Department of Environmental Health, University of Washington, 4225 Roosevelt Way NE #100, Seattle, Washington 981056099
Received October 20, 1999; accepted January 6, 2000
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ABSTRACT |
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Key Words: biomarkers; blood cells; cytochrome P450 (CYP); human quantitative competitive reverse transcriptase coupled polymerase chain reaction (QC RT-PCR) assay.
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INTRODUCTION |
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While the liver is the major organ involved in biotransformation, many drug-metabolizing enzymes also are present within extrahepatic tissues, including human peripheral lymphocytes (Raucy et al., 1999). Since blood is a readily accessible tissue, an appealing concept is to use peripheral blood cells as a surrogate, or sentinel model for CYP activities that manifest in internal organs. In fact, several previous studies have attempted to study the suitability of CYP expression and induction in human lymphocytes for use as a biomarker (Carcillo et al., 1996
; Cosmaet al.,1992
; Jacquet et al.,1997
; Raucy et al.,1997
; Rojas et al.,1992
; Rumsby et al.,1996
; Spencer et al.,1999
; Van den Heuvel et al.,1993
).
A number of investigators have examined the correlation between CYP1A1 enzyme activity and induction (assessed as aryl hydrocarbon hydroxylase [AHH] or ethoxyresorufin-O-deethylase [EROD] activity) in mitogen-stimulated human lymphocytes and lung tissue, especially with respect to the potential relationship between CYP1A1 induction by exposure to polycyclic aromatic hydrocarbons (PAHs) in cigarette smoke and the susceptibility to lung cancer (Jacquet et al., 1997; Karki et al., 1987
; Kiyohara et al.,1998
; McLemore et al., 1978
; Paigen et al., 1977
; Ward et al., 1978
). The results from these studies have been rather inconsistent. For example, Rojas and coworkers (1992) assessed CYP-dependent formation of benzo[a]pyrene-tetrols in lung microsomes and cultured lymphocytes and found no correlation between the two tissues in subjects with different smoking habits, whereas some modest effects of PAH exposure on CYP1A1 mRNA expression in fresh human lymphocytes have been reported (Cosma et al.,1992
; Rumsby et al.,1996
; Van den Heuvel et al.,1993
). Results of other recent investigations suggested that the mRNA concentrations of two CYP enzymes, CYP2D6 and CYP2E1, in human blood lymphocytes reflect the in vivo activity of their corresponding liver counterparts (Carcillo et al., 1996
; Raucy et al., 1997
). A study examining the expression of another biotransformation enzyme, microsomal epoxide hydrolase (mEH), indicated that basal activity levels in fresh lymphocytes were correlated with levels in liver and in lung (Omiecinski et al., 1993
).
In order for lymphocytes or other sentinel-cell types to serve as a general surrogate for CYP gene expression in the liver or other target organs, it is necessary that the levels and patterns of expression correlate between the respective cell or tissue sets. Historically, it has been difficult to accurately monitor biotransformation systems in extrahepatic cell types, since the typically low levels of their associated expression require highly sensitive and specific assays to enable their detection. In this study, we used an extremely sensitive quantitative-competitive (QC) RT-PCR assay that allowed measure of a battery of CYP and mEH gene expression levels in peripheral blood cells, established cell lines, and their direct comparison to absolute levels obtained previously from human liver (Andersen et al., 1998). We report that constitutive CYP and mEH expression profiles in blood lymphocytes are quite distinct from patterns characteristic of the liver.
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MATERIALS AND METHODS |
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Isolation of Peripheral Lymphocytes from Donors.
