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Biomedical subjects

A Madan

Publications and source records attributed to A Madan.

At least 37 records · Page 2Linked to original sources
PubMed2000

In vitro inhibition and induction of human hepatic cytochrome P450 enzymes by modafinil.

The ability of modafinil to affect human hepatic cytochrome P450 (CYP) activities was examined in vitro. The potential for inhibition of CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4/5, and CYP4A9/11 by modafinil (5-250 microM) was evaluated with pooled human liver microsomes. Modafinil exhibited minimal capacity to inhibit any CYP enzyme, except CYP2C19. Modafinil inhibited the 4'-hydroxylation of S-mephenytoin, a marker substrate for CYP2C19, reversibly and competitively with a K(i) value of 39 microM, which approximates the steady-state C(max) value of modafinil in human plasma at a dosage of 400 mg/day. No irreversible inhibition of any CYP enzyme was observed, and there was no evidence of metabolism-dependent inhibition. The potential for induction of CYP activity was evaluated by exposing primary cultures of human hepatocytes to modafinil (10-300 microM). Microsomes were then prepared and assayed for CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4/5 activities. The mean activities of microsomal CYP1A2, CYP2B6, and CYP3A4/5 from modafinil-treated hepatocytes were higher (up to 2-fold) than those in the solvent-treated controls but were less than those produced by reference inducers of these enzymes. At high concentrations of modafinil (>/=100 microM), the mean activity of CYP2C9 was decreased (up to 60%) relative to that in the solvent controls. Overall, modafinil was shown to have effects on human hepatic CYP1A2, CYP2B6, CYP2C9, CYP2C19, and CYP3A4/5 activities in vitro. Although effects obtained in vitro are not always predictive of effects in vivo, such results provide a rational basis for understanding drug-drug interactions that are observed clinically and for planning subsequent investigations.

Benzhydryl Compounds↗
PubMed2000

Biotransformation of the anti-angiogenic compound SU5416.

SU5416 [3-(3,5-dimethyl-1H-pyrrol-2-ylmethylene)-1, 3-dihydro-indol-2-one], an inhibitor of VEGF (vascular endothelial growth factor) receptor tyrosine kinase, Flk-1/KDR (fetal liver kinase 1/kinase insert domain-containing receptor), also known as VEGF receptor 2 (VEGFR2) is in advanced clinical trials for treatment of AIDS-related Kaposi's sarcoma and colorectal and nonsmall cell lung cancers. Since this chemical class has not been studied previously with therapeutic intent, the present study was designed to investigate the in vitro metabolism of SU5416 by mouse, rat, dog, monkey, and human liver microsomes and to identify the major metabolites of SU5416. An HPLC procedure was developed and validated to resolve and quantify SU5416 and its metabolites. To evaluate the in vitro metabolism of SU5416, pooled liver microsomes from mice, rats, dogs, monkeys, and humans were incubated with SU5416 (25 microM) in the presence of an NADPH-generating system. In the presence of NADPH, mouse, rat, dog, monkey, and human liver microsomes converted SU5416 to at least 12, 9, 9, 7, and 6 polar metabolites, respectively. Microsomal metabolism of SU5416 showed marked species differences in the levels of different metabolites formed. The overall rate of SU5416 metabolism by liver microsomes from the species examined followed the rank order: monkey > or = mouse approximately rat > dog > human. Two major metabolites of SU5416 were identified, a hydroxymethyl derivative of SU5416 (M12) and a carboxylic acid derivative of SU5416 (M6), by spectroscopic methods and comparison with authentic compounds. Both of these oxidative metabolites were further metabolized in vivo through glucuronidation. The metabolic fate of SU5416 in microsomes from various species as well as data from in vivo biotransformation in the rat are discussed.

Angiogenesis Inhibitors↗
PubMed1999

Effects of musk xylene and musk ketone on rat hepatic cytochrome P450 enzymes.

