Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells

Barbara C. Krovat, Julia H. Tracy, Curtis John Omiecinski

Research output: Contribution to journalArticle

55 Citations (Scopus)

Abstract

To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase- coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 105 molecules/μg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 105 and 1.8 x 105 molecules/μg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 104 molecules/μg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., β-naphthoflavone (β-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.

Original languageEnglish (US)
Pages (from-to)352-360
Number of pages9
JournalToxicological Sciences
Volume55
Issue number2
StatePublished - 2000

Fingerprint

Epoxide Hydrolases
Gene expression
Cytochrome P-450 Enzyme System
Blood Cells
Lymphocytes
Blood
Cells
Gene Expression
Cell Line
Cytochrome P-450 CYP3A
Cytochrome P-450 CYP2E1
Cytochrome P-450 CYP2D6
Cytochrome P-450 CYP1A1
RNA
Genes
Messenger RNA
Molecules
Assays
Cytochrome P-450 CYP1A2
Phenobarbital

All Science Journal Classification (ASJC) codes

  • Toxicology

Cite this

Krovat, Barbara C. ; Tracy, Julia H. ; Omiecinski, Curtis John. / Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells. In: Toxicological Sciences. 2000 ; Vol. 55, No. 2. pp. 352-360.
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abstract = "To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase- coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 105 molecules/μg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 105 and 1.8 x 105 molecules/μg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 104 molecules/μg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., β-naphthoflavone (β-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.",
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Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells. / Krovat, Barbara C.; Tracy, Julia H.; Omiecinski, Curtis John.

In: Toxicological Sciences, Vol. 55, No. 2, 2000, p. 352-360.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Fingerprinting of cytochrome P450 and microsomal epoxide hydrolase gene expression in human blood cells

AU - Krovat, Barbara C.

AU - Tracy, Julia H.

AU - Omiecinski, Curtis John

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N2 - To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase- coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 105 molecules/μg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 105 and 1.8 x 105 molecules/μg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 104 molecules/μg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., β-naphthoflavone (β-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.

AB - To examine the character and variability of human cytochrome P450 (CYP) and microsomal epoxide hydrolase (mEH) gene expression in human blood cells, we used a highly sensitive, quantitative, competitive reverse transcriptase- coupled polymerase chain reaction (QC RT-PCR) assay to assess mRNA profiles for a battery of 8 genes, in peripheral lymphocytes isolated from 10 healthy donors. Of the genes profiled, in lymphocytes CYP2D6 was typically expressed at the highest levels (3.8 x 105 molecules/μg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 105 and 1.8 x 105 molecules/μg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 104 molecules/μg total RNA), followed by CYP2F1 and CYP3A levels that were near the detection limit of the assay. CYP1A2 and CYP2A6/7 mRNAs were not detected in any of the lymphocyte samples. Overall, relatively low levels of inter-individual variation (2- to 6-fold) existed among these endpoint parameters in the subjects tested. To test whether established human blood cell lines were suitable models to assess basal expression and chemical induction responsiveness of these genes, we determined that constitutive CYP and mEH mRNA profiles were essentially conserved across 4 established human blood cell lines, and highly analogous to the basal expression patterns identified in freshly isolated peripheral lymphocytes. mEH protein was detected in all of the cell lines using Western immunoblotting and chemiluminescent visualization, whereas CYP1A1, CYP2D6, CYP2E1 or CYP3A proteins were not detected in these analyses. When blood cell-derived cultures were exposed to the prototypical CYP1A and CYP3A inducers, i.e., β-naphthoflavone (β-NF), dexamethasone (DEX) or phenobarbital, generally little or no inductive response was manifested. Thus, the data obtained from this investigation indicate that, although human blood cell lines in general exhibit poor responsiveness to prototypical inducer exposures, the constitutive patterns of CYP and mEH expression in peripheral lymphocytes appear to exhibit relatively low levels of variation among individuals. In addition, these in vivo patterns of expression are well maintained in established cultured blood-cell lines.

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