Experimental Procedures

Materials.

Murine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α were purchased from BioSource International (Camarillo, CA). Sodium phenobarbital (PB) was obtained from the Wyeth-Ayerst Research compound room (Princeton, NJ). Dexamethasone (DEX) and 3-methylcholanthrene (3-MC) were obtained from Sigma Chemical Co. (St. Louis, MO). TRIzol reagents, oligonucleotide primers (through custom synthesis), Superscript II, and RNase inhibitor were purchased from Life Technologies (Rockville, MD). TaqMan PCR Core Reagent Kit, TaqMan ribosomal RNA control reagent kit, and the oligonucleotide probes (custom synthesis) were purchased from PE Biosystems (Foster City, CA).

Methods.

Animal treatment

Male CD1 mice (8–10 weeks old; ∼30 g) were obtained from Charles River Laboratories (Raleigh, NC). Animals were allowed free access to food and water and were subjected to a 12-h light/dark cycle for 1 week before the experiment. Animals (five per group) received a single i.p. injection (0.3 ml) of IL-6 (100 ng/mouse), IL-1β (500 ng/mouse), TNF-α (2 μg/mouse), PB (80 mg/kg), 3-MC (80 mg/kg), or DEX (100 mg/kg; Kiyohara et al., 1990; Chen et al., 1992; Corcos, 1992). The vehicle control animals received 0.9% NaCl/0.1% BSA, or corn oil (for 3-MC). Animals were sacrificed 24 h after the treatment. Livers were excised and immediately frozen on dry ice and stored at −80°C before total RNA preparation.

RNA preparation.

Frozen livers were thawed on ice, and total RNA was isolated using TRIzol reagent. The quality of the isolated RNA was assessed by electrophoresis on 1% agarose gels based on the integrity of 28S and 18S bands after ethidium bromide staining.

Design of primers and probes.

The cDNA sequences of Cyp3a11, 2b10, 2d9, 2e1, and 1a2 were obtained from GenBank (accession numbers X60452, M21856, M27168,L11650, and X00479, respectively). PCR primers and fluorescent probe sequences were designed using PrimerExpress software (PE Biosystems) and shown in Table 1. The specificity ofCyp primers and probes was confirmed through alignment within the Cyp subfamily and by sequencing each RT-PCR amplicon. To avoid amplification of contaminating genomic DNA, one of the primers or the probe was placed at the junction between two exons. For example, Cyp1a2 forward primer crosses the junction of exons 3 and 4, whereas Cyp2d9 forward primer is from exon 5, and the probe and reverse primer are from exon 6.

Real-time RT-PCR.

RT and PCR were conducted in one step using TaqMan PCR Core Reagent Kit in a TaqMan 7700 Sequence Detector (PE Applied Biosystems) according to the manufacturer's protocol with slight modifications. Briefly, Master Mix solution was prepared at a final concentration of 5.5 mM MgCl₂; 0.025 U/μl Taq DNA polymerase (Amplitaq Gold; PE Applied Biosystems); 0.15 U/μl Superscript II; 1× reaction buffer (PE Biosystems) containing 500 mM KCl, 100 mM Tris-HCl (pH 8), 0.1 M EDTA, and 600 nM passive reference 1; 0.3 mM concentration each of dATP, dGTP, and dCTP; 0.6 mM dUTP; and 0.1 U/μl RNase inhibitor. The primers and fluorescent probes were used at a final concentration of 200 and 100 nM, respectively. Probes used 6-carboxy-fluorescein as the reporter dye and 6-carboxy-tetramethyl-rhodamine as the quencher dye at the 5′ and 3′ ends, respectively. The reaction also contained the primers and probe of 18S rRNA, as a housekeeping gene, for normalization of target mRNA results. The quantitative RT-PCR for target template and 18S rRNA was run in the same plate but separate tubes. RT reaction mixtures were preincubated at 60°C for 30 min and then at 95°C for 5 min to activate AmpliTaq Gold DNA polymerase. Two-step thermocycling was performed for 40 cycles: denaturation at 95°C for 20 s and anneal/extension at 58–60°C for 1 min. Increases in fluorescence, which were due to the cleavage of the reporter dye as the PCR proceeded relative to the starting values of delta normalized reporter fluorescence (ΔRn), were determined and plotted by the instrument against cycle number (Fig. 1). Ct values (the PCR cycle number required for fluorescence intensity to exceed an arbitrary threshold in the exponential phase of the amplification) were then determined for a series of standards. A standard curve was generated by plotting Ct versus the log of the amount of total RNA added to the reaction (0.3–100 ng; Bulletin 2, PE Applied Biosystems) and used to compare the relative amount of a particular Cyp mRNA in the samples from control and cytokine-treated animals. Calculations of Ct and ΔRn, standard curve preparation, and quantification of mRNA in the samples were performed by the software provided with the ABI 7700 system (Heid et al., 1996).