The purpose of the present work was to characterize the effect of musk xylene (MX) and musk ketone (MK) treatment on rat hepatic cytochrome P450 enzymes. Male F344 rats were dosed orally with MX (10, 50 or 200 mg/kg) or MK (20, 100 or 200 mg/kg) for 7 days, after which CYP1A, 2B and 3A enzyme activities and protein levels were determined. MX treatment resulted in a two- to four-fold increase in the activity of CYP1A, 2B and 3A enzymes. For CYP1A and 3A, these changes were consistent with small increases in immunoreactive proteins. However, for CYP2B, despite only a three-fold increase in enzyme activity, protein levels were increased nearly 50-fold relative to control. This induction occurred by transcriptional activation of the CYP2B1 gene as evidenced by increased steady state CYP2B1 mRNA levels. In contrast to MX, MK treatment increased CYP2B activity, protein and mRNA levels. However MK treatment also increased CYP1A enzyme activity nearly 30-fold higher than control rats, a profile that was markedly different from MX, and very different from its effects in mice (Stuard, S.B., Caudill, D., Lehman-Mc-Keeman, L.D., 1997. Characterization of the effects of musk ketone on mouse cytochrome P450 enzymes. Fund. Appl. Toxicol. 40, 264-271). These results indicate that in rats, MX is an inducer of CYP2B enzymes, but these enzymes are not functionally active. In contrast, MK also induces CYP2B enzymes, with no concurrent inactivation. MK also exhibits a unique pattern of cytochrome P450 induction by increasing both CYP1A and CYP2B in rats.

Animals↗
PubMed1999

Induction of hepatic xenobiotic metabolizing enzymes in female Fischer-344 rats following repeated inhalation exposure to decamethylcyclopentasiloxane (D5).

Decamethylcyclopentasiloxane (D5) is a cyclic siloxane with a wide range of commercial applications. The present study was designed to investigate the effects of D5 on the expression and activity of selected rat hepatic phase I and phase II metabolizing enzymes. Female Fischer-344 rats were exposed to 160 ppm D5 vapors (6 h/day, 7 days/week, for 28 days) by whole-body inhalation. Changes in the activity and relative abundance of hepatic microsomal cytochromes P450 (CYP1A, CYP2B, CYP3A, and CYP4A), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were measured. Repeated inhalation exposure of rats to D5 increased liver size by 16% relative to controls by day 28. During a 14-day post-exposure period, liver size in D5-exposed animals showed significant recovery. Exposure to D5 did not change total hepatic P450, but increased the activity of hepatic NADPH-cytochrome c reductase by 1.4-fold. An evaluation of cytochrome P450 (CYP) enzymes in hepatic microsomes prepared from D5-exposed rats revealed a slight (1.8-fold) increase in 7-ethoxyresorufin O-deethylase (EROD) activity, but no change in immunoreactive CYP1A1/2 protein. A moderate increase (4.2-fold) in both 7-pentoxyresorufin O-depentylase (PROD) activity and immunoreactive CYP2B1/2 protein (3.3-fold) was observed. Testosterone 6beta-hydroxylase activity was also increased (2.4-fold) as was CYP3A1/2 immunoreactive protein. Although a small increase in 11- and 12-hydroxylation of lauric acid was detected, no change in immunoreactive CYP4A levels was measured. Liver microsomal epoxide hydrolase activity and immunoreactive protein increased 1.7- and 1.4-fold, respectively, in the D5-exposed group. UDPGT activity toward chloramphenicol was induced 1.8-fold, while no change in UDPGT activity toward 4-nitrophenol was seen. These results suggest that the profile for enzyme induction following inhalation exposure of female Fischer-344 rats to D5 vapors is similar to that reported for phenobarbital, and therefore D5 may be described as a weak "phenobarbital-like" inducer.

Administration, Inhalation↗
PubMed1999

CAP3: A DNA sequence assembly program.

