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L S Kaminsky

Publications and source records attributed to L S Kaminsky.

At least 37 records · Page 2Linked to original sources
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

The role of the CYP2C9-Leu359 allelic variant in the tolbutamide polymorphism.

Tolbutamide undergoes hydroxylation in humans via a cytochrome P450-mediated pathway. The primary P450 isozyme responsible for this metabolism is thought to be CYP2C9. Population studies have indicated the existence of slow metabolizers of tolbutamide (approximately 1 in 500) suggesting a rare polymorphism associated with 2C9. Several allelic variants of 2C9 have been identified; however, the effect of these allelic variations on metabolism in vivo is not established. In the present study, the coding regions, intron-exon junctions, and upstream region of CYP2C9 were amplified by PCR and sequenced in two slow metabolizers. One individual was homozygous for Leu359/Leu359 and the other individual was heterozygous for Arg144/Cys144 and for Ile359/Leu359. No other genetic variations in 2C9 were detected in these individuals. PCR-RFLP tests showed that Arg144 Tyr358 Ile359 Gly417 is the principle CYP2C9 allele. Frequencies of the rarer Leu359 and Cys144 alleles were 0.06 and 0.08, respectively, in a Caucasian-American population and 0.005 and 0.01 respectively in African-Americans. The frequency of the Leu359 allele was 0.026 in Chinese-Taiwanese, but the Cys144 allele was not detected in this population. Studies in a recombinant yeast expression system showed that the Leu359 variant had the highest Km and the lowest Vmac for hydroxylation of tolbutamide of all the CYP2C9 allelic variants. This allelic variant also had the highest Km for the 7-hydroxylation of S-warfarin. The present data suggest that the incidence of the Leu359 allelic variant of CYP2C9 may account for the occurrence of poor metabolizers of tolbutamide.

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

Characterization of human cytochromes P450 involved in theophylline 8-hydroxylation.

Studies were undertaken to determine which human P450 enzymes catalyze the metabolism of theophylline to 1,3-dimethyluric acid (1,3-DU), to facilitate predictions of theophylline drug-drug interactions, and to develop a noninvasive test for human P4501A2. Microsomes from a human cell line transfected individually with human P450 cDNAs for P4501A1, 1A2, 2A6, 2B6, 2C9, 2D6, 2E1, or 3A4 were used to demonstrate that only P4501A2 exhibited catalytic activity for theophylline metabolism to 1,3-DU with high affinity and low capacity (Km = 0.6 mM, Vmax = 37.8, pmol/min/mg), while P4502D6, 2E1, and 3A4 (Km = 14.4, 19.9, and 25.1 mM, respectively, and Vmax = 219.8, 646.4, and 20.8 pmol/min/mg, respectively) exhibited activities with low affinity and variable capacities. Correlations of rates of theophylline 8-hydroxylation to 1,3-DU with other P450 form-specific activities, in a series of ten human liver microsomal preparations, at 5 and 40 mM theophylline concentrations, revealed that at low concentrations the metabolism was catalyzed primarily by P4501A2, while at high substrate concentrations P4502E1 was primarily responsible for catalysis. The results with individually expressed P450s and hepatic microsomal preparations were consistent, indicating that the former system provides a qualitatively accurate reflection of the function of the heterogeneously expressed liver P450s. At pharmacologic theophylline concentrations achieved in vivo, its metabolism must thus be catalyzed primarily by P4501A2.

Cell Line↗
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↗
PubMed1994

Site-directed mutagenesis of putative substrate recognition sites in cytochrome P450 2B11: importance of amino acid residues 114, 290, and 363 for substrate specificity.

