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Technical Briefs |
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Industrial Toxicology and Occupational Medicine Unit, Catholic University of Louvain, Clos Chapelle-aux-Champs, 30.54, 1200 Brussels, Belgium
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Endocrinology and Nutrition and
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Applied Molecular Technologies, Catholic University of Louvain Hospital, 1200 Brussels, Belgium
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Queen Astrid Military Hospital, 1120 Brussels, Belgium
aauthor for correspondence: fax 32-2-764-3228, e-mail haufroid{at}toxi.ucl.ac.be
Assessing the activity of a particular drug-metabolizing enzyme (i.e., its phenotypic status) has practical implications in the fields of therapeutic drug monitoring and clinical toxicology as well as for the biological monitoring of environmental and/or occupational exposure to chemical agents. This report describes a novel, convenient, and rapid analytical method for phenotyping cytochrome P4502E1 (CYP2E1), an important drug-metabolizing enzyme in industrial toxicology (1). The measurement of CYP2E1 expression also has applications for monitoring alcoholic patients (2).
Although the most accurate assessment of CYP2E1 activity is based on direct hepatic measurement, which requires a liver biopsy, alternative noninvasive approaches have been developed. Until now, the "gold standard" method for CYP2E1 phenotyping has used a probe drug, chlorzoxazone (CZX), administered orally (500 or 250 mg) to fasting individuals and metabolized to 6-hydroxychlorzoxazone (HCZX) by CYP2E1. Various CZX pharmacokinetic measures have been proposed to reflect CYP2E1 activity, as reviewed recently (3): plasma CZX t1/2ß, HCZX renal excretion, CZX oral clearance, fractional clearance to HCZX, and HCZX area under the concentration curve (AUC). However, these procedures are time-consuming, laborious (e.g., urine collection), and require subject compliance and catheterization. Furthermore, recent in vitro studies suggest that CYP1A1, CYP1A2, and possibly CYP3A are involved in CZX 6-hydroxylation, limiting the specificity of CZX for CYP2E1 phenotyping (3).
CYP2E1 phenotyping for screening or follow-up of large numbers of individuals, in epidemiological studies, or even as an adjunct to improve biological monitoring programs requires the implementation of more convenient methods. Circulating lymphocytes have been used as surrogates because CYP2E1 expression in these cells appears to parallel the concentration of the hepatic enzyme, suggesting that both cell types are influenced by the same factors, including xenobiotics and physiological state (4). In a recent report involving alcoholics and controls (2), pharmacokinetic parameters for CZX hydroxylation (CZX clearance rate and CZX AUC) were compared with CYP2E1 protein and mRNA content in human peripheral blood lymphocytes (HPBLs). Alcoholics exhibited a twofold increase in lymphocyte CYP2E1 mRNA and protein compared with nonalcoholics. CZX clearances rates were 1.9-fold higher and CZX AUC values were 1.8-fold lower in alcoholic individuals compared with nonalcoholics. Furthermore, CZX clearance rates correlated (r = 0.55; P <0.01) with lymphocyte CYP2E1 mRNA content, whereas transcription also correlated (r = 0.52; P <0.001) with CYP2E1 protein content in lymphocytes (2). However, determination of CYP2E1 protein content required large amounts of blood (320 mL). Determination of lymphocyte CYP2E1 expression by the measurement of specific mRNA may therefore provide a noninvasive alternative for estimating hepatic activity of this enzyme. Accordingly, practical and easier methods for CYP2E1 mRNA measurement in HPBLs could be developed for routine use. We report here a method for real-time quantification of CYP2E1 mRNA in HPBLs using TaqMan® (Perkin-Elmer Biosystems) technology (5).
The cDNA sequence of CYP2E1 was obtained from GenBank (Accession No. J02625). The PCR primers (forward primer, 5'-AACTGTCCCCGGGACCTC-3', nucleotides 790–807; reverse primer, 5'-GCGCTCTGCACTGTGCTTT-3', nucleotides 858–840) and fluorescent probe (5'-CCATTTCCACGAGCAGGCAGTCG-3', nucleotides 832–810) were designed using PrimerExpress software (PE Biosystems). To avoid amplification of possible contaminating genomic DNA, the selected forward and reverse primers were designed from exons 5 and 6, with the predicted size of the amplified cDNA product being 69 bp.
The plasmid was constructed using the TOPO TA Cloning® Kit (Invitrogen) according to the manufacturer’s protocol. Briefly, the pair of primers was designed to amplify a 458-bp fragment spanning CYP2E1 exons 4 and 6, including the 69-bp amplicon (forward primer, 5'-TTTCGACCCCACCTTCCTCATCGG-3', nucleotides 507–530; reverse primer, 5'-ATTTCATGAGAATCAGGAGCCCA-3', nucleotides 964–942). The cDNA sequence was cloned into the pCR®2.1-TOPO® vector. The Escherichia coli strain HB101 was chemically transformed, and colonies were selected according to the manufacturer’s instructions. The recombinant plasmid was assessed by sequence analysis. Analysis was carried out in both orientations on an automated ABI 377 A apparatus (Perkin-Elmer Applied Biosystems), using the Taq Dye Deoxy Terminator Cycle Sequencing reagent set from the same manufacturer and according to its instructions. A stock solution of recombinant plasmid DNA was prepared using Nucleospin Plasmid® (cat. no. 740588-50; Macherey-Nagel), according to the manufacturer’s protocol, and stored at -20 °C until use. The remaining plasmid was conserved in glycerol for long-term storage (-80 °C in a cryovial).
