominantly E2 2-hydroxylation. In MCF-10A, MCF-7, T-47D, ZR-75-1 and BT-20 cells, which expressed varying levels of ER alpha mRNA, both CYP1A1 and CYP1B1 mRNA levels and rates of both E2 2- and 4-hydroxylation were highly elevated following exposure to TCDD. In MDA-MB-157, MDA-MB-231 and MDA-MB-436 cells, which did not express detectable ER alpha mRNA and generally displayed fibroblastic or mesenchymal rather than epithelial morphology, CYP1B1 induction was favored, and the rate of E2 4-hydroxylation exceeded that of 2-hydroxylation in TCDD-treated cells. These results show that breast epithelial cells and tumor cells vary widely with regard to AhR-mediated CYP1A1 and CYP1B1 induction, suggesting that factors in addition to the AhR regulate CYP1A1 and CYP1B1 gene expression. In these cell lines, significant CYP1A1 inducibility was restricted to cultures displaying epithelial morphology, whereas CYP1B1 inducibility was observed in cells of both epithelial and mesenchymal morphology.

Aryl Hydrocarbon Hydroxylases↗
PubMed1997

Alternative splicing of CYP2D mRNA in human breast tissue.

The human cytochrome P450 (CYP) 2D subfamily comprises the CYP2D6 gene and four pseudogenes, CYP2D7P1 and 2 and CYP2D8P1 and 2. The CYP2D6 gene product is a prominent drug-metabolizing enzyme, which is probably constitutive and has no known inducing agents. Alternative splicing of the pre-mRNAs of these genes has been detected in human liver and breast tissue. RNA-PCR, competitive RNA-PCR, Southern blotting, cDNA sequencing, and gene-specific PCR have been used to fully characterize the alternatively spliced forms of CYP2D mRNA in human breast tissue in the region of exon 5 to 8. Such alternative splicing could regulate the expression of CYP2D6 protein. A full-length mRNA (exons 5 to 8), and variants c (exon 6 deleted), b' (3' portion of exon 6 deleted), e (3' portion of exon 6 deleted, 3' 57-bp portion of intron 6 included), d (3' 57-bp portion of intron 6 included), and b (intron 6 included) were characterized and quantitated. Variant c was derived from CYP2D6, variants d, e, and b were from CYP2D7P, and variant b' and full-length mRNA were derived from both CYP2D6 and 2D7P. Full-length mRNA was a minor form in human breast tissue where variants b' and c predominated. Human breast tumor MCF-7 cells had CYP2D mRNA splice variant patterns similar to those of human breast tissue, while human liver tumor HepG2 cells had wild-type mRNA predominating. These results suggest that CYP2D6 could be regulated tissue specifically using tissue-specific alternative mRNA splicing.

Alternative Splicing↗
PubMed1997

Comparisons of CYP2D messenger RNA splice variant profiles in human lung tumors and normal tissues.

Allelic variants of the CYP2D6 gene, a member of the cytochrome P450 gene superfamily, have been implicated in susceptibility to lung carcinogenesis. Human breast CYP2D6 and CYP2D7P (from a pseudogene) mRNAs were previously reported to be expressed as a series of splice variants. In this study, the expression of full-length and splice variants of these mRNAs in human lung tissue and tumors are reported for the first time and are compared in order to probe the potential for differential CYP2D6 regulation in lung normal tissue and tumors. The splice variant profiles differed within the same individual, but no consistent differences were detected.

Adult↗
PubMed1997

Quantitation of estrogen receptor mRNA and its alternatively spliced mRNAs in breast tumor cells and tissues.

Estrogen receptor (ER) mRNA exists as wild-type (full-length) and alternatively spliced variants in cell lines, normal tissues, and tumors. Most of the alternatively spliced variants discovered so far are missing one or more complete exons. RNase protection and RNA-PCR assays used previously to determine the relative concentration of a particular ER spliced-variant mRNA to wild-type mRNA have produced equivocal results because the probes/primers targeted only small regions within the nucleotide sequence. Variant ER mRNAs missing an exon outside the probe/primer region will react as if they were wild-type and any alternatively spliced variants containing a deletion at the probe/primer annealing site(s) will not be detected. A highly sensitive, competitive RNA-PCR assay has been developed that is quantitative with respect to the relative composition of wild-type ER and its alternatively spliced-mRNA forms, and semiquantitative with respect to their concentrations in cells and tissues. Separation and quantitation of the products are rapidly and accurately achieved by, respectively, capillary electrophoresis and laser-induced fluorescence. Wild-type ER mRNA concentration can be measured independently of all the reported exon deletion forms in a single PCR assay. Specific exon deletion forms can be measured by ER cDNA amplification with overlapping primer sets. Results obtained with RNAs isolated from two MCF-7 cell lines, a T-47D cell line, and five breast tumor tissues are presented.