Results

Development of Real-Time RT-PCR Assay.

Standard curve and reproducibility

Figure 1A shows a representative amplification plot for Cyp3a11 that was established by plotting the ΔRn versus PCR cycle number. There was no increase in fluorescence in the control samples either without the template or without the reverse transcriptase, indicating the absence of DNA contamination in the system and the samples (Fig.1A). The resulting standard curve of Ct against log nanograms of total RNA input was highly linear, as shown by a correlation coefficient (R²) value of >0.99 over a wide range of total RNA input (0.3–100 ng; Fig. 1B). Similar amplification plots and standard curves were obtained for Cyp2b10, 2d9, 2e1, and 1a2 (data not shown).

In addition, the method was highly reproducible, as demonstrated by the small standard deviation (0.03–0.16) and coefficient of variation (<1) of quadruplicate determinations in Table2 (1 and 100 ng of total RNA input).

Method validation with classic Cyp inducers.

The real-time RT-PCR method that had been developed forCyp3a11, 2b10, 2d9, 2e1, and 1a2 was further validated by measuring mRNA expression in livers of the mice treated with classic inducers (PB, 3-MC, and DEX), and the results were compared with those for control (uninduced) animals (Fig. 2). PB induced Cyp3a11, 2b10, and 1a2 mRNA by 4-, 22-, and 3-fold, respectively. 3-MC potently induced Cyp1a2 mRNA by 17-fold, whereas DEX induced both Cyp3a11 and 2d9 mRNA by 5-fold. Cyp2e1 mRNA was moderately, but significantly, suppressed (∼30%) by PB but unaffected by DEX or 3-MC. The result of Western blot analysis using rat CYP2B1/2, CYP2E1, and CYP3A2 antibodies, respectively, showed that Cyp2b-like andCyp3a-like proteins were dramatically induced by PB treatment, whereas Cyp2e1 protein was suppressed by 50% (data not shown).

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Figure 2

Effect of classic inducers on expression of Cyp mRNA assayed by real-time RT-PCR.

CD1 mice were injected once i.p. for 24 h with PB, 3-MC, and DEX, respectively. Control mice were injected with 0.9% NaCl/0.1% BSA, or corn oil only. Total RNA (10 ng) was loaded for one-step RT-PCR real-time assay of Cyp3a11, 2b10, 2d9, 2e1, and 1a2 mRNAs.

Modulation of Mouse Cyp mRNA by Cytokines.

After establishing the real-time RT-PCR method, it was subsequently used to study the effect of proinflammatory cytokines on CypmRNA expression in the mouse. IL-1β and IL-6 significantly suppressed the expression of constitutive Cyp2b10 mRNA to 40 and 15% of control values, respectively (Table3). IL-6 also suppressedCyp1a2 mRNA levels by 67%. These cytokines had little or no effect on the expression of the other mouse Cyp mRNAs that were studied (Cyp3a11, 2d9, and 2e1). TNF-α decreasedCyp1a2 mRNA by 59% and also significantly suppressedCyp2d9 mRNA to 70% of control values. TNF-α did not affect Cyp3a11, 2b10, and 2e1 mRNA levels.

Discussion

To our knowledge, this is the first application of real-time RT-PCR TaqMan method to study CYP mRNA expression. In the present study, we demonstrated that this method is highly reproducible, as demonstrated by low coefficients of variation as well as day-to-day reproducibility (data not shown). This method also works over an extended dynamic range (0.3–100 ng of total RNA input). In addition, the TaqMan method is exquisitely sensitive (0.3 ng of total RNA input or less) and quantitative. This is in contrast to existing methodologies that often require micrograms of total RNA (e.g., Northern blot, slot-blot, and RNase protection assays). This methodology could be therefore particularly useful in studies where the amount of target mRNA may be very low; such as in extrahepatic tissues or in cultured cells. In addition, the method does not require electrophoretic gels to be run, and the assay can be performed in a high throughput format, such as a 96-well microtiter plate. Furthermore, this method contains fluorescence probe as well as the use of gene-specific PCR primers, which provides additional specificity in gene superfamilies, because a positive fluorescent signal can be generated from a PCR product only if both the primers and the probe hybridize to the template cDNA.