Blood lymphocytes were isolated from EDTA-anticoagulated blood (7.5 mL) by centrifugation using a sterile lymphocyte separation medium (d = 1.077; ref. no. 85.89.837; Inter Medical). The lymphocyte layer was washed with phosphate-buffered saline (cat. no. 17-517Q; BioWhittaker). Total RNA was then isolated using TRIzol® reagent (cat. no. 15596-018, Life Technologies) followed by the classic chloroform extraction and isopropanol precipitation method, and quantified by measuring its absorbance at 260 nm. Total RNA (2.5 µg) was reverse transcribed with random hexamers (ref. no. OL-0021; Eurogentec) and SuperScript RT RNase H- reverse transcriptase (cat. no. 18053-017; Life Technologies) in a final reaction volume of 45 µL.
PCR was performed using TaqMan Universal PCR Master Mix reagent set (cat. no. 4304447; PE Biosystems) in a TaqMan 7700 Sequence Detection System (PE Biosystems) according to the manufacturer’s protocol. The primers and fluorescent probes were used at final concentrations of 300 and 200 nmol/L, respectively. The probe contained a 5'-terminal fluorescein "reporter" [6-carboxyfluorescein (FAM)] and a 3'-terminal 6-carboxytetramethylrhodamine (TAMRA) "quencher". The AmpliTaq Gold DNA polymerase was included in the TaqMan Universal PCR Master Mix. The reaction was initiated at 50 °C for 2 min to allow the incorporation of dUTP and incubated at 95 °C for 10 min to activate the polymerase, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 1 min. We verified that genomic DNA (theoretical fragment size, 951 bp) did not amplify under these PCR conditions.
A calibration curve was generated from serial dilutions of the CYP2E1
cDNA plasmid (Fig. 1A
). This calibration curve was linear, as shown by a correlation
coefficient of >0.99 over four orders of magnitude. Moreover, the
slope (-3.400) was close to the theoretical optimum (-3.322),
which indicates a very high PCR efficiency. The y-intercept
(38.457 cycles) for the highly diluted plasmid preparation was
comparable to the threshold cycle (Ct) value for a negative
control. The absolute number of CYP2E1 cDNA copies contained in 0.28
µg of total RNA (5 µL from 45 µL containing 2.5 µg of total
RNA) could be calculated using the calibration curve. A similar
approach was conducted for the cyclophilin housekeeping gene (data not
shown), and final results were expressed as the ratio CYP2E1 cDNA
copies/µg of total RNA or as the ratio CYP2E1 cDNA copies/cyclophilin
cDNA copies.
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To verify that this method specifically and quantitatively measures
mRNA, we ran a series of CYP2E1 reverse transcription (RT)-PCR assays
on undiluted and serially diluted (1:5, 1:25, 1:125) PBL RNA extract.
The number of copies measured was strictly proportional to the
dilution, as illustrated in Fig. 1B
, and the slope was equivalent to
the number of copies in the undiluted sample (r =
0.9989; P = 0.0011). The CV of the whole RT-PCR
method was 19% (n = 4). Comparison of quantitative data
on CYP2E1 and cyclophilin mRNA from blood samples processed immediately
or stored at room temperature for up to 6 h gave similar results
(data not shown).
Our real-time RT-PCR assay was applied to the measurement of CYP2E1
mRNA expression in HPBLs from subjects with poorly controlled diabetes
[glycohemoglobin (HbA1c) >6%] and
controls (HbA1c <6%) matched for age and sex
(Table 1
). All subjects gave informed consent. The lymphocytes were
processed for RNA extraction and reverse transcription within a
maximum of 6 h after sampling. Results presented in Table 1
clearly show that CYP2E1 mRNA expression is notably higher in HPBLs
from subjects with poorly controlled diabetes, a result in agreement
with a report by Song et al. (6), who noted a similar
increase in the protein concentration. Furthermore, results obtained
for control subjects (expressed as the ratio CYP2E1 cDNA copies/µg of
total RNA) were in accordance with those observed by Raucy et al.
(2) (expressed as CYP2E1 mRNA copies/µg of total RNA).
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The present method is rapid and convenient and allows phenotyping of large populations for CYP2E1 expression in epidemiological studies, biomonitoring programs, and for monitoring alcoholic patients.
Acknowledgments
This study was supported by the National Fund for Scientific Research, Belgium, and by the European Commission (Environment and Health, 5th Framework Program, Contract QLRT-1999-01368). The expert assistance of M. Heusterspreute and I. Millard is gratefully acknowledged.
References
The following articles in journals at HighWire Press have cited this article:
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  P. M. Vyas, S. Roychowdhury, F. D. Khan, T. E. Prisinzano, J. Lamba, E. G. Schuetz, J. Blaisdell, J. A. Goldstein, K. L. Munson, R. N. Hines, et al. Enzyme-Mediated Protein Haptenation of Dapsone and Sulfamethoxazole in Human Keratinocytes: I. Expression and Role of Cytochromes P450 J. Pharmacol. Exp. Ther., October 1, 2006; 319(1): 488 - 496. [Abstract] [Full Text] [PDF]
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 P. Soucek, P. Anzenbacher, I. Skoumalova, and M. Dvorak Expression of Cytochrome P450 Genes in CD34+ Hematopoietic Stem and Progenitor Cells Stem Cells, September 1, 2005; 23(9): 1417 - 1422. [Abstract] [Full Text] [PDF]
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 M. R. Sharma, P. Periandythevar, and B. H. Shapiro SPURIOUS OBSERVATION OF SPLENIC CYP2B1 EXPRESSION Drug Metab. Dispos., September 1, 2003; 31(9): 1074 - 1076. [Abstract] [Full Text] [PDF]
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