0090-9556/00/2805-0608–616$03.00/0
DRUG METABOLISM AND DISPOSITION
Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics
DMD 28:608–616, 2000 /1789/817857
Vol. 28, No. 5
Printed in U.S.A.
DETECTION OF CHEMICAL-INDUCED DIFFERENTIAL EXPRESSION OF RAT HEPATIC
CYTOCHROME P450 MRNA TRANSCRIPTS USING BRANCHED DNA SIGNAL
AMPLIFICATION TECHNOLOGY
DYLAN P. HARTLEY
AND
CURTIS D. KLAASSEN
Environmental and Occupational Medicine Center, Department of Pharmacology, Toxicology, and Therapeutics, University of Kansas Medical
Center, Kansas City, Kansas
(Received September 3, 1999; accepted January 21, 2000)
This paper is available online at http://www.dmd.org
The importance of the cytochrome P450 (CYP) enzyme family in
xenobiotic metabolism, as well as their differential expression and
activity in response to a wide range of environmental chemicals
and pharmaceuticals, is well documented. The objective of this
study was to evaluate the specificity of the branched DNA (bDNA)
signal amplification technique for the detection of multiple rat
CYPs from hepatocellular RNA. Oligonucleotide probe sets were
designed to various chemically inducible rat CYP mRNA transcripts, including CYP1A1, CYP1A2, CYP2B1/2, CYP2E1, CYP3A1/
23, and CYP4A2/3. The robustness of the bDNA assay was assessed with the CYP2B1/2-specific probe set, and total RNA was
isolated from control and phenobarbital (PB)-treated rats. Analysis
of these RNA samples by bDNA signal amplification resulted in a
linear quantifiable range of RNA detection that spanned three
orders of magnitude (0.1–100 ␮g of total RNA). The fidelity of the
bDNA assay was evaluated within a single assay and between
assays where repeated measurements of a single sample were
reproduced reliably. The specificity of individual CYP probe sets
was evaluated with five typical CYP-inducing chemicals on the
expression of specific hepatic CYP mRNA transcripts. Male
Sprague-Dawley rats were administered 3-methylcholanthrene,
PB, isoniazid, pregnenolone-16␣-carbonitrile, or clofibric acid to
induce transcription of CYP1A1, CYP1A2, CYP2B1/2, CYP2E1,
CYP3A1/23, and CYP4A2/3 mRNA, respectively. Analysis of chemical-induced differences in gene expression by bDNA signal amplification indicated that 3-methylcholanthrene induced CYP1A1
and CYP1A2 mRNA levels 670- and 11-fold, respectively; PB induced CYP2B1/2 expression 71-fold; pregnenolone-16␣-carbonitrile induced CYP3A1/23 expression 34-fold; and clofibric acid induced CYP4A2/3 expression 4.7-fold. Overall, these data support
the use of bDNA signal amplification technology as a robust, reproducible, and efficient means of monitoring the differential expression of multiple isoforms of the CYP enzyme family.
In two decades, hundreds of cytochrome P450 (CYP)1 enzymes
have been cloned, isolated, and functionally characterized from numerous species, including humans. The efforts of scientists in the
CYP field have given rise to advances by other drug metabolism
researchers in the identification of novel proteins that play an essential
role in the absorption, activation, deactivation, excretion, and toxicity
of various chemicals. This wealth of knowledge has led to the realization that many enzymes cannot be categorized according to substrate specificity, due to the multiplicity of enzymes/proteins with
apparent functional redundancy. However, when compared at the
level of amino acid sequence, proteins can be categorized into large
families of related molecules, such as the CYPs, UDP-glucuronosyltransferases, sulfotransferases, organic-anion transporters, multidrug
resistance proteins, etc. Each of these encompassing enzyme families
contains multiple enzyme subfamilies; each of these may contain
numerous and highly similar homologous genes. As a result, it becomes increasingly clear that new and novel methods that allow us to
monitor these enzymes simultaneously will be important in our efforts
to glean new information on the differential regulation of homologous
genes and their respective protein products.
One technology that will assist in the concerted effort of scientists
to evaluate the CYPs at the mRNA level, as well as many other gene
products, is the branched DNA (bDNA) signal amplification assay.
The bDNA signal amplification assay is a nonpolymerase chain
reaction (PCR) and nonradioactive based method of RNA analysis
that resembles the well established enzyme-linked immunosorbent
assay (ELISA); the design of the bDNA signal amplification assay is
shown in Fig. 1. It is evident from this figure that the bDNA assay is
designed as a multioligonucleotide approach, wherein three types of
gene transcript-specific oligonucleotides are designed against each
mRNA target of interest. These three oligonucleotides are termed
capture extender, label extender, and blocker probes. Capture extender
oligonucleotide probes (Fig. 1, dark blue) consist of a mRNA-specific
This study was supported by U.S. Public Health Service Grant ES-03192. D.H.
was supported by an National Institute of Environmental Health Sciences Training
Grant ES-07079 and by the Kansas Health Foundation (KHF-411606).
1
Abbreviations used are: CYP, cytochrome P450; bDNA, branched deoxyribonucleic acid; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; 3MC,
3-methylcholanthrene; PB, phenobarbital; ISO, isoniazid; CLO, clofibric acid;
PCN, pregnenolone-16␣-carbonitrile; RLU, relative luminescence units; ELISA,
enzyme-linked immunosorbent assay; PCR, polymerase chain reaction; RT-PCR,
reverse transcription-polymerase chain reaction.
Send reprint requests to: Curtis D. Klaassen, Ph.D., Dept. of Pharmacology,
Toxicology, and Therapeutics, University of Kansas Medical Center, 3901 Rainbow Blvd., Kansas City, KS 66160-7417. E-mail: [email protected]
608
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
ABSTRACT:
CYTOCHROME P450 ANALYSIS USING QUANTIGENE TECHNOLOGY
FIG. 1. Two-dimensional illustration of the multimeric complex formed with a
target mRNA transcript during the bDNA signal amplification assay.
sequence, and have an additional 3⬘concatenated nucleotide sequence
that is complementary to a nucleotide sequence that is fixed to the
solid support phase (Fig. 1, short black lines). Thus, the extension on
the capture probe functions to anchor the capture probe-mRNA target
hybrid to the solid support phase. Label extender oligonucleotide
probes (Fig. 1, red) are also specific to a gene product of interest, but
have an additional 3⬘concatenated nucleotide sequence extension that
facilitates hybridization to a cognate sequence on the bDNA molecule.
Blocker oligonucleotide probes (Fig. 1, green) are used to span gaps
between capture and label extender probes in the probe set. Blocker
probes function to minimize RNase-mediated sample degradation in
the mRNA target region as well as to stabilize the secondary structure
of the mRNA target region. In practice, the bDNA assay uses this
novel oligonucleotide chemistry and solution hybridization that functions to both capture the RNA molecule of interest to the solid support
phase, and label the RNA with “amplifier” bDNA molecules (Fig. 1,
light blue). Detection of the RNA molecule is similar to an indirect
ELISA, where an enzyme (e.g., alkaline phosphatase; Fig. 1, red
diamonds) is conjugated to an oligonucleotide (label probe; Fig. 1,
orange), which hybridizes to the branches of the bDNA molecules,
and on addition of substrate, dioxetane, a chemiluminescent signal is
produced and measured. So, unlike PCR-based methodologies that
amplify the target RNA molecule, this methodology amplifies the
hybridization of a single RNA molecule such that the signal generated
is proportional to the amount of RNA input into the system. The
ELISA-like format and the ability to measure signal from the transcript itself are two major advantages of the bDNA assay, and lend to
the increased efficiency of mRNA analysis and reproducibility of
results.