Peripheral blood (40ml) was obtained by venapuncture from 10 normal healthy volunteers, 5 female and 5 male Caucasians between the ages of 22 and 56. Mononuclear cells were isolated using Ficoll density gradient centrifugation (Pharmacia, Inc., Piscataway, NJ) according to manufacturer's instructions. Isolated cells were resuspended in RPMI 1640 medium (Life Technologies Inc., Grand Island, NY) supplemented with 100 U/ml penicillin and 100 ng/ml streptomycin (Sigma-Aldrich Inc., Saint Louis, MO), placed into tissue culture flasks, and incubated at 37°C for thirty min, allowing monocytes to adhere. Following incubation, non-adherent lymphocytes were recovered from the medium. Purity of these cell populations was determined by fluorescent activated cell sorting (FACS) using a Coulter Epics Elite ESP (Coulter Corp., Miami, FL) analysis with human CD3-, CD19-, and CD14-specific antibodies to label T lymphocytes, B lymphocytes, and monocytes, respectively. The cell isolates typically contained less than 4% monocytes, about 6% B lymphocytes, and about 70% T lymphocytes.
Cell Culture and Treatments.
Human blood cell lines were cultured in suspension in RPMI 1640 medium (Life Technologies) supplemented with 10% Nu-serum (Becton Dickinson Inc., Franklin Lakes, NJ), 100 U/ml penicillin, and 100 ng/ml streptomycin (Sigma-Aldrich) at 37°C and 5% CO2. HepG2 cells were cultured in DMEM/F12 medium (Life Technologies), supplemented with 10% Nu-serum, 100 U/ml penicillin, and 100 ng/ml streptomycin at 37°C and 5% CO2, except for one control induction experiment where they were grown in the RPMI 1640 medium. For chemical treatments, logarithmically growing cells were pre-cultured in medium supplemented with 2% Nu-serum and antibiotics for 24 h. These culture conditions were derived empirically as optimal for the cell lines in use. Cells were incubated with 22 µM ß-naphthoflavone (ßNF), 22 µM ßNF plus 25 nM dexamethasone (ßNF + DEX), 10 µg/ml Arochlor 1254 (ARO), 10 µM dexamethasone (DEX) or 1 mM phenobarbital (PB) in medium containing 2% Nu-serum. The concentrations of inducers used were taken from general literature values. Control cultures were incubated with DMSO (typically <0.05%). Cell viability after treatment was assessed by Trypan blue exclusion and was typically >85%.
RNA Isolation and Quantitative-Competitive (QC) RT-PCR Assay.
Total cellular RNA was isolated from cell preparations using the Trizol Reagent (Life Technologies) following the manufacturer's instructions. The concentrations of RNA were determined spectrophotometrically by monitoring UV absorbance at 260 nm. Between 0.2 and 5 µg of total RNA were used for quantification of mRNA levels for CYP 1A1, 1A2, 2A6/7, 2D6, 2E1, 2F1, 3A, and microsomal EH, using a QC RT-PCR assay described previously (Andersen et al., 1998).
DNA Sequencing.
Sequencing of PCR products was performed using the ABI PRISM BigDyeTM DNA sequencing kit from PE Applied Biosystems (Foster City, CA). Samples were analyzed on an ABI PRISM 310 Genetic Analyzer (PE Applied Biosystems).
Fluorescent Activity Assay.
Ethoxyresorufin-O-deethylase (EROD) activity was measured in whole cell suspensions. Briefly, cells were washed and resuspended at a concentration of 106 cells/ml in Earle's balanced salt solution (Life Technologies) supplemented with 15 mM HEPES, 5 mM MgCl2, 2.5 mM CaCl2, pH 7.5, gassed with 95% O2/5% CO2 and supplemented with 25 mM dicumarol. Cells were kept at 37°C. The reaction was initiated by adding 1 µl of 5 mM 7-ethoxyresorufin in DMSO to a 1-ml cell suspension. Fluorescence intensity was measured over the period of 5 min and at 10-, 15-, 20-, and 30-min time points, using a wavelength set of 547 nm (excitation) and 584 nm (emission) with a Perkin Elmer LS50 fluorescent detector. Rates were calculated from the slopes in the linear range of increase in fluorescence intensity. The results were quantified using resorufin as a standard (Sidhu et al, 1993).
Preparation of Microsomal Proteins and Western Blot Analysis.
