Short Communication SPURIOUS OBSERVATION OF SPLENIC CYP2B1 EXPRESSION

Phenobarbital (PB) induction of the CYP2B subfamily was studied in the livers and spleens of male and female rats. Animals were treated with either PB (10 mg/kg) or vehicle for 4 consecutive days. A reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative Northern blotting, Western blotting, and a radioenzymatic assay were used to observe differential levels of CYP2B1 and CYP2B2 mRNAs, proteins, and catalytic activities. CYP2B2 expression was limited to the livers of PB-treated male and female rats and was not detected in spleen. Low constitutive levels of CYP2B1 mRNA were markedly induced 7to 17-fold in the livers of PBtreated male and female rats, respectively. However, using the same standard oligonucleotide probe for CYP2B1 mRNA, we observed considerably greater constitutive concentrations of the transcript in spleen than in liver. Putative splenic CYP2B1 mRNA was significantly elevated by the PB treatment, although not as profoundly as the hepatic response. In contrast, only the livers of the barbiturate-treated rats expressed CYP2B1 proteins or specific catalytic activity (androstenedione 16 -hydroxylase). Protein and catalytic activities of the isoforms were undetectable in spleen of either male or female vehicleand PB-treated rats. In agreement, RT-PCR was unable to demonstrate the expression of splenic CYP2B1 mRNAs. Investigating the possibility that the Northern probe for CYP2B1 was identifying a similar sequence isoform, we performed RT-PCR using primers for CYP2B12 and CYP2B15. Since neither of these isoforms was expressed in spleen, we conclude that the spurious results using the Northern probe for CYP2B1 mRNA were due to the presence of a cross-reacting, PB-responsive transcript not currently identifiable in existing databases. The cytochrome P450 mixed-function oxygenases are a family of hemoenzymes that catalyze a large variety of biotransformation reactions. The liver contains the highest specific content of total P450, but P450s are present in extrahepatic tissues as well (Gonzalez, 1988). Hepatic P450s play a major role in the detoxification and/or activation of xenobiotics. Extrahepatic tissues are often target sites for bioactivation-mediated toxicities involving cancers, mutations, and birth defects (Hall et al., 1999; Maliakal et al., 2002). Consequently, the tissue-specific regulation of P450 gene expression may represent an important parameter determining the susceptibility of individual tissues to the toxic effects of’ chemicals undergoing bioactivation. P450-mediated metabolism in extrahepatic organs (such as lung, kidney, skin, and nasal epithelium) have been recognized in laboratory animals (Gram et al., 1986) and humans (McLemore et al., 1990). Transcription of individual P450 genes can be markedly stimulated by a variety of agents. In the rat, phenobarbital (PB) induces members of at least three distinct P450 subfamilies [i.e., CYP2B and CYP2C (Hardwick et al., 1983; Omiecinski et al., 1990) and CYP3A (Gonzalez et al., 1986)]. The PB-responsive P450 genes also are subject to highly tissue-specific and developmentally regulated programs (Gonzalez, 1988; Hall et al., 1999). This study was designed to investigate baseline expression and PB-induction of CYP2B isoforms in spleen. Materials and Methods Reagents. Phenobarbital and most of the other reagents were purchased from Sigma-Aldrich (St. Louis, MO). Monoclonal anti-rat CYP2B1 and 2B2 were purchased from Oxford Biomedical Research, Inc. (Oxford, MI). Enhanced chemiluminescence kits were purchased from Amersham Biosciences UK, Ltd. (Little Chalfont, Buckinghamshire, UK). TRIzol reagent was purchased from Invitrogen (Carlsbad, CA). Enzymes used in RT-PCR reactions were purchased from Promega (Madison, WI). Rat cytochrome P450 competitive RT-PCR kits were purchased from PanVera Corp. (Madison, WI). Nytran N (nylon) membrane was purchased from Schleicher and Schuell (Dassel, Germany). [4-C]Androstenedione (53.9 mCi/mmol) was purchased from PerkinElmer Life Sciences Inc. (Boston, MA). All other reagents were of the highest quality. Animal Treatment. Fischer 344 and Sprague-Dawley rats were purchased from Charles River Laboratories, Inc. (Wilmington, MA) and housed in the University of Pennsylvania Laboratory Animal Resources facility, under the supervision of certified Laboratory Animal Medicine veterinarians, and were treated according to a research protocol approved by the University Institutional Animal Care and Use Committee. Rats were injected daily, intraperitoneally, with