The bDNA signal amplification system was initially developed, and
has been used extensively, in clinical settings as a tool to monitor HIV
and hepatitis (B and C) viral load in patients (Hendricks et al., 1995;
Pachl et al., 1995; Detmer et al., 1996). For these applications, bDNA
analysis has proven to be an efficient, sensitive, and reliable tool for
clinicians. The assay is beginning to find an audience among basic
science researchers, where it has been used successfully to monitor
insulin RNA processing (Wang et al., 1997), and in the measurement
of cytokine mRNA from peripheral blood cells (Shen et al., 1998). In
these studies as well as the present study, the bDNA assay had a wide
dynamic range of response (more than three orders of magnitude).
Comparisons of the bDNA system with quantitative reverse transcription-polymerase chain reaction (RT-PCR) have demonstrated that the
bDNA system is comparable with PCR-based strategies with regard to
sensitivity (Collins et al., 1997; Guenthner and Hart, 1998).
The enzymatic assays used to measure changes in chemically
inducible forms of P450 enzymes (Table 1) are well characterized,
and are still the standard for assessing chemical-mediated changes in
CYP activity (Parkinson, 1996). However, the capacity to rapidly
monitor changes in the transcriptional regulation of specific gene
products, including the CYPs, could benefit a wide array of researchers, clinicians, and commercial organizations. From the knowledge of
the nucleotide sequence for any given gene/mRNA, the specificity
inherent with oligonucleotide-based techniques such as Northern-blot
analysis, RT-PCR, and now bDNA signal amplification can be used to
differentiate between highly related molecules at the level of mRNA
transcript expression. In an attempt to increase the efficiency, reliability, and reproducibility of analyzing multiple mRNA transcripts
within homologous enzyme families, the bDNA signal amplification
technology was evaluated in the present study with the major inducible CYP enzymes.
Although there are now at least 53 rat CYP genes in the Unigene
database (Schuler et al., 1996), there are four CYP gene families (i.e.,
CYP1, CYP2, CYP3, and CYP4) that encompass eleven CYPs commonly used to characterize drug-drug interactions. These eleven enzymes (CYP1A1, CYP1A2, CYP2B1, CYP2B2, CYP2E1, CYP3A1,
CYP3A2, CYP3A23, CYP4A1, CYP4A2, and CYP4A3) span four
CYP gene subfamilies (i.e., CYP1A, CYP2B, CYP2E, CYP3A, and
CYP4A). As a general rule, within a CYP subfamily, CYP enzymes
respond similarly to a class of compounds (i.e., CYP1A1 and
CYP1A2 gene expression is induced by polychlorinated hydrocarbons). However, between CYP subfamilies, each subfamily of P450s
are differentially up-regulated by chemicals such as dioxin, phenobarbital (PB), rifampicin, ethanol, and clofibric acid (CLO; Porter and
Coon, 1991; Parkinson, 1996; Dogra et al., 1998). In this report, the
well characterized response of a particular CYP to a specific chemical
was used to evaluate the use of the bDNA assay as a high-throughput
technique for assessing xenobiotics as CYP inducers.
Our rationale for assessing the bDNA assay as a technique for
monitoring xenobiotic-mediated enzyme induction originated from
the specificity, sensitivity, reliability, and efficiency of the bDNA
assay. These characteristics were indicative of a technology that could
serve as a primary screening tool for xenobiotic induction of the CYP
enzymes. Therefore, in this report the bDNA signal amplification
assay was used to monitor the expression of CYP1A1, CYP1A2,
CYP2B1/22, CYP2E1, CYP3A1/232, and CYP4A2/32 in rats treated
with classical enzyme-inducing chemicals. From this study, the validity of the bDNA system in assessing CYP induction was determined to use this system to predict potential drug-drug interactions.
Thus, the intention of this report is to provide the research community
with a data set derived from applying the bDNA system to a well
known chemical response paradigm, chemical induction of specific
CYPs.
2
For CYP2B1/2, CYP3A1/23, and CYP4A2/3 oligonucleotide probe sets were
not designed to distinguish between these subfamily members (e.g., CYP2B1 and
CYP2B2 transcript expression levels were detected together and were not differentiated by the bDNA assay).
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
The drawing is a representation of the final complex formed in the assay. The
long solid black line represents the single-stranded mRNA target, blue lines represent capture probes, red lines represent label probes, and green lines represent
blocker probes. Oligonucleotides complementary to the tail of the capture probes are
shown as short black bars attached to the solid support phase. The bDNA molecule
(light blue), with its 15 branch points, is shown hybridized to the tail of the label
probe. Alkaline phosphatase (AP; red diamonds) conjugated oligonucleotides (orange) are shown hybridized to the branches of the bDNA molecule (3 AP-oligonucleotide conjugate molecules/branch). The signal generated by AP-mediated
dioxetane chemiluminescence is shown in yellow.
609
610
HARTLEY AND KLAASSEN
TABLE 1
Genes, GenBank accession numbers, Unigene accession numbers, and target sequences used for oligonucleotide probe design
a
b
CYP Gene
GenBank No.a
1A1
1A2
2B1, 2B2
2E1
3A1, 3A23
4A2, 4A3
I00732
X01031
J00719
S48325
D13912, X96721
M57719, M33936
Unigene No.
Rn.
Rn.
Rn.
Rn.
Rn.
Rn.
10352
5563
2287
1372
11291
33492
References
Targetb
Oeda et al., 1988
Yabusaki et al., 1984
Fujii-Kuriyama et al., 1982
Richardson et al., 1992
Kirita and Matsubara, 1993; Komori and Oda, 1994
Kimura et al., 1989a,b
570–972
501–998
566–982
501–963
616–914
501–865
Accession numbers were downloaded from GenBank into ProbeDesigner Software Version 1.0.
Target sequences were selected from nonhomologous sequences after nucleotide sequence alignments were performed in OMIGA (Cambridge, UK).
Materials and Methods
3
Capture and label oligonucleotide probes were synthesized with an additional 3⬘-concatenated nucleotide sequence where the 3⬘-sequence is constant
for either capture probes (ctcttggaaagaaagt) or label probes (aggcataggacccgtgtct) and designates each probe as a capture or label probe. This 3⬘-nucleotide
sequence extension on capture and label probes is separated from the genespecific oligonucleotide sequence by a stretch of five thymidines (TTTTT). Blocker
probes do not contain a nucleotide sequence extension. Each oligonucleotide is
listed with the sequence information for each probe in Table 2.