Microsomes from cell lines were prepared by differential centrifugation. Briefly, cells were washed, resuspended, and sonicated in ice-cold homogenization buffer (10mM KH2PO4, 1.15% KCL). Cell homogenates were centrifuged at 9000 x g for 20 min. Supernatants were collected and centrifuged for 1 h at 100,000 x g at 4°C. The resulting pellets were resuspended in storage buffer (10 mM KH2PO4, 1mM EDTA, 20% glycerol). Protein contents were assessed by the BCA reagent assay (Pierce Inc., Rockford, IL).
Twenty µg of microsomal protein from each cell line and 10 µg of human liver microsomes were electrophoretically separated on 10% sodium dodecyl sulfate-polyacrylamide gels and electroblotted to Sequi-Blot PVDF membranes (Bio-Rad Inc., Hercules, CA) according to standard methods. The membranes were subsequently blocked in 1 x TTBS (10 mM Tris base, 0.9% NaCl, pH 7.4, 0.1% Tween 20) plus 1% bovine serum albumin (Boehringer Mannheim, Inc., Germany), and 1% dry milk (Bio-Rad Inc., Hercules, CA). Primary antibodies, diluted from 1:2000 to 1:10,000 were applied to the membranes for 2 h at room temperature. After 3 15-min washes with 1x TTBS, the membranes were exposed to secondary antibodies for 1 h (horseradish peroxidase-conjugated IgGs at a 1:4000 dilution) and washed as described. Chemiluminescent visualization of the resulting immunoblots was achieved with ECL system (Amersham, Inc., Arlington Heights, IL).
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RESULTS |
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Overall, the profiles appeared to be substantially conserved among individuals. The degree of variation among individuals ranged from 2.1-fold (CYP1A1) to 5.7-fold (CYP2E1). CYP2D6 and mEH exhibited 4.0- and 3.8-fold variations among individuals, respectively. No gender related differences in expression profiles could be ascertained. Together, these results portray a comprehensive picture of basal CYP and mEH gene-expression patterns in human peripheral lymphocytes and demonstrate an essentially low level of interindividual variation within these cell types.
Profiles of CYP and mEH Gene Expression in Human Cell Lines
To compare CYP and mEH gene expression in established human cell lines with those found in freshly isolated human lymphocytes, total RNA from the following human cell lines was subjected to analysis with QC RT-PCR: the erythroblastic leukemia cell line, HEL 92.1.7; the B lymphoblastic leukemia cell line, IM9; the promyelocytic leukemia cell line, HL60; the monocytic leukemia cell line, THP-1; and the hepatoma cell line, HepG2. Constitutive expression profiles of logarithmically growing cells are shown in Figure 2.
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Overall, profiles of CYP and mEH basal gene expression were largely conserved among cells from different lineages and the differentiation stage of human hematopoiesis. Moreover, both the levels and the profiles of gene expression, measured in all the blood cell lines, resembled those exhibited in freshly isolated lymphocytes. The latter observation indicates that these established blood cell lines possess a set of basic features resembling that of freshly isolated lymphocytes and therefore may serve as suitable in vitro models for xenobiotic biotransformation in human blood.
Inducibility of CYP1A and CYP3A Gene Expression in Human Cell Lines
To test whether human blood cell lines are capable of induction responses subsequent to xenobiotic challenge, we cultured the various lines in the presence of several prototypical inducers and examined the resulting mRNA expression levels.
For evaluation of CYP1A gene responsiveness, logarithmically growing cells were treated with DMSO (vehicle control), 22 µM ßNF, 22 µM ßNF + 25nM DEX, or 10 µg/ml ARO, respectively, for 24 h. Total RNA was isolated from these cells and analyzed for CYP1A1 and CYP1A2 gene expression using QC RT-PCR. The results are summarized in Figure 3A.
