Fingerprinting of Cytochrome P450 and Microsomal Epoxide Hydrolase Gene Expression in Human Blood Cells

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 10(5) molecules/microg total RNA), with CYP2E1 and mEH also maintained at relatively high abundance (1.2 x 10(5) and 1.8 x 10(5) molecules/microg total RNA, respectively). CYP1A1 levels were approximately an order of magnitude lower (3.9 x 10(4) molecules/microg 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., beta-naphthoflavone (beta-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.

Transcription Factor and Drug-Metabolizing Enzyme Gene Expression in Lymphocytes from Healthy Human Subjects

“…In addition, we also found CYP2A6 in contrast to previous studies (Koskela et al, 1999;Krovat et al, 2000). As for the lack of expression of CYP3A4 and CYP3A5 in our subjects, these findings are not in agreement with others previously reported (Nakamoto et al, 2000;Finnström et al, 2001;Gashaw et al, 2003).…”

“…As for the lack of expression of CYP3A4 and CYP3A5 in our subjects, these findings are not in agreement with others previously reported (Nakamoto et al, 2000;Finnström et al, 2001;Gashaw et al, 2003). Krovat et al (2000), who were not able to detect CYP3A5 and whose detection of CYP3A4 was near the detection limit of the assay, proposed as an explanation the preferential localization of CYP3A in B cells, which constitute only a small part of total lymphocytes.…”

“…Epoxide hydrolase (EH), GST, N-acetyltransferase, and ST also have been described (Raucy et al, 1997;Baron et al, 1998;Dassi et al, 1998;Spencer et al, 1999;Stärkel et al, 1999;Takeda et al, 1999;Boucher et al, 2000;Krovat et al, 2000;Nakamoto et al, 2000;Nguyen et al, 2000;Smart and Daly, 2000;Finnström et al, 2001Finnström et al, , 2002Hannon-Fletcher et al, 2001;Asghar et al, 2002;Carcillo et al, 2003;Gashaw et al, 2003;Landi et al, 2003Landi et al, , 2005Lin et al, 2003;Toide et al, 2003;Tuominen et al, 2003;Furukawa et al, 2004;Lampe et al, 2004;Yamamoto et al, 2004;Haas et al, 2005;Liangpunsakul et al, 2005;Miura et al, 2006). However, study of the expression of both DME and TF is rarely conducted at the same time.…”

Interindividual Variability and Tissue-Specificity in the Expression of Cytochrome P450 3A mRNA

“…The numbers in parentheses depict the number of organs from which total RNA was isolated and pooled by the manufacturer of the panel. (Janardan et al, 1996;Hukkanen et al, 1997;Sempoux et al, 1999;Starkel et al, 1999;Krovat et al, 2000;Nakamoto et al, 2000;Asghar et al, 2002;Finnstrom et al, 2002;Nowakowski-Gashaw et al, 2002). With the specific and sensitive TaqMan assays we developed, none of the CYP3A transcripts was detectable in peripheral blood leukocytes.…”

“…In addition, since CYP3A genes appear to be regulated mostly at the transcription level (Telhada et al, 1992;Schuetz et al, 1993;Li et al, 1995b), mRNA quantification should give a fair estimate of the variability of their expression. Interestingly, some, albeit not all, workers in the field have reported the expression and even the induction of CYP3A genes in peripheral blood leukocytes (Janardan et al, 1996;Hukkanen et al, 1997;Sempoux et al, 1999;Starkel et al, 1999;Krovat et al, 2000;Nakamoto et al, 2000;Asghar et al, 2002;Finnstrom et al, 2002;Nowakowski-Gashaw et al, 2002). These results have raised the exciting prospect of prediction of hepatic CYP3A expression of an individual based on an assay performed on a blood sample.…”