either 10 mg/kg body weight of phenobarbital sodium or an equivalent sodium concentration of NaCl diluent, pH 9.1. On the morning following the fourth injection of the barbiturate, the rats were decapitated, and livers and spleens were quickly removed and minced; a portion reserved for mRNA determinations was plunged into liquid nitrogen and subsequently stored at 70°C. The remaining minced liver and spleen were used for microsomal preparations. Isolation and Purification of Total RNA. Total hepatic and splenic RNA was isolated using TRIzol reagent according to the protocol of the manufacturer. The purity of RNA was estimated by the absorbance ratio 260:280 nm. RNA was incubated for 10 min at 37°C with RQ1 RNase-free DNase (1 unit/ g) according to the recommendations of the supplier, followed by thermal inactivation of the enzyme (65°C for 10 min) and rapid cooling down to 4°C. Northern Blotting Analysis. Ten micrograms of RNA were electrophoresed under formaldehyde denaturing conditions on 1.2% agarose containing 0.2 mol/l formaldehyde and transferred to Nytran membranes. The blots were This work was supported by National Institutes of Health Grants HD16358 and GM45758. 1 Abbreviations used are: P450, cytochrome P450; PB, phenobarbital; RT-PCR, reverse transcriptase-polymerase chain reaction; AMV, avian myeloblastosis virus. Address correspondence to: Dr. Meena R. Sharma, Laboratories of Biochemistry, School of Veterinary Medicine, University of Pennsylvania, 3800 Spruce Street, Philadelphia, PA 19104-6048. E-mail: [email protected] 0090-9556/03/3109-1074–1076$7.00 DRUG METABOLISM AND DISPOSITION Vol. 31, No. 9 Copyright © 2003 by The American Society for Pharmacology and Experimental Therapeutics 1066/1084195 DMD 31:1074–1076, 2003 Printed in U.S.A. 1074 at A PE T Jornals on Sptem er 8, 2017 dm d.aspurnals.org D ow nladed from probed with P-labeled CYP2B1 and 2B2 oligonucleotide probes (Omiecinski et al., 1990). Evidence that RNA was equally loaded and transferred was obtained by equivalent intensity of ethidium bromide staining of 18S and 28S rRNA bands (Schuetz et al., 1990). Furthermore, the 18S rRNA oligonucleotide probe was used as a control to verify the consistency and integrity of RNA loading (Ramsden et al., 1993). RT-PCR Assay. RT was performed using 2 g of total RNA from liver and spleen, which used the AMV reverse transcriptase, with one cycle of the RT reaction (30°C for 10 min, 55°C for 20 min, and 95°C for 5 min). Negative controls contained either no sample RNA or no AMV RT. The cDNA was stored at 20°C. The PCR reaction was conducted containing 10 l of cDNA. A primer mix for cyclophilin was used as an external standard. The rat CYP2B1/2 and CYP2E1 primer mix was used from the rat cytochrome P450 competitive RT-PCR set identified above. Primers for 2B12 and 2B15 were designed using the Primer3 tool (http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi/). The sequences were 2B12 (forward, 5 -CAGTGCTCCACGAGATTTCA-3 ; reverse, 5 -TGGACTTTCTCTGCAACGTG-3 ) and 2B15 (forward, 5 TATTAGCCAGCCAAAATCGG-3 ; reverse, 5 -TGCATGAAAGAACGCAGAAG-3 ). PCR conditions were heat denatured at 94°C for 2 min, followed by 24 cycles of 94°C for 30 s, 56°C for 30 s, and 72°C for 30 s on a GeneAmp PCR system 2400 (PerkinElmer). Amplified PCR products were separated on 2% agarose gel and stained with ethidium bromide. Western Blotting Analysis. Hepatic and splenic microsomes were prepared according to our reported method (Shapiro et al., 1989). Five and 40 g of microsomal protein of liver and spleen, respectively, were electrophoresed on SDS-polyacrylamide gels and electroblotted onto nitrocellulose filters. The blots were probed with monoclonal anti-rat CYP2B1 and CYP2B2 mouse IgG (dilution 1:100). The primary antibody was located with horseradish peroxidase conjugated to anti-mouse IgG and detected with minor modification (Pampori et al., 1995) using an enhanced chemiluminescence kit. Catalytic Activity. Androstenedione 16 -hydroxylation, a specific catalytic activity of CYP2B1 and 2B2 (Waxman and Azaroff, 1992) was assayed using [4-C]androstenedione, and the hydroxylated products were resolved by thin layer chromatography (Shapiro et al., 1994; Sharma et al., 1996) and quantitated by the Storm 860 PhosphorImager (Amersham Biosciences Inc., Piscataway, NJ). Statistics. Data were subjected to analysis of variance, and differences were determined with t statistics and the Bonferroni procedure for multiple comparisons. Results and Discussion In agreement with earlier findings (Agrawal and Shapiro, 1996), we observed that a fraction of the