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Animals. Male Sprague-Dawley rats (200 g; Harlan Sprague-Dawley, Inc.,
Indianapolis, IN) were acclimated to the housing facility (2–3 rats/cage, 50%
relative humidity, 12-h light/dark cycle) for 1 week before the initiation of the
study. Animals were given free access to water and rat chow (Teklad; Harlan
Sprague-Dawley, Inc.).
Chemical Treatment of Animals. Rats were randomly grouped into seven
treatment groups (4 rats/chemical treatment). Animals were injected (1 injection/day for 4 days; i.p.; 5 ml/kg) with PB (80 mg/kg), 3-methylcholanthrene
(3MC; 27 mg/kg), pregnenolone-16␣-carbonitrile (PCN; 50 mg/kg), isoniazid
(ISO; 200 mg/kg), or CLO (200 mg/kg). Control animals were given the
appropriate vehicle (corn oil for 3MC and PCN, or saline for PB, ISO, and
CLO).
Isolation of RNA. Total RNA was isolated using RNAzol B reagent
(Tel-Test Inc., Friendswood, TX) as per the manufacturer protocol. Each RNA
pellet was resuspended in 0.2 ml of 10 mM Tris-HCl buffer, pH 8.0. The
concentration of total RNA in each sample was quantified spectrophotometrically at 260 nm. Each RNA sample was analyzed by formaldehyde-agarose
(1.2% agarose, 2.1 M formaldehyde in 1⫻ MOPS, ethidium bromide 0.5
␮g/mg) gel electrophoresis. The quality of each RNA sample was visualized
under ultraviolet light by fluorescence of ethidium bromide intercalated into
18S and 28S rRNA. The presence of these bands indicated the RNA was of
sufficient quality for use in subsequent analyses.
Development of Specific Oligonucleotide Probe Sets for bDNA Analysis
of CYPs. The CYP gene sequences of interest accessed from GenBank are
listed in Table 1. Each of these CYPs has multiple GenBank accession
numbers, where an accession number corresponding to the complete coding
sequence was used for probe development; the Unigene accession numbers are
given for simplicity. Before development of each probe set, the nucleotide
sequences were aligned using CLUSTALW (Thompson et al., 1994) with
software provided by OMIGA (Oxford Molecular Group, Inc., Oxford, UK) to
determine specific target regions (i.e., nucleotide regions of dissimilarity
between CYP sequences) for oligonucleotide probe development (Table 1).
These target sequences were analyzed by ProbeDesigner Software Version 1.0
(Bayer Diagnostics, formerly Chiron Diagnostics Corp., Emeryville, CA and
East Walpole, MA). Multiple and specific probes were developed to each CYP
mRNA transcript. Oligonucleotide probes designed in this manner were either
specific to a single mRNA transcript (i.e., CYP1A1, CYP1A2, or CYP2E1) or
to multiple transcripts within a CYP subfamily (i.e., CYP2B1/2, CYP3A1/23,
or CYP4A2/3; Table 2). All oligonucleotide probes were designed with a Tm
of approximately 63°C. This feature enables hybridization conditions to be
held constant (i.e., 53°C) during each hybridization step and for each oligonucleotide probe set. Every probe developed in ProbeDesigner was submitted
to the National Center for Biotechnological Information (NCBI) for nucleotide
comparison by the basic logarithmic alignment search tool (BLASTn; Altschul
et al., 1997) to ensure minimal cross-reactivity with other rat sequences.
Oligonucleotides with a high degree of similarity (ⱖ80%) to other rat gene
transcripts were eliminated from the design. All probes were synthesized (i.e.,
50-nmol synthesis scale) by Operon Technologies (Palo Alto, CA), and obtained desalted and lyophilized. A total of 107 oligonucleotide probes were
generated3 to the different CYP mRNA transcripts (Table 2). A schematic map
of the location and function of all of these probes is graphically illustrated in
Fig. 2. Based on the analysis of these probe sets in BLASTn, the probe sets
were deemed specific and sufficient to detect differential expression of CYP
mRNA. All probes (i.e., blocker probes, capture extenders3, and label extend-
ers3) were diluted in 1.0 ml of 10 mM Tris-HCl, pH 8.0, with 1 mM EDTA and
stored at ⫺20°C. As commonly used in Northern blot analysis, glyceraldehyde
3-phosphate dehydrogenase (GAPDH) was used for normalization of mRNA
expression between wells. The probe set to rat GAPDH was directed toward a
400-nucleotide region (basepair 112–512; GenBank accession numbers
AF106860 and M17701), and consisted of four capture extender, five label
extender, and one blocker oligonucleotide probes. The GAPDH probe set
information was generously donated by Jeff Donahue of Bayer Diagnostics
(East Walpole, MA).
bDNA Assay. Specific CYP oligonucleotide probe sets (i.e., blocker
probes, capture probes, and label probes) were combined and diluted to 50
fmol/␮l in the lysis buffer supplied in the Quantigene bDNA Signal Amplification Kit (Bayer Diagnostics, East Walpole, MA). The GAPDH probe set was
used at 50, 100, and 200 fmol/␮l for capture probes, blocker probes, and label
probes, respectively. All reagents for analysis (i.e., lysis buffer, capture hybridization buffer, amplifier/label probe buffer, washes A and D, and substrate
solution) were supplied in the Quantigene bDNA Signal Amplification Kit; the
components of these reagents were published previously (Wang et al., 1997).
Total RNA (1 ␮g/␮l; 10 ␮l) was added to each well of a 96-well plate
containing capture hybridization buffer and 100 ␮l of each diluted probe set.
Total RNA was allowed to hybridize to each probe set containing all probes for
a given transcript (blocker probes, capture probes, and label probes) overnight
at 53°C in a Quantiplex bDNA Heater (Bayer Diagnostics). Subsequently, the
plate was removed from the heater, cooled to room temperature, and rinsed
with wash A. Samples were hybridized with a solution containing the bDNA
amplifier molecules (50 ␮l/well) diluted in amplifier/label probe buffer and
incubated for 30 min at 53°C. The plate was again cooled to room temperature.
The amplifier solution was aspirated and wells were washed with wash A
(3⫻). Label probe, diluted in amplifier/label (same as above) probe buffer, was
added to each well (50 ␮l/well), and hybridized to the bDNA-RNA complex
for 15 min at 53°C. The plate was cooled to room temperature, and each well
was rinsed with wash A (2⫻), followed by wash D (3⫻). Alkaline phosphatase-mediated luminescence was triggered by the addition of a dioxetane
substrate solution (50 ␮l/well). The enzymatic reaction was allowed to proceed
for 30 min at 37°C, and luminescence was measured with the Quantiplex 320
bDNA Luminometer (Bayer Diagnostics) interfaced with Quantiplex Data
Management Software Version 5.02 (Bayer Diagnostics) for analysis of luminescence from 96-well plates.
Statistical Analysis. All values are either relative luminescence units
(RLU) or the ratio of expression for a specific CYP isoform to that of GAPDH.
Final determinations are the mean ⫾ S.E. for an n of 4 animals. One-way
ANOVA was used (P ⬍ .05). Post hoc comparisons were made using Duncan’s multiple range analysis.