We describe the third generation of the CAP sequence assembly program. The CAP3 program includes a number of improvements and new features. The program has a capability to clip 5' and 3' low-quality regions of reads. It uses base quality values in computation of overlaps between reads, construction of multiple sequence alignments of reads, and generation of consensus sequences. The program also uses forward-reverse constraints to correct assembly errors and link contigs. Results of CAP3 on four BAC data sets are presented. The performance of CAP3 was compared with that of PHRAP on a number of BAC data sets. PHRAP often produces longer contigs than CAP3 whereas CAP3 often produces fewer errors in consensus sequences than PHRAP. It is easier to construct scaffolds with CAP3 than with PHRAP on low-pass data with forward-reverse constraints.

Algorithms↗
PubMed1999

Effect of cryopreservation on cytochrome P-450 enzyme induction in cultured rat hepatocytes.

In the present study, we evaluated the inducibility of cytochrome P-450 (CYP) CYP1A, CYP2B, CYP3A, and CYP4A by beta-naphthoflavone, phenobarbital, dexamethasone, and clofibric acid, respectively, in primary hepatocyte cultures prepared from both fresh and cryopreserved rat hepatocytes. Rat hepatocytes were successfully thawed and cultured after cryopreservation in liquid nitrogen for up to 1 month. Percentage of total recovery, viable cell recovery, and final viability of the cells were 68%, 72%, and 85%, respectively. Regardless of whether they were cryopreserved or not, cultured hepatocytes exhibited near-normal morphology. Treatment of cryopreserved hepatocytes with beta-naphthoflavone caused an 8-fold increase in 7-ethoxyresorufin O-dealkylase (CYP1A1/2) activity, with an EC50 of 1.5 microM; treatment with phenobarbital caused a 26-fold increase in 7-pentoxyresorufin O-dealkylase (CYP2B1/2) activity, with an EC50 of 10 microM; treatment with dexamethasone caused a 10-fold increase in testosterone 6beta-hydroxylase (CYP3A1/2) activity, with an EC50 of 1.3 microM, whereas treatment with clofibric acid caused a 3-fold increase in lauric acid 12-hydroxylase (CYP4A1-3) activity, with an EC50 of 170 microM. The induction of CYP1A, CYP2B, CYP3A, and CYP4A enzymes by these inducers was confirmed by Western immunoblotting. The patterns of P-450 induction in cryopreserved rat hepatocytes, in terms of concentration response, reproducibility, magnitude, and specificity of response, were similar to those observed in freshly isolated hepatocytes. Additionally, the magnitude and specificity of induction was similar to that observed in vivo in rats. In conclusion, under the conditions examined, cryopreserved rat hepatocytes appear to be a suitable in vitro system for evaluating xenobiotics as inducers of P-450 enzymes.

Animals↗
PubMed1999

Role of CYP2B6 and CYP3A4 in the in vitro N-dechloroethylation of (R)- and (S)-ifosfamide in human liver microsomes.

The central nervous system toxicity of ifosfamide (IFF), a chiral antineoplastic agent, is thought to be dependent on its N-dechloroethylation by hepatic cytochrome P-450 (CYP) enzymes. The purpose of this study was to identify the human CYPs responsible for IFF-N-dechloroethylation and their corresponding regio- and enantioselectivities. IFF exists in two enantiomeric forms, (R) - and (S)-IFF, which can be dechloroethylated at either the N2 or N3 positions, producing the corresponding (R,S)-2-dechloroethyl-IFF [(R, S)-2-DCE-IFF] and (R,S)-3-dechloroethyl-IFF [(R,S)-3-DCE-IFF]. The results of the present study suggest that the production of (R)-2-DCE-IFF and (S)-3-DCE-IFF from (R)-IFF is catalyzed by different CYPs as is the production of (S)-2-DCE-IFF and (R)-3-DCE-IFF from (S)-IFF. In vitro studies with a bank of human liver microsomes revealed that the sample-to-sample variation in the production of (S)-3-DCE-IFF from (R)-IFF and (S)-2-DCE-IFF from (S)-IFF was highly correlated with the levels of (S)-mephenytoin N-demethylation (CYP2B6), whereas (R)-2-DCE-IFF production from (R)-IFF and (R)-3-DCE-IFF production from (S)-IFF were both correlated with the activity of testosterone 6beta-hydroxylation (CYP3A4/5). Experiments with cDNA-expressed P-450 and antibody and chemical inhibition studies supported the conclusion that the formation of (S)-3-DCE-IFF and (S)-2-DCE-IFF is catalyzed primarily by CYP2B6, whereas (R)-2-DCE-IFF and (R)-3-DCE-IFF are primarily the result of CYP3A4/5 activity.

