Introduction

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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 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.

View larger version (110K): [in this window] [in a new window]   Fig. 1.   Two-dimensional illustration of the multimeric complex formed with a target mRNA transcript during the bDNA signal amplification assay. 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.

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.

	Materials and Methods

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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 µg/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 T_m 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 (80%) 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 extenders³) 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).

View larger version (25K): [in this window] [in a new window]   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.

bDNA Assay. Specific CYP oligonucleotide probe sets (i.e., blocker probes, capture probes, and label probes) were combined and diluted to 50 fmol/µl 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/µl 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 µg/µl; 10 µl) was added to each well of a 96-well plate containing capture hybridization buffer and 100 µl 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 µl/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 µl/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 µl/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.

	Results

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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 µg of total RNA (range, 0.27-20 RLU; Fig. 3A). However, the response attained from low levels (less than 5 µg) 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 µg 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).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Linearity and sensitivity of response of the bDNA assay with the CYP2B probe set and RNA isolated from rat livers. Top, concentration response curve of total liver RNA (0.1-100 µg) from a control (untreated) rat versus RLU observed for CYP2B1/2 expression. Bottom, concentration response curve of total liver RNA (0.1-100 µg) 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 µg of total RNA.

Sensitivity. CYP2B1/2 transcripts were detected reliably above background noise with concentrations of total RNA from 5 µg isolated from control rats and as low as 0.1 µg from PB-treated rat livers (Fig. 3). Therefore, we routinely analyzed 10 µg 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).

View larger version (43K): [in this window] [in a new window]   Fig. 4.   Specificity of the bDNA signal amplification system for CYP transcripts is apparent in chemical-induced differential expression of CYP mRNA transcripts. 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 (*). 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).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

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 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 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 chemical-induced 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 PB-induced 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. 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 µg of total RNA. This is impressive as it is generally assumed that mRNA constitutes approximately 1 to 2% of total RNA. Therefore, 0.1 µg 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 µg 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 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.