611
CYTOCHROME P450 ANALYSIS USING QUANTIGENE TECHNOLOGY
TABLE 2
List of oligonucleotide probes generated for analysis of CYP expression by bDNA signal amplification
Probe ID
Targeta
Functionb
Probe Sequencec
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
I00732
570–591
658–679
680–699
700–721
722–745
746–766
767–787
788–811
812–833
834–860
861–885
887–910
912–931
932–953
954–972
CE
LE
LE
BL
CE
LE
LE
BL
BL
BL
CE
BL
LE
LE
CE
gtattcagcctctttgctcacgTTTTTctcttggaaagaaagt
agatgacattggccactgacacTTTTTaggcataggacccgtgtct
tctgccaaagcatatggcacTTTTTaggcataggacccgtgtct
cttggtcatcgtggtcataacg
ttagattgactatgctgagcagctTTTTTctcttggaaagaaagt
taacctccccaaactcattgcTTTTTaggcataggacccgtgtct
cagctgggtatccagaaccagTTTTTaggcataggacccgtgtct
ggtaacggaggataggaatgaagt
gcatccagggaagagttaggga
tagaacttcttattcaagtccttgaag
tttgattagcttcttcatgaaactgTTTTTctcttggaaagaaagt
ccttctcaaatgtcctgtagtgct
ctgtgatgtcccggatgtggTTTTTaggcataggacccgtgtct
tgacaatgctcaatgaggctgtTTTTTaggcataggacccgtgtct
ctcgtccagcctcctgtccTTTTTctcttggaaagaaagt
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
CYP1A1
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
X01031
501–522
569–591
592–612
613–631
632–651
652–671
672–695
696–716
717–734
757–776
777–799
800–821
822–841
842–861
862–880
881–903
904–921
922–937
938–955
956–978
979–998
LE
CE
LE
CE
BL
CE
CE
BL
LE
CE
BL
LE
BL
BL
LE
BL
BL
BL
CE
CE
CE
ctgtttcaaatccagctccaaaTTTTTaggcataggacccgtgtct
catgaatcttcctctgcaccttgTTTTTctcttggaaagaaagt
caatcaccgtgtccagctcctTTTTTaggcataggacccgtgtct
cgtggctgccgatctctgcTTTTTctcttggaaagaaagt
gctggggtctgtcagaaagc
gaaggcctccagatatggcaTTTTTctcttggaaagaaagt
tgtgtatcggtagatctccaggatTTTTTctcttggaaagaaagt
gatggtgaaggggacaaagga
cctcgttgtgctgtggggTTTTTaggcataggacccgtgtct
gcagcactccttgggaatgtTTTTTctcttggaaagaaagt
acctgccactggtttatgaagat
ccactgcttctcatcatggttgTTTTTaggcataggacccgtgtct
cggaacacaaaggggtcttt
tggtaagaaaccgctctggg
tcgatggccgtgttgtcatTTTTTaggcataggacccgtgtct
tcaccttctcactcagggtcttg
ttcccaagccgaagagca
ccaatgcaccggcgct
cacttggccgggatctccTTTTTctcttggaaagaaagt
tggctaagaagaggaagacttccTTTTTctcttggaaagaaagt
ctccagctgatgcaggaggaTTTTTctcttggaaagaaagt
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
CYP1A2
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
J00719
566–584
585–606
607–629
630–652
653–675
676–699
700–722
723–744
745–763
764–785
786–806
807–828
829–852
853–876
877–895
896–915
916–937
938–958
959–982
LE
BL
CE
BL
BL
BL
CE
LE
CE
BL
BL
LE
CE
CE
BL
BL
BL
LE
LE
tccaacaggcgcaggaactTTTTTaggcataggacccgtgtct
ggaaaaggtccggtagaacagc
tggctggagaatgaacttaggagTTTTTctcttggaaagaaagt
acccagagaagaactcaaacacc
ggcaccaggaaagtatttcagga
gaggtttttggagatttgtctgtg
ccaatgtaatcgaggatttcctgTTTTTctcttggaaagaaagt
cctgtgcttctccacaatatggTTTTTaggcataggacccgtgtct
cgcttgggtctaaggtggcTTTTTctcttggaaagaaagt
gtgtcgatgaagtctcgtggag
ttctccatgcgcagaaggtaa
tgtgtggtggttcgacttctccTTTTTaggcataggacccgtgtct
catgaggttctcatgatggaactcTTTTTctcttggaaagaaagt
aaagaagagagagagcagggagatTTTTTctcttggaaagaaagt
tgctggtctcagtgccagc
accatagcggagtgtggtgc
ggtacttgagcatcagcaggaa
ggactttctctgcgacatgggTTTTTaggcataggacccgtgtct
cgatcacctgatcaatctccttttTTTTTaggcataggacccgtgtct
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1
CYP2B1/2
CYP2B1/2
CYP2B1/2
CYP2B1/2/3/12/15
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
S48325
501–525
526–548
549–574
575–594
598–616
617–639
640–663
664–690
691–710
711–727
728–751
752–771
772–796
797–818
819–842
843–865
866–888
889–914
915–940
941–963
LE
CE
BL
BL
BL
LE
LE
BL
BL
LE
LE
LE
BL
CE
BL
BL
BL
BL
CE
CE
ctccatctctatgaggagacagtcaTTTTTaggcataggacccgtgtct
ggttcttggctgtgtttttccttTTTTTctcttggaaagaaagt
cagaaacattttccattgtgtacatg
gaacaggtcggccaaagtca
tggtggtctcagttcctgc
gagcccatatctcagagttgtgcTTTTTaggcataggacccgtgtct
ttctgggtatttcatgaggatcagTTTTTaggcataggacccgtgtct
aatttcttcatgaagtttctcttcaat
cttggcccaataaccctgtc
tgacagcagggacgcggTTTTTaggcataggacccgtgtct
tgtagggcatatccagtctgtctcTTTTTaggcataggacccgtgtct
ctcatgcaccacagcatccaTTTTTaggcataggacccgtgtct
ggacaagattgatgaatctctggat
gcttcatggggtaggttggaagTTTTTctcttggaaagaaagt
ccttggaacacagtatctctggtt
ctgtacccttggggatgacatat
ggagtccagagttggaatcacaa
ggaaactcatggctgtcatataagag
gctcaggtttaaacttctctggatctTTTTTctcttggaaagaaagt
cttcccattttcattcaggaaatTTTTTctcttggaaagaaagt
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
CYP2E1
Specificity
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
CYP1A1
CYP1A1.2001
CYP1A1.2032
CYP1A1.2033
CYP1A1.2080
CYP1A1.2003
CYP1A1.2034
CYP1A1.2035
CYP1A1.2081
CYP1A1.2082
CYP1A1.2083
CYP1A1.2004
CYP1A1.2084
CYP1A1.2036
CYP1A1.2037
CYP1A1.2005
CYP1A2
CYP1A2.2038
CYP1A2.2006
CYP1A2.2039
CYP1A2.2007
CYP1A2.2088
CYP1A2.2008
CYP1A2.2009
CYP1A2.2089
CYP1A2.2040
CYP1A2.2010
CYP1A2.2091
CYP1A2.2041
CYP1A2.2092
CYP1A2.2093
CYP1A2.2042
CYP1A2.2094
CYP1A2.2095
CYP1A2.2096
CYP1A2.2011
CYP1A2.2012
CYP1A2.2013
CYP2B1/2
CYP2B.2044
CYP2B.2099
CYP2B.2014
CYP2B.2100
CYP2B.2101
CYP2B.2102
CYP2B.2015
CYP2B.2045
CYP2B.2016
CYP2B.2103
CYP2B.2104
CYP2B.2046
CYP2B.2017
CYP2B.2018
CYP2B.2105
CYP2B.2107
CYP2B.2108
CYP2B.2047
CYP2B.2048
CYP2E1
CYP2E1.2049
CYP2E1.2019
CYP2E1.2109
CYP2E1.2110
CYP2E1.2106
CYP2E1.2050
CYP2E1.2051
CYP2E1.2111
CYP2E1.2112
CYP2E1.2052
CYP2E1.2053
CYP2E1.2054
CYP2E1.2113
CYP2E1.2020
CYP2E1.2114
CYP2E1.2115
CYP2E1.2116
CYP2E1.2117
CYP2E1.2021
CYP2E1.2022
GenBank ID
612
HARTLEY AND KLAASSEN
TABLE 2
Continued.