Animals↗
PubMed1999

Influence of extracellular matrix overlay and medium formulation on the induction of cytochrome P-450 2B enzymes in primary cultures of rat hepatocytes.

The effect of medium formulation, composition of extracellular matrix overlay, and culture dish material on liver microsomal cytochrome P-450 (CYP) 2B induction by phenobarbital (PB) was investigated in primary cultures of rat hepatocytes. When hepatocytes were maintained on Permanox dishes with an overlay of either collagen (type I) or Matrigel, Williams' E medium was superior to other medium formulations in terms of the magnitude of induction of CYP2B on a per milligram microsomal protein basis. Modified Chee's medium (MCM) and hepatocyte culture medium were intermediate in their capacity to sustain induction of CYP2B by PB, and Dulbecco's modified Eagle's medium was slightly less effective. The overall induction of CYP2B activity by PB was, on average, 50% lower in hepatocytes cultured on polystyrene dishes (LUX). Little or no difference was observed between hepatocytes overlaid with collagen and those overlaid with Matrigel. MCM was superior to Williams' E medium in terms of the yield of microsomal protein and the ultrastructural features of the hepatocyte monolayers. CYP2B induction by PB was optimal after 3 days of treatment in either medium. CYP1A, CYP3A, and CYP4A activities could be induced in vitro by prototypical inducing agents in hepatocytes cultured on Permanox dishes with MCM and a Matrigel overlay to comparable levels observed in vivo. The results of these studies show that medium formulation and culture vessel material, but not the type of extracellular matrix overlay, have significant effects on the induction of CYP enzymes in cultured rat hepatocytes maintained in a sandwich configuration.

Animals↗
PubMed1999

Cloning, expression and purification of the DNA binding domain of RFX protein.

The RFX DNA binding domain (DBD) is a novel highly conserved motif belonging to a large number of dimer DNA binding proteins which have diverse regulatory functions in eukaryotic organisms. To characterize this novel motif, a 78mer polypeptide corresponding to the DBD of human hRFX (hrfX1/DBD), a prototypical member of the RFX family has been cloned and overproduced in Escherichia coli. A purification procedure using cation exchange chromatography has also been developed.

Amino Acid Sequence↗
PubMed1998

Genetic evaluation of suspected cases of transient HIV-1 infection of infants.

Detection of human immunodeficiency virus-type 1 (HIV-1) on only one or a few occasions in infants born to infected mothers has been interpreted to indicate that infection may be transient rather than persistent. Forty-two cases of suspected transient HIV-1 viremia among 1562 perinatally exposed seroreverting infants and one mother were reanalyzed. HIV-1 env sequences were not found in specimens from 20; in specimens from 6, somatic genetic analysis revealed that specimens were mistakenly attributed to an infant; and in specimens from 17, phylogenetic analysis failed to demonstrate the expected linkage between the infant's and the mother's virus. These findings argue that transient HIV-1 infection, if it exists, will only rarely be satisfactorily documented.

DNA, Viral↗
PubMed1998

Evaluation of octamethylcyclotetrasiloxane (D4) as an inducer of rat hepatic microsomal cytochrome P450, UDP-glucuronosyltransferase, and epoxide hydrolase: a 28-day inhalation study.