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To examine induction of CYP1A1 enzyme activity, EROD assays were performed on DMSO and ßNF + DEX (48 h) treated cells. However, blood cell line, control or induced, exhibited EROD activity higher than the background of the assay (data not shown). In contrast, induced HepG2 cells exhibited relatively large increases in EROD levels, 10-fold higher than controls (data not shown), reflective of the large increases of CYP1A1 mRNA measured independently.
For evaluation of CYP3A gene responsiveness, logarithmically growing cells were treated with DMSO (vehicle control), 1.0 mM PB, 10 µM DEX or 10 µg/ml ARO, respectively, for 24 h. Total RNA was isolated from the cells and analyzed for CYP3A gene expression using QC RT-PCR. The results are summarized in Figure 3B.
Constitutive CYP3A gene expression in HEL, HL60, and THP cells was very low (less then 5.0 x 103 molecules/µg total RNA) and this level was not increased subsequent to treatment with the 3 prototypic inducers assayed. A slight increase in CYP3A, reflecting approximately 2.5-fold induction, was detected in IM9 cells after chemical challenge. The constitutive CYP3A7 mRNA level in HepG2 cells was relatively high and increased moderately (approximately 3- to 5-fold) after treatments. All values presented were an average of measurements obtained from at least 2 independent experiments, with <15% variation between replicate analyses. Induction experiments performed using serum-free culture conditions or extending treatments for 48 h did not result in altered cellular responsiveness (data not shown).
Overall, the blood cell lines tested in this study exhibited only weak responsiveness to prototypic inducers of CYP1A and CYP3A. Therefore, their use as models to assess the response of chemical exposure in humans or to study mechanistic aspects of xenobiotic CYP gene induction occurring in the liver appears limited.
Protein Expression in Human Cell Lines
To determine functional levels of CYP and mEH protein, microsomes were isolated from all the cell lines described above and were subjected to analysis by Western immunoblotting. Antibodies specific for CYP1A1, CYP2D6, CYP3A, CYP2E1, mEH, and P450 reductase were assessed in these studies. The results of these experiments are presented in Figure 4.
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These results demonstrate the existence of mEH protein in human blood cell lines but suggest that the level of CYP biotransformation enzymes is, at best, extremely low. Correlating well with the positive results of the Western blot signals, mEH mRNA levels were similarly of highest abundance in these sample preparations.
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DISCUSSION |
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Initial studies were conducted using fresh lymphocyte preparations from 10 individual donors. The studies were repeated with separate blood samples obtained approximately 1 month later from the same individuals. Overall, we observed a remarkably low inter-individual variation of CYP and mEH gene expression in lymphocytes, ranging from 2- to 6-fold in our sample of 10 individuals. To date, most studies have indicated less than 10-fold inter-individual variation of basal expression of CYP genes in freshly isolated lymphocytes (Dassi et al., 1998; Carcillo et al., 1996
; Van den Heuvel et al., 1993
). In contrast, in our previous analysis of 8 human livers, the variation in RNA expression ranged from 13-fold (CYP2E1) to 220-fold (CYP2D6), likely explained by a combined set of factors including genetic polymorphism and possible influences of nutritional deprivation, medication, and ischemic conditions during organ harvest (Andersen et al., 1998
).
More specifically, CYP1A1 mRNA was detectable in the lymphocyte preparations from all 10 of the individual donors, albeit at low levels (Fig. 1). This CYP was undetected in 5 of 8 human liver samples using the same highly sensitive assay procedure (Andersen et al., 1998
). The presence of CYP1A1 mRNA in fresh lymphocytes also has been reported in other studies (Van den Heuvel et al., 1993
; Lang et al., 1998
; Omiecinski et al., 1990
; Rumsby et al., 1996
and Wei et al., 1998
, 1999
). In contrast, CYP1A2, like CYP2A6/7, was highly abundant in liver (Andersen et al., 1998
) but was not detectable in fresh lymphocytes (Fig. 1
). This latter finding appears to be in agreement with previous reports (Hukkanen et al. (1997), Koskela et al. (1999) and Raunio et al. (1998)).