Probe ID
Targeta
Functionb
Probe Sequencec
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
D13912
616–637
638–661
662–681
683–706
707–729
730–755
756–780
781–802
804–831
832–851
852–873
874–896
897–923
924–946
947–968
970–994
LE
LE
LE
CE
BL
BL
LE
BL
BL
CE
LE
BL
LE
CE
CE
LE
ccaaatgatgtgctggtgatcaTTTTTaggcataggacccgtgtct
ttgttgagggaatcaacattcactTTTTTaggcataggacccgtgtct
ccacaaaaggatccttcgggTTTTTaggcataggacccgtgtct
attcttaagagcttcttggctttcTTTTTctcttggaaagaaagt
agaacaacggatcaaaaaaatca
gaggaatggaaagagtactactgaca
tgtttaacatctcatatactggcgtTTTTTaggcataggacccgtgtct
gaatcttttgggaacatgcaga
tgtacacaaatttttgaaaaattctat
caggcgggtttccttcattcTTTTTctcttggaaagaaagt
ctcgatgcttctgcacagaatcTTTTTaggcataggacccgtgtct
catcatcagctgaagaaaatcca
tttgtctttagaatcattatgagcattTTTTTaggcataggacccgtgtct
tcggatagggctgtatgagattcTTTTTctcttggaaagaaagt
tgactgggctgtgatctccataTTTTTctcttggaaagaaagt
tcatatccagcaaaaataaaaatgaTTTTTaggcataggacccgtgtct
CYP3A1/2/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CY3A1/2/23
CYP3A1/2/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
CYP3A1/23
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
M57719
501–526
527–548
549–575
576–601
602–621
622–646
647–667
669–694
695–716
717–742
743–761
762–778
779–801
802–823
824–845
846–865
CE
LE
BL
BL
CE
BL
BL
BL
LE
LE
LE
BL
LE
CE
CE
CE
tctcccatttgtctagcattatattgTTTTTctcttggaaagaaagt
gggtgatcctggtcatcaagctTTTTTaggcataggacccgtgtct
aaggagacatagtggaagatctctaga
acttcataacagtgtccagtgtcatc
gccctgatggctgaaagcacTTTTTctcttggaaagaaagt
tggaatttacatccaactgaacact
cgacagccttggtgtaggacc
gaaagaaagtcaggttgtttagatcc
ccataaaaggcactcctcacacTTTTTaggcataggacccgtgtct
aggacatattgtagatgatgctgttcTTTTTaggcataggacccgtgtct
cgggacaaacggccatcagTTTTTaggcataggacccgtgtct
caatctggcaggcccgg
cactccatctgtgtgctcatgagTTTTTaggcataggacccgtgtct
gctgagccttcctcattttgatTTTTTctcttggaaagaaagt
tgaagctcttcctcattctgcaTTTTTctcttggaaagaaagt
gcctcttcttcctggccttcTTTTTctcttggaaagaaagt
CYP4A3
CYP4A2/3
CYP4A2/3
CYP4A2/3
CYP4A3
CYP4A3
CYP4A3
CYP4A3
CYP4A2/3
CYP4A2/3
CYP4A2/3
CYP4A2/3
CYP4A3
CYP4A2/3
CYP4A2/3
CYP4A2/3
Specificity
a
Target relates to the target sequence of the mRNA transcript and the numbers signify the first nucleotide of the target sequence and extend through the last nucleotide of the target sequence.
Function relates to the function of the oligonucleotide probe in the bDNA assay (CE, capture probe; LE, label probe; BL, blocker probe).
Probe sequence relates to the oligonucleotide probe sequence that includes a poly T spacer with or without a CE or LE extension. All oligonucleotide probe sequences are the reverse complement
of the respective mRNA target sequence.
b
c
Results
Linearity of Response. To determine the RNA concentration range
in which bDNA analysis could be used in this study, various concentrations of total RNA were analyzed with the CYP2B1/2 probe set to
generate a standard curve of luminescence versus amount of RNA
added to each well (Fig. 3). When total RNA was increased, the
bDNA assay yielded a linear response for CYP2B1/2 RNA from
control (Fig. 3A) and PB-treated rats (Fig. 3B). The CYP2B1/2 signal
from total RNA isolated from control animals was linear from 0.1 to
100 ␮g of total RNA (range, 0.27–20 RLU; Fig. 3A). However, the
response attained from low levels (less than 5 ␮g) of control RNA was
less reliable than at higher concentrations of control RNA. A much
more robust signal was observed using 0.1 to 100 ␮g of total RNA
isolated from PB-treated rats (range, 13–2500 RLU; Fig. 3B). Very
similar results, in regard to linearity, were obtained with the CYP1A1
probe set and total RNA from control or 3MC-treated rats (data not
shown).
Sensitivity. CYP2B1/2 transcripts were detected reliably above
background noise with concentrations of total RNA from 5 ␮g isolated from control rats and as low as 0.1 ␮g from PB-treated rat livers
(Fig. 3). Therefore, we routinely analyzed 10 ␮g of total RNA from
control and treated rats. Furthermore, the level of background noise in
the assay was very low in which relative luminescence was consistently ⬍0.2 RLU.
Reproducibility of Response. Individual samples of total RNA
from a control and a PB-treated rat were analyzed repeatedly within
an experiment on 1 day (i.e., on the same plate) or between experiments (i.e., different plates on different days). When a single sample
from a control and a PB-treated rat was analyzed repeatedly, the
resulting data was reproducible between sample wells on a single
plate and between days (Table 3). Replicates within an experiment
were very reproducible (c.v. 8 –15%). Experimental reproducibility
was also reliable between days where the c.v. for raw luminescence
values for both control and treated samples were 25% (Table 3).