Repeated inhalation exposure to octamethylcyclotetrasiloxane (D4) produces a reversible and dose-related hepatomegaly and proliferation of hepatic endoplasmic reticulum in rats. However, the effects of D4 on the expression of cytochrome P450 enzymes have not been evaluated. In the present study, the time course for changes in hepatic microsomal cytochrome P450 enzyme expression following repeated inhalation exposure to D4 vapors was determined in male and female Fischer 344 rats. Animals were exposed to D4 vapor at concentrations of 70 and 700 ppm, via whole body inhalation for 6 h/day, 5 days/week for 4 weeks. Specified animals were euthanized on exposure days 3, 7, 14, 21, and 28. Microsomal fractions were prepared from fresh liver by differential centrifugation. Enzyme activity as well as immunoreactive protein levels of several cytochrome P450 enzymes (CYP), epoxide hydrolase, and UDP-glucuronosyltransferase (UDPGT) were evaluated. The time course for enzyme induction was monitored by measuring 7-ethoxyresorufin O-deethylase (EROD) and 7-pentoxyresorufin O-depentylase (PROD) activities on days 3, 7, 14, 21, and 28. CYP1A1/2 activity, as determined by EROD activity, was increased approximately 2- to 3-fold over the exposure period. However, an examination of immunoreactive protein revealed no induction of CYP1A1 and a suppression of CYP1A2 in the 700 ppm D4 group. In comparison, CYP2B1/2 enzyme activity, as determined by PROD, was significantly increased as early as day 3 in both the 70 and 700 ppm D4 groups of male and female rats. Overall, PROD activity on day 28 was induced more than 10-fold in the 70 ppm D4 groups and more than 20-fold in the 700 ppm D4 groups. The increase in PROD activity was paralleled by a comparable increase in CYP2B1/2 immunoreactive protein. There was a modest (2- to 3-fold) increase in CYP3A1/2 activity and immunoreactive protein, as determined by 6 beta-hydroxylation of testosterone and Western blot analysis. Expression of CYP enzymes was at or near maximum by day 14 and remained relatively constant throughout the exposure period. On day 28, epoxide hydrolase activity and immunoreactive protein were induced (2- to 3-fold) in a dose-dependent manner. Only slight changes in the expression and activity of UDPGT were detected, and these did not appear to be dose related. Thus, repeated inhalation exposure to D4 induces CYP enzymes and epoxide hydrolase in a manner similar to that observed for phenobarbital (PB). Therefore, D4 can be described as a "PB-like" inducer of hepatic microsomal enzymes in the Fischer 344 rat.

Adjuvants, Immunologic↗
PubMed1998

Regulation of human erythropoietin gene induction by upstream flanking sequences in transgenic mice.

Human erythropoietin (Epo) gene expression is inducible by hypoxia or anaemia in the kidney and liver. Previous transgenic mouse experiments have demonstrated that sequences required for Epo gene induction in the kidney reside in a 7 8 kb Barn HI fragment located 6 kb upstream of the gene. To sublocalize these sequences, we performed Desoxyribonuclease I (DNAse I) mapping studies using transgenic mice which carried this DNA fragment. These studies revealed a DNAse I hypersensitive site (DNAse I HS) located 4 6 kb from the upstream end of the 7.8 kb fragment in anaemic kidney and liver samples. Sequence analysis of the region encompassing the DNAse I HS revealed an element with remarkable homology to the 3' Epo gene hypoxia-inducible enhancer. This suggested the presence of an additional regulatory element that contributes to the control of hypoxia-inducible Epo gene expression in kidney and liver. We constructed transgenic mice containing the human Epo gene linked to either the 5 kb upstream or 2.5 kb downstream portion of the 7.8kb fragment. Inducible expression was limited to the liver. Thus, neither fragment was alone sufficient to confer kidney inducible expression. These findings indicate that sequences more than 8.5 kb upstream of the Epo gene are required for kidney-specific induction. They suggest that either those sequences reside in an 0.3 kb Hind III fragment located between the 5 kb and the 2.5 kb fragments or that sequences in the 5 kb or 0.3 kb fragments must interact with sequences in the 2.5 kb fragment to allow Epo gene induction in the kidney.