It is noteworthy that we observed only very low abundance of CYP3A mRNA in fresh lymphocytes (Fig. 1), near the detection limits of our assay. However, through DNA sequence analysis, we clearly identified CYP3A4 as the specifically expressed CYP3A form in the lymphocyte-derived PCR products. It was reported recently that CYP3A is selectively expressed only in B lymphocytes (Sempoux et al., 1999
). This information may explain the generally low levels of CYP3A detected in our cell preparations since they contained only about 6% of this cell type as assessed by FACS analysis (see Materials and Methods). In contrast, Janardan and coworkers (1996) were unable to detect CYP3A4 mRNA in mononuclear blood cell preparations, although they did report detection of CYP3A5-specific mRNA in one out of 6 individuals. It should be emphasized that the CYP3A-selective forward and reverse primers used in this investigation, and Andersen's study (1998), exhibit perfect homology with endogenous CYP3A4 and CYP3A7 sequences but mismatch slightly (2/20 and 2/22 bases, respectively) with the corresponding CYP3A5 sequence and thus may have affected our ability to robustly amplify CYP3A5 sequences. In comparison, it appears that all human adult livers express CYP3A4, whereas CYP3A5 and CYP3A7 are present in only about 25% of all postnatal livers (Aoyama et al.,1989
; Schuetz et al.,1994
; Wrighton et al.,1989
, 1990
).
CYP2F1 is considered a lung specific protein (Nhamburo et al., 1989), expressed in bronchial epithelial cells and in alveolar macrophages (Hukkanen et al., 1997
; Willey et al., 1996
). Therefore, the weak CYP2F1 signals in our samples may be ascribed to the monocytic population (approximately 2.4%) in our cell preparation. Although CYP2D6 and mEH mRNAs are expressed at relatively high levels in lymphocytes, they are still approximately one-tenth as abundant as that in the human liver (Andersen et al., 1998
). The difference for CYP2E1 is even more dramatic, with lymphocytes expressing approximately 1/580 the levels of liver (Andersen et al., 1998
).
Taken together, we conclude that with respect to expression of phase I biotransformation enzymes, human liver and peripheral lymphocytes are rather distinctly equipped, both with respect to overall levels as well as inherent profiles of gene expression. This result suggests that the applicability of blood cells as surrogates for liver biotransformation capacities should be evaluated cautiously.
Additional objectives of this investigation were to test whether established human blood cell lines could appropriately model basal expression occurring in primary blood lymphocytes, and to assess whether xenobiotic induction responses of blood cells may serve as a biomarker of induction responses occurring in the liver. Human leukemia cell lines express major blood cell-specific markers and are widely used models for the study of hematopoesis, cell differentiation, and CYP expression/regulation (Auwerx, 1991; Collins 1987
; Jakob et al., 1995
; James et al., 1999
; Masten et al., 1996). Our data demonstrate that, with respect to constitutive CYP and mEH RNA levels, the established cell lines we studied exhibited expression character that resembled freshly isolated lymphocytes remarkably well (Fig. 2
). This result is somewhat surprising considering the significant differences in profiles between human liver (Andersen et al., 1998
) and the hepatoma HepG2 cells (Fig. 2
). Interestingly, no substantive differences in CYP and mEH profiles were noted among the representatives of the different lineages of human hematopoesis, i.e., erythrocytic, lymphocytic, mylocytic, or monocytic cell line derivatives (Fig.2
).