Chemical Responsiveness. The chemical responsiveness of the
CYP family of drug-metabolizing enzymes is the standard measurement for assessing drug/chemical CYP interactions, where specific
chemicals are known to induce specific CYP genes (Table 4). In this
study, we used this classic response paradigm to evaluate the bDNA
system. The detection of the chemical effects on specific CYP enzymes indicates that the oligonucleotide probe sets and the bDNA
system detect the classical differential response of specific CYPs to
various microsomal enzyme-inducing chemicals (Fig. 4).
The CYP1A CYP gene family is largely inducible by polyaromatic
hydrocarbons, like 3MC (Table 4). From bDNA analysis for CYP1A1
and CYP1A2 mRNA transcripts, results demonstrated that in the
absence of a chemical stimulus, constitutive levels of hepatic
CYP1A1 mRNA were very low (2-fold above background), whereas
a much greater level of constitutive CYP1A2 mRNA expression was
detected (Fig. 4). In stark contrast, in rats treated with 3MC, hepatic
expression of CYP1A1 and CYP1A2 was increased 670- and 11-fold,
respectively (Fig. 4 and Table 5). In general, results obtained from rats
treated with 3MC were consistent with previous reports for CYP1A1
and CYP1A2 mRNA expression.
The CYP2B CYP family is classically known to be inducible by PB
(Table 4). The oligonucleotide probes used to detect CYP2B1/2
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
CYP3A1/23
CYP3A.2059
CYP3A.2060
CYP3A.2061
CYP3A.2023
CYP3A.2120
CYP3A.2121
CYP3A.2062
CYP3A.2122
CYP3A.2123
CYP3A.2024
CYP3A.2063
CYP3A.2124
CYP3A.2064
CYP3A.2025
CYP3A.2026
CYP3A.2065
CYP4A2/3
CYP4A.2027
CYP4A.2066
CYP4A.2125
CYP4A.2126
CYP4A.2028
CYP4A.2127
CYP4A.2128
CYP4A.2129
CYP4A.2067
CYP4A.2068
CYP4A.2069
CYP4A.2130
CYP4A.2070
CYP4A.2029
CYP4A.2030
CYP4A.2031
GenBank ID
CYTOCHROME P450 ANALYSIS USING QUANTIGENE TECHNOLOGY
613
FIG. 2. Schematic mapping the location and function of individual
oligonucleotide probes to individual CYP transcripts.
Each single stranded CYP mRNA transcript is represented by a black line,
blocker probes are represented by green lines, capture probes are represented by
blue lines, label probes are represented by red lines, and gaps in the probe set are
indicated by arrows and the nucleotide position at which the gap occurs.
mRNA levels were specific for this subfamily of CYP mRNA transcripts, demonstrating that PB caused a significant increase in the
levels of CYP2B1/2 (Fig. 4). PCN also significantly increased
CYP2B1/2 mRNA levels. Overall, PB and PCN induced a 70- and
5.7-fold increase in hepatic CYP2B1/2 mRNA, respectively (Table 5).
The CYP2E1 enzyme is induced in chronic disease states like that
observed in diabetes and chronic alcoholism. This enzyme is also
up-regulated by acetone, pyrazole, and ISO (Table 4). However, the
mechanism by which CYP2E1 is up-regulated is primarily due to
stabilization of the protein and, secondarily, to transcriptional induction. Therefore, this gene is generally less responsive to chemical
stimuli. An oligonucleotide probe set to CYP2E1 was developed and
used to monitor chemical modulation of hepatic CYP2E1 mRNA by
bDNA analysis. Our results for CYP2E1 mRNA levels indicated that
this CYP maintains a high level of constitutive expression (i.e.,
control levels), and that the CYP2E1 gene is largely refractory to
chemical-mediated induction (Fig. 4). The effect of ISO on CYP2E1
was not statistically significant, but was 1.6-fold greater than salinetreated controls; this result agrees with ISO being a weak transcriptional inducer of CYP2E1 (Table 5).
The CYP3A family is known to be inducible by PCN (Table 4) and
other steroid-like compounds. Analysis of CYP3A1/23-mRNA expression by bDNA analysis demonstrated that PCN and PB significantly increased the expression of hepatic CYP3A1/23 (Fig. 4). PCN
and PB increased CYP3A1/23 expression 34- and 15-fold, respectively (Table 5). These results are consistent with PCN effects on
CYP3A expression (Table 4).
The CYP4A family of CYP enzymes is induced at the transcrip-
Top, concentration response curve of total liver RNA (0.1–100 ␮g) from a
control (untreated) rat versus RLU observed for CYP2B1/2 expression. Bottom,
concentration response curve of total liver RNA (0.1–100 ␮g) from a PB-treated rat
versus relative luminescence observed for CYP2B1/2 expression. The inset concentration response curves in the top and bottom graphs are provided to illustrate the
concentration response curves in the range of 0.1 to 10 ␮g of total RNA.
tional level by a number of plasticizers and antihyperlipidemic drugs.
We used the peroxisome proliferator, CLO, to evaluate the CYP4A2/
3-oligonucleotide probe set during bDNA analysis. Expression of
CYP4A2/3 is very low in control liver and the CYP4A2/3-mRNA
levels were specifically increased by CLO (Fig. 4). Compared with
control levels of CYP4A2/3, in response to CLO, CYP4A2/3 was
induced nearly 5-fold (Table 5).
Discussion
The ability to evaluate rapidly and accurately drug/chemical-elicited changes in drug metabolism enzymes would be a valuable commodity to pharmaceutical companies as well as to clinical investigators. The inducible CYP enzymes are markers of drug-mediated gene
expression and predictors of potential drug-drug interactions. To date,
analysis of P450 activity has been the standard for assessing such
interactions (Parkinson, 1996). In this report, bDNA signal amplification technology (Fig. 1) was used to assess the differential expression of CYPs as an alternative approach to enzymatic assays. We
developed oligonucleotide probe sets to the inducible CYPs and
monitored the mRNA expression of these enzymes in response to five
different chemicals known to induce specific CYP enzymes by bDNA
signal amplification technology. The present results demonstrate that
the bDNA signal amplification assay can be used as a specific,
efficient, and reproducible assay to monitor chemical-induced
changes in CYP expression.
A critical component of the bDNA signal amplification system is
that multiple and highly specific oligonucleotides are generated to a
target region of a mRNA transcript. This novel approach is advantageous as it uses the specificity inherent with short oligonucleotide
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
FIG. 3. Linearity and sensitivity of response of the bDNA assay with the CYP2B
probe set and RNA isolated from rat livers.
614
HARTLEY AND KLAASSEN
TABLE 3
Reproducibility of the bDNA assay for CYP2B2 mRNA quantification within and between experiments
CYP2B2 mRNA
Exp (10 ␮g/well)
Control RLUa
PB RLU
Replicates
Mean
Range
S.D.
c.v.
Replicates
Mean
Range
S.D.
c.v.
#
5
3
7.3
9.4
5.9–8.8
7.3–11.8
1.1
2.3
%
15.2
24.5
#
5
3
413.6
328.4
379–458.5
238.9–413.6
32.2
87.4
%
7.8
26.6
Within
Between
a
RLU signifies relative luminescence units. In all cases in wells without RNA, luminescence was less than 0.1 RLU (probe set only), and in wells with RNA in the absence of probe sets,
luminescence was less than 0.2 RLU.