Anemia↗
PubMed1998

Serial serum C-reactive protein levels in the diagnosis of neonatal infection.

OBJECTIVE: To evaluate serial serum C-reactive protein (CRP) levels for diagnosis of neonatal infection. SETTING: A regional intensive care nursery and two community intensive care nurseries. METHODS: All neonates treated for suspected bacterial infection were prospectively evaluated using a standardized clinical pathway. Infants were categorized as having proven sepsis (bacteria isolated from blood, cerebrospinal fluid, or urine culture), probable sepsis (clinical and laboratory findings consistent with bacterial infection without a positive culture), or no sepsis (findings not consistent with sepsis), without consideration of CRP levels. Infants whose blood cultures yielded skin flora but who demonstrated no other signs of bacterial infection were not considered to have sepsis. CRP levels were determined at the initial evaluation and on each of the next two mornings. Sensitivity, specificity, predictive values, and likelihood ratios were calculated for the first (CRP #1), second (CRP #2), higher of the second and third (CRP #2 and #3), or highest of all three CRP levels (CRP x 3). RESULTS: Sepsis was suspected within the first 3 days after birth in 1002 infants (early-onset) and on 184 occasions in 134 older infants (late-onset). There were 20 early-onset and 53 late-onset episodes of proven sepsis, and 74 early-onset and 12 late-onset episodes of probable sepsis. CRP #1 had sensitivities of 39.4% and 64.6% for proven or probable sepsis and 35.0% and 61.5% for proven sepsis in early-onset and late-onset episodes, respectively. CRP levels on the morning after the initial evaluation (CRP #2) had higher sensitivities (92. 9% and 85.0% for proven or probable sepsis and 78.9% and 84.4% for proven sepsis in early-onset and late-onset episodes, respectively), and normal results were associated with lower likelihoods of infection (likelihood ratios for normal results of 0.10 and 0.19 for proven or probable sepsis and 0.27 and 0.21 for proven sepsis, in early-onset and late-onset episodes, respectively). Three serial serum CRP levels had sensitivities of 97.8% and 98.1% for proven or probable sepsis and 88.9% and 97.5% for proven sepsis in early-onset and late-onset episodes, respectively. The negative predictive values for CRP x 3 were 99.7% and 98.7% for both proven or probable sepsis and for proven sepsis in early-onset and late-onset episodes, respectively. A CRP level obtained at the time of the initial evaluation can be omitted without significant loss of sensitivity or negative predictive value: the sensitivities of CRP #2 and #3 were 97.6% and 94.4% for proven or probable sepsis and 88.9% and 96.4% for proven sepsis in early-onset and late-onset episodes, respectively; negative predictive values were 99.7% both for proven and for proven or probable early-onset sepsis, 97.6% for proven or probable late-onset infection, and 98.8% for proven late-onset infection. Serial normal CRP levels were associated with a markedly reduced likelihood of infection as compared with that in the entire population before testing, with likelihood ratios ranging from 0.03 to 0.16 for the various subgroups. Maximum CRP levels >3 mg/dL had positive predictive values >20% for proven or probable early-onset infections and for proven or probable and proven late-onset infections, but only those >6 mg/dL had such a high positive predictive value for proven early-onset sepsis. CONCLUSIONS: Serial CRP levels are useful in the diagnostic evaluation of neonates with suspected infection. Two CRP levels <1 mg/dL obtained 24 hours apart, 8 to 48 hours after presentation, indicate that bacterial infection is unlikely. The sensitivity of a normal CRP at the initial evaluation is not sufficient to justify withholding antibiotic therapy. The positive predictive value of elevated CRP levels is low, especially for culture-proven early-onset infections.