With respect to induction, of the 4 blood-cell lines analyzed, only HEL cells exhibited considerable CYP1A1 gene responsiveness to the prototypical inducers ßNF and ARO (Fig. 3A). Although we attempted EROD activity measurements on all the cell lines, it was curious that none of the lines, including HEL cells, exhibited EROD levels that were distinguishable from background controls (data not shown). Perhaps this result attests to the sensitivity of the QC RT-PCR assay used here. In comparison, using HepG2 cells, ßNF treatment resulted in 100-fold induction in CYP1A1 mRNA levels that paralleled a 10-fold increase in induced EROD activity (data not shown). The presence of the aryl hydrocarbon receptor (AhR) and the aryl hydrocarbon receptor nuclear translocator protein (ARNT), which are prerequisites for CYP1A1 induction, have been demonstrated in HEL, IM9, HL60, and THP cells (Hayashi et al., 1995
; Masten et al., 1995). However, Hayashi et al. (1995) also demonstrated a positive correlation between the stage of cell differentiation, expression level of AhR and induction of CYP1A1 gene expression for several human leukemia cell lines. Thus, although HL60 cells expressed high levels of AhR, they were functionally responsive to xenobiotics only after treatment with stimulators of monocytic differentiation (Hayashi et al., 1995
). Similarly, blood lymphocytes exhibit CYP1A1 gene induction only subsequent to stimulation with mitogens (Kouri et al., 1974
). In mitogen-stimulated human blood lymphocytes, CYP1A1 mRNA expression is induced 10- to 20-fold by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), a response that is also correlated with induction of EROD activity (Van den Heuvel et al., 1993
; Lang et al., 1998
).
In addition to the low CYP1A1 responsiveness, the lack of CYP3A inducibility (Fig. 3B) is a further indication of the limited suitability of these cell lines to function as appropriate models to study hepatic responses or mechanistic aspects of xenobiotic CYP gene induction, or to assess the risk of chemical exposure in humans. Interestingly, our PCR product DNA sequence analysis indicated that only the HEL cells expressed CYP3A4, the adult-specific isoform of this gene subfamily. This finding, coupled with the observation that HEL cells exhibit CYP1A1 inducibility, suggest that these cells may reflect a more advanced stage of differentiation than the other cell lines. In IM9, HL60, and THP cells, using sequence analysis, we specifically identified CYP3A7, the fetal form of CYP3A that is also found in dedifferentiated HepG2 hepatoma cells (Schuetz et al., 1993
and 1994
).
Finally, in this study we investigated the correlation between mRNA and protein levels. Western blot analysis revealed that CYP proteins are present in very low abundance in these cell lines (Fig. 4). As a comparison, in human lymphocytes, constitutive CYP3A protein was detectable (Janardan et al., 1996
; Sempoux et al., 1999
, Starkel et al., 1999
), whereas CYP2E1 protein was detectable only under inducing conditions (e.g., in diabetic patients) (Song et al., 1990
). However, the relatively high abundance of mEH mRNA correlates with detectable mEH protein levels in all the cell lines (Fig. 4
) suggesting that a mRNA level of 5 x 105 copies per µg total RNA reflects the approximate detection limit for corresponding protein levels. Again, these results likely attest to the extremely sensitive nature of detection afforded by the QC RT-PCR assay used in this investigation.
In summary, given the relatively low levels of both mRNA and protein expression in blood cells associated with a battery of selected drug metabolizing enzymes, we conclude that the blood-cell lines characterized in this study have only limited value with respect to modeling the basal or inducible gene expression character of biotransformation in the liver, or, in this sense, as a suitable biomarker of assessing risk of chemical exposure in humans. However, the QC RT-PCR assay used in this study was demonstrated to be a powerful tool to characterize different cell types for their respective CYP gene-expression patterns and induction behavior. Our laboratory is currently extending and adapting this type of quantitative assay for the TaqmanTM-based real time-PCR technology to create an even more powerful tool for quantitative analysis of gene expression. Although the effectiveness of blood cells as models of liver expression is called into question by these studies, importantly, our data do suggest that established human blood cell lines reflect the constitutive CYP and mEH expression status of primary blood lymphocytes, and therefore, are appropriate models for studying blood cell-related xenobiotic metabolism. Since the biotransformation properties of lymphocytes, as well as many other nonhepatic cells, importantly contribute to cell-specific and tissue-specific toxic events, it is desirable to more precisely assess these contributions, using sensitive means of detection such at those used in the current investigation.
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ACKNOWLEDGMENTS |
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NOTES |
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