TABLE 4
Effect of chemicals on the expression of specific CYP mRNAs
Major CYP Isoform(s) Up-Regulated
3MC
PB
ISO
PCN
CLO
CYP1A1, CYP1A2
CYP2B1, CYP2B2
CYP2E1
CYP3A1, CYP3A23, CYP3A2
CYP4A1, CYP4A2, CYP4A3
probes and the sensitivity of longer oligonucleotides or cDNA probes.
Designing specific probe sets to individual CYP mRNA transcripts
can be challenging as there are multiple and highly identical homologs
within the P450 superfamily. As summarized in Table 2, probe sets to
CYP1A1, CYP1A2, and CYP2E1 were designed to be specific to each
of these individual CYP transcripts. Individual oligonucleotide probe
sets for the CYP1A subfamily of CYP transcripts were developed to
both CYP1A1 and to CYP1A2, where these two transcripts share 76%
nucleic acid identity when analyzed by pair-wise sequence alignment
(Smith et al., 1996). The results obtained with these CYP1A probe
sets in the bDNA assay were consistent with previous reports for
CYP1A1- and CYP1A2-mRNA expression in response to chemical
stimuli. In the absence of a chemical stimulus, constitutive levels of
hepatic CYP1A1 mRNA were very low, whereas a much greater level
of constitutive CYP1A2 mRNA expression was detected (Fig. 4;
Table 5). In addition, both CYP1A1 and CYP1A2 mRNA levels were
substantially induced by 3MC (Fig. 4; Table 5). These results demonstrate that the bDNA system can be useful to detect differences
between highly similar homologs, like CYP1A1 and CYP1A2, which
share greater than 75% nucleic acid identity.
Like the CYP1A family, the CYP2E1 probe set was developed to
be specific to the CYP2E1 transcript. The bDNA data for hepatic
CYP2E1-mRNA expression in control and ISO-treated rats is in
accord with previous reports. Results for CYP2E1 gene expression as
measured by bDNA analysis demonstrate that this transcript is expressed constitutively at high levels, and that the CYP2E1 gene is
relatively insensitive to chemical stimuli. Our data (Fig. 4 and Table
5) suggest that ISO can mediate a mild induction of CYP2E1 gene
expression (1.6-fold; Table 5), whereas others have reported a similar
range (2–5-fold increase) for ISO induction of CYP2E1 mRNA (Parkinson, 1996).
Unlike the probe sets designed for CYP1A1, CYP1A2, and
CYP2E1, which were generated to delineate between these transcripts, other probe sets were designed to be specific to individual
CYP subfamilies. This was due to the high nucleotide similarity
between the CYP transcripts in the CYP2B, CYP3A, and CYP4A
subfamilies. Within each of these subfamilies, there are at least two
highly similar transcripts that share greater than 95% nucleotide
identity (e.g., CYP2B1 and CYP2B2 are 97% identical). In designing
oligonucleotide probes within each of these three CYP subfamilies, a
sufficient number of specific capture and label probes could not be
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
Chemical Inducer Administered
developed to differentially measure these transcripts. Therefore, in the
probe sets developed to the CYP2B, CYP3A, and CYP4A subfamilies,
probes were designed to hybridize to multiple CYP isoforms within each
subfamily, where CYP2B1/2, CYP3A1/23, and CYP4A2/3 are detected
(Table 2). In each case, it is known that the subfamily members respond
to the same chemical stimulus as described below (Table 4).
As alluded to, probes were developed to the CYP2B subfamily
because CYP2B1 and CYP2B2 transcripts are 97% identical at the
nucleotide level, and both genes are induced by PB (Parkinson, 1996).
One label probe in the CYP2B probe set, specifically CYP2B.2048,
also interacts with CYP2B3, CYP2B12, and CYP2B15 (Table 2).
However, these transcripts are not targets for any capture probes, and
as such, only the signals from CYP2B1 and CYP2B2 were measurable. When the CYP2B1/2 probes were used to analyze chemicalinduced differential expression of CYP mRNA, PB was shown to
elicit a 70-fold increase in hepatic CYP2B1/2 mRNA as compared
with control, and indicates that this probe set was sensitive to PBinduced changes in CYP2B1/2 mRNA levels. These results complement those reported by LeCluyse and colleagues (1999) for PB
induction of CYP2B activity, where PB caused a 61-fold increase in
CYP2B-mediated 7-pentoxyresorufin O-dealkylation.
As with the CYP2B subfamily, probe sets to the CYP3A subfamily
encompass both CYP3A1 and CYP3A23. These transcripts are 98.6%
identical, and both genes are up-regulated by PCN (Kirita and Matsubara 1993; Komori and Oda, 1994). Although CYP3A2 is 90%
identical with CYP3A1 and CYP3A23, only three probes,
CYP3A.2120, CYP3A.2121, and CYP3A.2122 (Table 2) interact with
CYP3A2 in BLAST analyses, and none of these three probes are
capture probes. Therefore, in theory CYP3A2 mRNA levels were not
measurable in these experiments. The nearly 34-fold increase (compared with control) in CYP3A1/23 signal obtained from samples
isolated from rats treated with PCN were consistent with the known
effects of this compound on CYP3A expression (Table 4). Additional
studies with a probe set developed to CYP3A2 combined with a
comparison of male and female CYP3A2 expression could indicate
whether our probe set to CYP3A1/23 is specific only for CYP3A1/23,
as CYP3A2 is expressed predominately in male rats.
Similarly, an oligonucleotide probe set that recognized the CYP4A2
and CYP4A3 isoforms of the CYP4A subfamily was developed. These
two distinct transcripts are 96% identical and both genes are up-regulated
by peroxisome proliferators like CLO (Kimura et al., 1989a,b). Although
a probe set was not developed to CYP4A1 (a.k.a. CYPLA-omega), which
is also inducible by peroxisome proliferators, a specific probe set could
easily be generated against CYP4A1 as it is only 68% identical with
CYP4A2 and CYP4A3. In this study, a 5-fold induction of CYP4A2/3
transcripts was observed (Table 5). This lower level of CYP4A induction
in response to CLO treatment is likely related to the specificity of the
probe set to the CYP4A2 and CYP4A3 mRNA transcripts. Sundseth and
Waxman (1992) reported that the CYP4A2 and CYP4A3 genes are much
less sensitive to CLO induction as compared with the CYP4A1 gene.
CYTOCHROME P450 ANALYSIS USING QUANTIGENE TECHNOLOGY
615
A to F, bar graphs depicting the CYP/GAPDH ratios for CYP1A1-, CYP1A2-, CYP2B1/2-, CYP2E1-, CYP3A1/23-, and CYP4A2/3-mRNA expression levels,
respectively, in response to five typical CYP inducers. Results are means ⫾ S.E. for n ⫽ 4 animals. Statistically significant differences (P ⬍ .05) are designated by an
asterisk (ⴱ). Vehicle controls, saline and corn oil, were not significantly different from each other, and are represented as a composite control for each CYP to which all
treatment groups are compared (filled columns).