Bacteria↗
PubMed1998

Development of a non-high pressure liquid chromatography assay to determine testosterone hydroxylase (CYP3A) activity in human liver microsomes.

The major pathway of testosterone oxidation by human liver microsomes is the formation of 6beta-hydroxytestosterone, which is catalyzed by CYP3A4/5 and which accounts for 75-80% of all metabolites formed. In the present study, we describe a non-high pressure liquid chromatography assay (HPLC) of CYP3A4/5 activity based on the release of tritium (with formation of tritiated water) upon incubation of [1,2,6,7-3H]testosterone with human liver microsomes and NADPH. Unreacted testosterone and its metabolites were quantitatively extracted from the incubation mixture with activated charcoal under conditions that resulted in no extraction of tritiated water. The amount of tritiated water formed was quantified by liquid scintillation spectrometry and compared with the amount of hydroxylated testosterone metabolites formed, as determined by HPLC. Rates of tritium release from [1,2,6, 7-3H]testosterone paralleled rates of testosterone 6beta-hydroxylation as a function of incubation time, the amount of microsomal protein, and the concentration of substrate (which yielded identical apparent Km and Vmax values). The sample-to-sample variation in tritium release from [1,2,6,7-3H]testosterone with a panel of human liver microsomes was highly correlated with rates of testosterone 6beta-hydroxylation and terfenadine metabolism, two commonly used markers of CYP3A activity. Several recombinant human P450 enzymes were incubated with [1,2,6,7-3H]testosterone, and only cDNA-expressed CYP3A4 catalyzed a high rate of tritium release. The close agreement between the tritium-release assay and HPLC procedure for measuring testosterone oxidation indicates that tritium release from [1,2,6,7-3H]testosterone provides a simple and rapid alternative to the HPLC procedure for measuring CYP3A4/5 activity in human liver microsomes. However, the tritium-release assay may have limited value in measuring CYP3A activity in liver microsomes from other species due to the presence of other P450 enzymes that can catalyze tritium release from [1,2,6,7-3H]testosterone.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Development of a non-high pressure liquid chromatography assay to determine [14C]chlorzoxazone 6-hydroxylase (CYP2E1) activity in human liver microsomes.

The activity of liver microsomal CYP2E1 is commonly measured as the rate of 5-chloro-2-benzoxazolone (chlorzoxazone) 6-hydroxylation, which requires separation of 6-hydroxychlorzoxazone and chlorzoxazone by high pressure liquid chromatography (HPLC). In the present study, we describe a solvent extraction (non-HPLC) assay for measuring CYP2E1 activity, based on the 6-hydroxylation of [14C]chlorzoxazone. When [14C]chlorzoxazone was incubated with human or rat liver microsomes in the presence of NADPH, the major product formed was 6-[14C]hydroxychlorzoxazone. Unreacted [14C]chlorzoxazone was quantitatively extracted from the incubation mixture with dichloromethane under conditions that resulted in approximately 45% extraction of 6-[14C]hydroxychlorzoxazone. The amount of 6-[14C]hydroxychlorzoxazone remaining in the aqueous incubation mixture ( approximately 55% of the total amount formed) was quantified by liquid scintillation spectrometry. The limit of detection for this assay was 100 pmol of 6-[14C]hydroxychlorzoxazone. The solvent extraction procedure was validated by comparing the rates of formation of 6-[14C]hydroxychlorzoxazone with those determined by HPLC under a variety of experimental conditions. The close correspondence between the two analytical methods suggests that the extraction procedure for measuring 6-[14C]hydroxychlorzoxazone provides a simple, sensitive, and rapid alternative to the HPLC procedure for measuring CYP2E1 activity. In rats, the assay is not specific for CYP2E1 because CYP1A1 also catalyzes the 6-hydroxylation of chlorzoxazone. Recombinant human CYP1A1 also catalyzed the 6-hydroxylation of chlorzoxazone (at (1)/(5) the rate of CYP2E1), although CYP1A1 is not expressed in human liver microsomes. The non-HPLC assay was used to investigate the postulated role of CYP1A2 in the 6-hydroxylation of chlorzoxazone by human liver microsomes. Recombinant CYP1A2 did not catalyze the 6-hydroxylation of chlorzoxazone, and studies with 1-[(3,4-dimethoxyphenyl)methyl]-6,7-dimethoxyisoquinoline, which inhibits CYP1A2 but not CYP2E1, indicated that, in human liver microsomes, the 6-hydroxylation of chlorzoxazone is catalyzed by CYP2E1 with little or no contribution from CYP1A2 enzymes over a wide range of substrate concentrations.