Eleven amino acid residues unique to dog cytochrome P450 (P450) 2B11, compared with rat 2B1 and 2B2, rabbit 2B4 and 2B5, and mouse 2B10, in the putative substrate recognition sites [J. Biol. Chem. 267:83-90 (1992)] were mutated to the residues found in 2B1 or 2B5. The mutants were expressed initially in COS cells and screened for activity toward androstenedione and 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). P450 2B11 mutants V107I, M199L-N200E-V204R, V234I, A292L, Q473R, and I475S showed no differences from wild-type P450 2B11 in metabolite profiles with either substrate. Mutants V114I, D290I, and L363V exhibited altered androstenedione metabolite profiles and were expressed in Escherichia coli for further study with androstenedione, testosterone, 7-ethoxycoumarin, (R)- and (S)-warfarin, and 245-HCB. With V114I, hydroxylation of steroids and warfarin and 2-hydroxylation of 245-HCB were decreased, whereas 7-ethoxycoumarin O-dealkylation and 3-hydroxylation of 245-HCB were unaltered. For D290I, activities toward all substrates were decreased, except for 16 beta-hydroxylation of testosterone. The activity of L363V was increased 5-6-fold for 16 alpha-hydroxylation of androstenedione and testosterone but was decreased to 40-50% of wild-type activity with 7-ethoxycoumarin and warfarin and to 6-8% of control for 2-hydroxylation of 245-HCB. Alignment of P450 2B11 with P450 101 and super-imposition of the 11 mutated 2B11 residues on a P450 101 three-dimensional model suggest that only residues 114, 290, and 363 represent substrate contact residues, in excellent agreement with the experimental results. The data indicate the importance of the three residues 114, 290, and 363 in substrate specificity and regio- and stereoselectivity of P450 2B11 and also demonstrate that the effects of the mutations vary considerably with different substrates.

Amino Acids↗
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↗
PubMed1993

Correlation of human cytochrome P4502C substrate specificities with primary structure: warfarin as a probe.

The regio- and stereoselectivity of warfarin metabolism have been used to assess structure-function relationships of human P4502C subfamily members. Metabolism was investigated using a yeast cDNA expression system in which full length cDNAs for P4502C8, -2C9 (alleles Arg144 Tyr358 Ile359 Gly417 and Arg144 Tyr358 Leu359 Gly417), -2C18 (alleles Thr385 and Met385), and -2C19 were expressed. Additionally, two mutations reported in other P4502C9/2C10 alleles were individually introduced into P4502C9 by site-directed mutagenesis, to yield Cys144 Tyr358 Ile359 Gly417, Arg144 Tyr358 Ile359 Asp417, and Arg144 Cys358 Ile359 Gly417, which were expressed in yeast; their ability to metabolize warfarin was then studied. Warfarin metabolism by purified preparations of P4502C9 allele Arg144 Tyr358 Ile359 Gly417 and its Leu359 mutant was also investigated in reconstituted systems. Both alleles of P4502C18 were regioselective for 4'-hydroxywarfarin, without any significant stereoselectivity. Both also metabolized warfarin at the 6-position, but to a lesser extent, and metabolism at this site was stereoselective for (R)-warfarin. P4502C8 metabolized warfarin at the 7-position and was stereospecific for (R)-warfarin. It also metabolized warfarin to a lesser extent at the 4'-position, and metabolism at this site was stereoselective for (R)-warfarin. P4502C19 was regioselective for 6- and 8-hydroxywarfarin and was stereoselective for (R)-warfarin. The highly conservative mutation of Ile359 to Leu359 in P4502C9 profoundly altered the regio- and stereoselectivity of warfarin metabolism, from regioselective for 7-hydroxywarfarin, with stereospecificity for (S)-warfarin, to regioselective for 4'-hydroxywarfarin, with stereoselectivity for (R)-warfarin, which was confirmed in a reconstituted system using purified recombinant enzymes. In contrast, individual mutations of P4502C9 of Arg144 to Cys, Tyr358 to Cys, and Gly417 to Asp did not markedly affect the regio- or stereoselectivity of warfarin metabolism, although the overall rates of warfarin metabolism were apparently increased by these changes. We conclude that residue 359 is at the substrate binding site of P4502C9, whereas residues 144, 358, and 417, and residue 385 of P4502C18, are not.

Alleles↗
PubMed1992

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.