Alternative Splicing↗
PubMed1997

The molecular epidemiology of lung cancer.

One in ten tobacco smokers develops bronchogenic carcinoma over a lifetime. The study of susceptibility of an individual and a population to lung cancer traditionally has been limited to the study of tobacco smoke dose and family history of cancer. New insights into lung carcinogenesis have made the study of molecular markers of risk possible in human populations in the emerging field of molecular epidemiology. This review summarizes data addressing the relationships of human lung cancer to polymorphisms of phase I procarcinogen-activating and phase II-deactivating enzymes and intermediate biomarkers of DNA mutation, such as DNA adducts, oncogene and tumor suppressor gene mutation, and polymorphisms. These parameters are reviewed as they relate to tobacco smoke exposure, procarcinogen metabolizing polymorphisms, and the presence of lung cancer. Problem areas in biomarker validation, such as cross-sectional data interpretation; tissue source, race, statistical power, and ethical implications are addressed.

Biomarkers, Tumor↗
PubMed1996

Characterization of purified human recombinant cytochrome P4501A1-Ile462 and -Val462: assessment of a role for the rare allele in carcinogenesis.

Human cytochrome P4501A1 (CYP1A1) occurs extrahepatically and is polymorphic, the common form having Ile at position 462 and the rare form having Val. The rare allele has been associated with enhanced susceptibility to lung cancer. To resolve its role in cancer we have constructed CYP1A1-Val462 cDNA by site-directed mutagenesis from CYP1A1-Ile462, as confirmed by sequencing and allele-specific PCR. Both alleles were expressed in Escherichia coli, and CYP1A1-Ile462 and -Val462 were purified to electrophoretic homogeneity. The secondary structures of both forms were virtually identical, with high alpha helix content, as assessed by circular dichroism. The P450s stereoselectively and regioselectively catalyzed the metabolism of (R)- and (S)- warfarin, in reconstituted systems, with very similar profiles. Both P450s produced (R)-6- and 8-hydroxy-warfarin with Km values of 0.40 +/- 0.06 and 0.43 +/- 0.05 mM, respectively, and Vmax values of 84.0 +/- 6.8 and 137.7 +/- 8.9 pmol/min/nmol CYP1A1-Val462, respectively, 1.0 +/- 0.1 and 1.0 +/- 0.1 mM, respectively, and 46.7 +/- 2.5 and 80.0 +/- 4.4 pmol/min/nmol CYP1A1-Ile462, respectively. Reconstituted CYP1A1-Val462 catalyzed ethoxyresorufin metabolism at a slightly but significantly higher rate than did CYP1A1-Ile462; Vmax values were 4.4 +/- 0.6 and 3.1 +/- 0.3 nmol/min/nmol CYP1A1, respectively. However, with the carcinogen benzo(a) pyrene as substrate, reconstituted CYP1A1-Ile462 together with epoxide hydrolase produced 7,8- and 9,10-dihydrodiols at comparable rates than did CYP1A1-Val462. Thus, the apparently greater susceptibility of the CYP1A1-Val462 genotype to lung cancer is probably not related to greater extents of carcinogen bioactivation.

Alleles↗
PubMed1996

Optimization of Dnase I removal of contaminating DNA from RNA for use in quantitative RNA-PCR.

In competitive RNA-PCR studies, contaminating DNA can produce incorrect results because of its potential to act as a second competitor. Preliminary studies using published methods for DNase I digestion of DNA as a contaminant of RNA, followed by thermal inactivation of the enzyme at 95 degrees C for 5 min before reverse transcription and PCR, suggested that the mRNA was also affected by these treatments. This investigation was undertaken to optimize DNase I treatment of RNA with respect to DNA removal and mRNA preservation. Competitive RNA-PCR of DT-diaphorase transcript was used to quantitate the effects of the various treatments. Other transcripts with varying initial concentrations were visually compared to ensure that the effects observed were not unique to specific mRNAs. With 1 U of DNase I/microgram RNA, thermal denaturation of the enzyme at 75 degrees C for 5 min preserved nearly all of the mRNA. Thermal denaturation at 95 degrees C for 5 min inactivated approximately 80% of the mRNA, whereas heating at 55 degrees C for 10 min did not completely denature the DNase I. For RNA-PCR of every transcript investigated, incubation of 1 microgram RNA with 1 U of DNase for 30 min at 37 degrees C followed by heat-denaturation of the enzyme for 5 min at 75 degrees C was sufficient to destroy all the contaminating DNA, while completely preserving the respective mRNAs. This treatment is highly recommended as a routine step in RNA-PCR and particularly with competitive RNA-PCR with human breast tissue samples (and presumably other human tissues), which are often contaminated with small amounts of genomic DNA.