usually administered inductive dose of PB was quite capable of inducing considerable levels of hepatic CYP2B1 and CYP2B2 mRNAs, proteins, and catalytic activity. The studies described herein employed two synthetic 18-mer hybridization probes widely used (Omiecinski et al., 1990; Waxman and Azaroff, 1992; Larsen et al., 1994; Agrawal and Shapiro, 1996; Sharma et al., 1998) in the assessment of PB-inducible CYP2B1 and CYP2B2 mRNAs. CYP2B2 expression was only detectable in the livers of barbiturate-treated males and females (Fig. 1). In contrast, putative constitutive CYP2B1 mRNA was present in spleen at an even higher concentration than liver, although less responsive than liver to PB induction (Fig. 1). Whereas hepatic CYP2B1 protein and catalytic activity was dramatically induced by the barbiturate (Fig. 1), its absence from the spleens of the same animals was worrisome but not without some precedence. At very low doses of PB (1 mg/kg/day), induced concentrations of the CYP2B1 transcript were not necessarily translated into new protein or catalytic activity (Agrawal and Shapiro, 1996). However, the added finding of undetectable levels of splenic CYP2B1 mRNA by RT-PCR (Fig. 2) strongly indicated that the oligonucleotide probe did not identify CYP2B1 mRNA in spleen. Accordingly, a Blast search showed that the 18-mer hybridization probe used in our study has sequence similarity with rat CYP2B1 (gi:203752), CYP2B12 (gi:56049), and CYP2B15 (gi:468482). Nevertheless, using highly selective primers, we were unable to detect by RT-PCR any of these isoform transcripts, including CYP2E1, in spleen (Fig. 2). In contrast, we did observe RT-PCR-measurable CYP2B1/2 (overlapping primers), CYP2B12 and CYP2E1 mRNAs in both male and female liver. We can only conclude that the spurious results using the 18-mer 2 Demonstrating that our findings were not necessarily strain specific, we observed the same pattern of splenic CYP2B1 and CYP2B2 mRNA and protein expression in Sprague-Dawley rats (data not shown) as found in Fischer 344 rats (Fig. 1). 3 Although exhibiting little sequence similarity with CYP2B1, CYP2E1 (gi:3126850) is widely distributed in the body (Haufroid et al., 2001; Zhu et al., 2002) and has been included as a biological control. 4 The cyclophilin gene was amplified, generating 266 base pairs of PCR product in all the samples at similar intensity, ensuring the effectiveness of the procedure and an equal quantity of mRNA in each group of animals. Sequence analysis of all RT-PCR products were in agreement with the known sequences of the designated P450 isoforms and cyclophilin (data not presented). FIG. 1. Relative hepatic and presumed splenic CYP2B2 and CYP2B1 mRNA, protein and specific androstenedione 16 -hydroxylase catalytic activities in vehicle-treated and PB-treated Fischer 344 male and female rats. A representative Northern blot analysis of a single liver and spleen per treatment group with a P-labeled oligonucleotide probe presumed specific for CYP2B1 mRNA is included in the bottom panel. Vehicle and PB treatments are described in the text. Protein and mRNA levels were determined by a Fluor-S MultiImager Bio-Rad image analysis system (Bio-Rad, Hercules, CA) of actual Northern autoradiographs and Western-enhanced chemiluminescence autoradiographs, and the 16 -hydroxylase product of C-androstenedione metabolism was isolated by thin layer chromatography and quantified by the Storm 860 PhosphorImager for at least four different livers and spleens in each group (mean S.D.). ND, not detected. , P 0.02, comparing the same tissue from vehicle-treated rats of the same sex. Results are presented as a percentage of the mRNA, protein, and catalytic values for the livers from the PB-treated males represented as 100%. FIG. 2. Hepatic and splenic mRNA from untreated Fischer 344 rats were amplified using RT-PCR as described in the text to detect CYP2B1/2, CYP2E1, and CYP2B12. Cyclophilin mRNA was amplified (the six columns on the right half of the photograph) from the same RNA samples. The results presented represent one or two of four tissues used in each group. M (male), F (female). Number of base pairs (bp) generated by PCR are identified in parentheses. 1075 SPURIOUS OBSERVATION OF SPLENIC CYP2B1 EXPRESSION at A PE T Jornals on Sptem er 8, 2017 dm d.aspurnals.org D ow nladed from Northern probe for CYP2B1 mRNA is due to the presence of a crossreacting, PB-inducible transcript not currently identifiable in existing databases. Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania MEENA R. SHARMA PARAMESWARAN PERIANDYTHEVAR BERNARD H. SHAPIRO