TABLE 5
Fold induction of CYP mRNA expression detected by bDNA signal amplification
analysis
Chemical Inducer
CYP
CYP1A1
CYP1A2
CYP2B1/2
CYP2E1
CYP3A1/23
CYP4A2/3
3MC
PB
ISO
PCN
CLO
671.4a,b
11.4a,b
0.9
0.8
1.8
1.2
2.9
0.6
71.0a,b
0.9
15.1b
1.2
4.8
2.3
1.0
1.7a
0.9
1.7
0.8
0.4
5.8b
0.6
33.6a,b
0.4
1.3
0.3
2.4
1.1
0.6
4.7a,b
a
Denotes those treatment by enzyme interactions that are considered to be the classical
responses. Values are relative to control values for expression of each CYP. These values are also
in bold text for visual effect.
b
Denotes significant differences P ⬍ .05.
Additional studies that use a CYP4A1-specific probe set could further
expand on this result.
The specificity and sensitivity that result from the probe design is
apparent in our ability to detect the differential expression of highly
similar CYP transcripts (Fig. 5). For the CYPs analyzed in this study,
the bDNA assay was quite specific, to at least the subfamily level.
Overall, the results obtained by bDNA analysis of CYP expression
were as expected and consistent with previously reported findings
from many other investigators in terms of the differential and specific
response of individual CYPs to enzyme-inducing chemicals (Porter
and Coon, 1991; Parkinson, 1996). Certainly, the robust response
elicited by each chemical inducer on specific CYPs (Tables 3 and 5)
supports the use of the bDNA technique as an excellent method to
detect drug/chemical modulation of drug-metabolizing enzymes.
In terms of obtaining reproducible data in gene expression experiments, the bDNA assay is as good as any other assay we have
routinely used in this laboratory, including Northern blot analysis, in
situ hybridization, quantitative RT-PCR, RNase protection assays, as
well as the newer cDNA or oligonucleotide array technologies. The
cumulative data from Table 3 and Fig. 4 support this statement, where
Table 3 demonstrates that the bDNA assay generated reproducible
data by both repeated measures of a sample over a number of days,
and in the same experiment. It is apparent from Table 3 that the c.v.
is relatively high (25%) for repeated analysis of samples between
days. However, it is important to point out that this c.v. was determined for non-normalized luminescence measurements, and, as such,
appears relatively high. Normalization of the luminescence values to
GAPDH expression reduces this value as it controls for slight differences in RNA input between days and day-to-day variation in overall
luminescence. Analysis of a given CYP transcript between animals
resulted in reproducible data as suggested by the S.E.M. shown in Fig.
4.
The bDNA assay is a very sensitive measure of gene expression.
From this data (Fig. 3), a measurable and reproducible signal for
CYP2B1/2 was detected with 0.1 ␮g of total RNA. This is impressive
as it is generally assumed that mRNA constitutes approximately 1 to
2% of total RNA. Therefore, 0.1 ␮g of total RNA would be equivalent
to 1 to 2 ng of poly A⫹ mRNA. From this calculation, bDNA analysis
would be at least 100 times more sensitive than cDNA or oligonucleotide array technologies, which require 1 ␮g of poly A⫹ RNA.
Others have reported that bDNA system is comparable in sensitivity
to PCR-based strategies (Collins et al., 1997; Guenthner and Hart,
1998).
The bDNA signal amplification system has many advantages over
other contemporary methods of gene expression analysis. One distinct
advantage is that the researcher can use total RNA (as in a Northern
blot) or cell extracts for the analysis. Use of total RNA in the bDNA
system resulted in experimentally reproducible and appropriate (in
terms of chemical-induced CYP expression) data. In this report, the
analysis of gene expression data was presented in a semiquantitative
Downloaded from dmd.aspetjournals.org at ASPET Journals on June 16, 2017
FIG. 4. Specificity of the bDNA signal amplification system for CYP transcripts is apparent in chemical-induced differential expression of CYP mRNA transcripts.
616
HARTLEY AND KLAASSEN
Acknowledgments. We thank Nicole Dockendorf and Robert
Bencher of Bayer Diagnostics (Emeryville, CA) for providing assistance in obtaining the necessary equipment and reagents for this study.
In addition, we thank Jeff Donahue (Bayer Diagnostics, East Walpole,
MA) for technical training with the ProbeDesigner Software and the
Quantigene bDNA Signal Amplification system, and for donating the
information on the GAPDH probe set.
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manner by expressing the level of CYP to that of GAPDH. This assay
can be made quantitative by incorporating known amounts of in vitro
transcripts to each gene of interest, and therefore, the bDNA assay is
an excellent quantitative measure of gene expression. This method of
quantifying gene expression is superior to quantitative RT-PCR for
several reasons, including: 1) curves generated are linear (not logarithmic); and 2) the product measured represents the actual amount of
a given transcript. Although the bDNA assay is not yet run in a
multiplex arrangement, where more than one gene transcript is detected in a single well, gene expression for many genes can be
monitored simultaneously in parallel wells. This approach was taken
in the current study, where six CYP probe sets and the GAPDH probe
set were used simultaneously to assess chemical-induced changes in
gene expression. Another distinct advantage to the bDNA assay is that
assay development time is not spent at the laboratory bench. Rather,
oligonucleotide probe sets are developed at the computer where the
combination of Bayer Diagnostics ProbeDesigner software and
BLAST search tools are used to generate gene-specific probe sets.
Lastly, the 96-well bDNA-plate format allows for thousands of samples to be analyzed simultaneously. For this study, two bDNA-plates
(192 samples) were typically analyzed per experiment. Together, the
time saved in sample preparation, assay development, and during
experimental analysis are significant advantages of the bDNA assay
when compared with other assays for gene expression.
With all the advantages of bDNA analysis over other orthologous
techniques, there are two distinct disadvantages to this technique,
including expense of analysis and potential difficulties in generating
oligonucleotide probe sets within highly homologous gene families.
Regarding expense, oligonucleotide probe sets can be a significant
initial expenditure. This is considered a one-time cost as many thousands of assays can be run from a single probe set. The Quantigene
bDNA Signal Amplification Kits are consumables and are quite
expensive. However, because assay development problems are negligible, the interim period between assay development and data collection is substantially shorter than with more traditional techniques of
mRNA analysis, thus, keeping associated costs (i.e., labor, reagents,
animals, etc.) lower. A recognized limitation of the bDNA assay is
that inherent in its design is the inability to easily develop probe sets
to highly homologous gene families. Alternatively, if known, the 5⬘and 3⬘-untranslated regions of a mRNA transcript can serve as an
excellent target region to design oligonucleotide probes sets that
differentiate between highly homologous gene products, as these
untranslated regions of the mRNA are generally less conserved between homologs.
In conclusion, the present data supports the use of the bDNA signal
amplification technology as an efficient, subfamily-specific, and reliable assay for the assessment of xenobiotic-mediated CYP induction.
The efficiency, reproducibility, and specificity of the bDNA system as
used in this report could serve to supplant or support enzyme assays
as a primary screening technology for drug-induced changes in CYP
mRNA expression.