We have confirmed the suitability of using the real-time RT-PCR method to study Cyp mRNA expression with several prototypic CYP inducers. As expected, we found that PB induced Cyp2b10 and 3a11 mRNA expression, as well as the levels of Cyp2b-like and 3a-like protein (data not shown), which is consistent with the literature (Corcos, 1992; Damon et al., 1996). We also demonstrated that PB appeared to induce Cyp1a2 mRNA expression (∼3-fold) in this strain of mouse (CD1). This observation is interesting because arylhydrocarbons (e.g., 3-MC), not PB, are generally considered to induce the CYP1A gene family. This finding is consistent with Sakuma et al. (1999), who recently demonstrated thatCyp1a2 mRNA is induced by PB and other barbiturates in aryl hydrocarbon-responsive (C57BL/6, C3H) and nonresponsive (DBA/2, AKR, NZB) mice. It was also interesting to find that PB markedly suppressedCyp2e1 mRNA. Suppression of mRNA encoding this enzyme has been observed in the Western blot analysis by using anti-rat CYP2E1 antibody. The finding that DEX markedly induced Cyp2d9 (∼5-fold) was also a new and intriguing finding in that this gene subfamily is generally considered to be refractory to induction. The magnitude of this effect (∼5-fold) was comparable with the expected effects of this agent (Corcos, 1992) on Cyp3a11 mRNA levels and suggests common or overlapping regulatory mechanisms for these two enzymes in this species.

The new quantitative RT-PCR technique was then used to study the effects of several proinflammatory cytokines on the expression of specific mouse Cyp mRNAs. Consistent with reported effects of these compounds on CYP expression in a number of other species (Chen et al., 1995; Monshouwer et al., 1996), including humans (Abdel-Razzak et al., 1993; Fukuda and Sassa, 1994), all three proinflammatory cytokines were found to down-regulate mouse Cyp mRNA. However, these effects were highly cytokine/isoenzyme-selective. For example, IL-6 and IL-1β suppressed Cyp2b10 mRNA without affecting the enzymes of Cyp3a11, 2e1, and 2d9. Although both of these cytokines are known to decrease CYP2B induction in response to PB (Clark et al., 1995), this is the first time that these cytokines have been shown to reduce constitutive expression of this gene subfamily. Similar cytokine/isoenzyme-specific effects have been noted in other species. For example, IL-6 down-regulates CYP2C11, but not CYP3A2, mRNA in the rat (Chen et al., 1995). This selectivity is important from both a clinical and toxicological perspective and because it implies that effects on CYP mRNA may occur via multiple mechanisms.

The finding that TNF-α suppressed Cyp1a2 mRNA levels was consistent with the noted down-regulation of Cyp1a activity and mRNA by this cytokine in cultured murine hepatoma cells (Paton and Renton, 1998). IL-6, as well as TNF-α, also markedly decreasedCyp1a2 mRNA in the present study. It is noteworthy that both TNF-α and IL-6 suppress CYP1A2 expression in cultured human hepatocytes (Abdel-Razzak et al., 1993; Guillen et al., 1998). Our finding that Cyp2d9 mRNA was down-regulated by TNF-α is consistent with data from an earlier study by Trautwein et al. (1992)in which a Cyp2d-like mRNA species was down-regulated by this cytokine in the C3H/HeJ mouse. Our finding that TNF-α suppressesCyp2d9 mRNA is also consistent with a recent study by Warren et al. (1999) in which it was noted that Cyp2d9 activity (testosterone 16α-hydroxytestostertone formation) is lower in TNF-α receptor knockout mice. In the same study, the effects of lipopolysaccharide on Cyp2b and 3a mRNAs were similar in knockout versus wild-type animals. This finding suggests that the effect of lipopolysaccharide on these enzymes occurs through a mechanism that is independent of TNF-α. The finding in the present study that Cyp2b10 and 3a11 mRNAs were unaffected by TNF-α supports this conclusion.

In summary, we have established a real-time quantitative RT-PCR technique to study Cyp mRNA expression in the mouse. This technique allows us to quantitatively measure a set of mouseCyp mRNAs and to study the effects of classic inducers and proinflammatory cytokines on Cyp mRNA expression in the mouse.