Animals↗
PubMed1998

Identification of the human P-450 enzymes responsible for the sulfoxidation and thiono-oxidation of diethyldithiocarbamate methyl ester: role of P-450 enzymes in disulfiram bioactivation.

Diethyldithiocarbamate methyl ester (DDTC-Me) is a precursorto the formation of S-methyl-N,N-diethylthiolcarbamate sulfoxide, the active metabolite proposed to be responsible for the alcohol deterrent effects of disulfiram. The present study investigated the role of human cytochrome P-450 (CYP) enzymes in sulfoxidation and thiono-oxidation of DDTC-Me, intermediary steps in the activation of disulfiram. Several approaches were used in an attempt to delineate the particular P-450 enzyme(s) involved in the sulfoxidation and thiono-oxidation of DDTC-Me. These approaches included the use of cDNA-expressed human P-450 enzymes, correlation analysis with sample-to-sample variation in human P-450 enzymes in a bank of human liver microsomes, and chemical and antibody inhibition studies. Multiple human P-450 enzymes (CYP3A4, CYP1A2, CYP2A6, and CYP2D6) catalyzed the sulfoxidation of DDTC-Me, as determined with cDNA-expressed enzymes. Several lines of evidence suggest that the sulfoxidation of DDTC-Me by human liver microsomes is primarily catalyzed by CYP3A4/5, including (1) a high correlation between DDTC-Me sulfoxidation and testosterone 6beta-hydroxylation; (2) increased DDTC-Me sulfoxidation in the presence of alpha-naphthoflavone, an activator of CYP3A enzymes; (3) inhibition of this reaction by inhibitors of CYP3A4/5 enzymes, such as troleandomycin and ketoconazole; and (4) inhibition of DDTC-Me sulfoxidation by antibodies against CYP3A enzymes. On the other hand, several lines of evidence suggested that the thiono-oxidation of DDTC-Me by human liver microsomes is catalyzed in part by CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5, including (1) these human P450 enzymes among others have the capacity to catalyze this reaction, as determined with cDNA-expressed enzymes; (2) a high correlation between DDTC-Me thiono-oxidation and testosterone 6beta-hydroxylation, weak inhibition by ketoconazole, troleandomycin, and anti-CYP3A antibodies suggested a minor role for CYP3A4; (3) a high correlation with immunoreactive CYP2B6 suggested involvement of this enzyme; (4) weak inhibition of DDTC-Me thiono-oxidation by furafylline and anti-CYP1A antibody suggested involvement of CYP1A2; and (5) inhibition of DDTC-Me thiono-oxidation by DDTC and anti-CYP2E antibodies suggested a role for CYP2E1. Collectively, these data suggested CYP3A4/5 enzymes are the major contributors to the sulfoxidation of DDTC-Me by human liver microsomes, and CYP1A2, CYP2B6, CYP2E1, and CYP3A4/5 contribute toward DDTC-Me thiono-oxidation by human liver microsomes. This study, in conjunction with others (Madan et al., Drug Metab. Dispos. 23:1153-1162, 1995), may help explain the variability in disulfiram's effectiveness as an alcohol deterrent.

Alcohol Deterrents↗