Base Sequence↗
PubMed1996

Expression of cytochromes P450 in human breast tissue and tumors.

In an effort to determine which members of the cytochrome P450 (CYP) superfamily are expressed in human breast tissue and tumors, RNA-polymerase chain reaction studies have been undertaken. Detection of expressed CYP mRNAs identifies those forms of the enzyme that are capable of expression in breast tissue, and provides insight into the potential for in situ xenobiotic and therapeutic drug metabolism. CYP1A1 mRNA was present in (5/11) breast tissues and (6/13) tumors. When normal and tumor tissues were from the same individuals, higher amplification occurred in normal tissues. CYP1B1 mRNA was present in all but one tissue, and CYP2C mRNA forms were present in all of the tissues. CYP3A4 mRNA was present in (8/11) normal breast tissues and (2/13) tumor tissues, and CYP3A5 mRNA was present in (9/11) normal tissues and (2/13) tumor tissues. The expression of the CYP3A mRNA forms was not coincident, suggesting differential regulation. CYP2D6 mRNA was present in (10/11) normal breast tissue and (10/13) tumors. Two splice variants of CYP2D6 mRNA were also detected; one with a 207 bp intron spliced in was detected in all of the normal tissue samples and (11/13) tumors, whereas another (which lacks a 3'-portion of exon 6) was detected in (9/11) normal breast tissues and (7/13) tumors. Thus, examples of each of the xenobiotic-metabolizing CYP1, CYP2, and CYP3 subfamilies were detected in low levels in human normal breast tissue and tumors. The machinery for possible in situ bioactivation of xenobiotics and modification of therapeutic drugs is thus present in human breast tissue.

Breast↗
PubMed1995

Determination of theophylline and its metabolites in rat liver microsomes and human urine by capillary electrophoresis.

A capillary electrophoretic (CE) method has been developed for the determination of theophylline and all of its identified and potential metabolites. The method is rapid, resolves all metabolites to baseline, and requires extraction of only some biological fluids. It has been applied to the analysis of theophylline metabolism by hepatic microsomes from rats treated with a variety of inducing agents for different forms of P450 enzymes which metabolize theophylline, and to human urine spiked with theophylline and its metabolites, and concentrated by solid-phase extraction.

Animals↗
PubMed1995

Quantitative RNA-polymerase chain reaction-DNA analysis by capillary electrophoresis and laser-induced fluorescence.

Quantitative RNA-polymerase chain reaction (RNA-PCR) is an extremely powerful analytical tool owing to its specificity and high level of sensitivity. Quantitative RNA-PCR is, however, highly labor intensive. No analytical method currently exists that can accurately and rapidly quantitate the small quantities of DNA in RNA-PCR reaction mixtures. We have developed a method using capillary electrophoresis and laser-induced fluorescence to detect YOYO-1 complexes of DNA produced by PCR. RNA-PCR mixtures can be analyzed either directly (without primer and protein removal) or by electrokinetic injection following desalting. Modified competitive and multiplex competitive RNA-PCR assays for glyceraldehyde-3-phosphate dehydrogenase and P4501A1 were tested in a series of mixtures containing equal concentrations, but different proportions, of RNA from untreated (essentially no P4501A1 mRNA) and 2,3,7,8-tetrachlorodibenzo-p-dioxin-treated (high levels of P4501A1 mRNA) HepG2 cells. Twofold differences in concentrations between two P4501A1 mRNA solutions could be detected by competitive RNA-PCR. Glyceraldehyde-3-phosphate dehydrogenase concentrations were constant throughout. Multiplex competitive PCR produced more variable results due to the presence of contaminating peaks, which hindered accurate area integration. These data demonstrate the potential usefulness of capillary electrophoresis in a variety of quantitative PCR applications.

Base Sequence↗
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↗