Acetaldehyde↗
PubMed1992

Azidowarfarin photoaffinity probes of purified rat liver cytochrome P4501A1.

Substrate specificity differences between various forms of cytochrome P450 (P450) are governed by substrate binding site amino acid residue differences. To determine the identities of these residues, four analogs of warfarin, a thoroughly investigated anticoagulant drug which is regio- and stereoselectively metabolized by many P450s, have been synthesized as photoaffinity probes. The probes 4'-, 6-, 7-, and 8-azidowarfarin were readily photolyzed in neutral solution by 254-nm light, with half-lives of less than 15 s. When the azidowarfarins were photolyzed in the presence of beta-naphthoflavone-inducible P4501A1 (2.5 microM) at -196 degrees C and the P450 was subsequently reconstituted for warfarin metabolism, 50% inactivation was achieved with 160 microM 4'-azidowarfarin, 64 microM 6-azidowarfarin, 127 microM 7-azidowarfarin, and 29 microM 8-azidowarfarin. This inactivation is irreversible. When these concentrations of the azidowarfarins were photolyzed prior to addition to P4501A1, less inhibition of P450 activity was detected and the inhibition was reversible. The CO-ferrous P450 spectrum of P4501A1 at 448 nm was diminished when photoactivated azidowarfarins bound to and inactivated the enzyme, with essentially no formation of P420 except in the case of 4'-azidowarfarin. The inactivation of P4501A1 by photoactivated 4'-azidowarfarin was prevented by 50% by 1.2 mM R-warfarin or 0.3 mM 4'-nitrowarfarin, consistent with the latter being a better P4501A1 substrate than R-warfarin. The photoinactivation of P4501A1 by each of the azidowarfarins was prevented to variable extents by R-warfarin or by 4'-, 6-, 7-, or 8-nitrowarfarin. Taken together these results demonstrate that all four azidowarfarins are potentially useful photoaffinity probes of the substrate binding site amino acid residues of P450s.

Affinity Labels↗
PubMed1992

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA.

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.

Breast Neoplasms↗
PubMed1992

Phase II metabolism of warfarin in primary culture of adult rat hepatocytes.

We used adult rat hepatocytes in primary culture (HPC) as a model system to study the hepatic phase II metabolism of the anticoagulant warfarin. Hepatocytes were isolated by a collagenase perfusion technique and maintained for 24 hr in Waymouth's medium containing 0.1 mM (R)-warfarin. When HPC medium was analyzed by reverse phase high performance liquid chromatography with diode-array detection, 4'-, 6-, and 7-hydroxywarfarin were identified. Several putative conjugates were observed eluting between 13 and 18 min. Treatment of hepatocyte medium with beta-glucuronidase and sulfatase resulted in the loss of five putative conjugates and concomitant increases in 4'-, 6-, and 7-hydroxywarfarin and warfarin, suggesting that these metabolites and warfarin were conjugated. Use of the beta-glucuronidase inhibitor saccharic acid 1,4-lactone enabled the determination of the relative extents of conjugation of each metabolite by glucuronic acid and sulfate. Glucuronidation was the predominant pathway for 4'-hydroxywarfarin, whereas 6-hydroxywarfarin and warfarin occurred mainly as sulfate conjugates. In contrast, 7-hydroxywarfarin was converted to both glucuronide and sulfate conjugates. Exposure of HPC to phenobarbital resulted in a decrease in cytochrome P-450-mediated production of hydroxylated warfarin metabolites; however, an increase in the production of 8-hydroxywarfarin was observed when HPC were exposed to beta-naphthoflavone. Unique conjugation patterns were found when hydroxylated warfarins were substituted for warfarin in HPC medium. Both 7- and 8-hydroxywarfarin were converted to one sulfate and two glucuronide conjugates, whereas 4'-hydroxywarfarin was converted to a single glucuronide conjugate. A spectral library of these conjugates was used to identify the major conjugates of warfarin formed by rat HPC.

Animals↗