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Abstract |
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 Top Abstract Materials and MethodsResults and DiscussionReferences |
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7- to 17-fold in the livers of PB-treated 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 16ß-hydroxylase). Protein and
catalytic activities of the isoforms were undetectable in spleen of either
male or female vehicle- and 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.
). 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.
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Materials and Methods |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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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/µg) 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 probed with 32P-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 µg 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 µl 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 µg 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 16ß-hydroxylation, a
specific catalytic activity of CYP2B1 and 2B2
(Waxman and Azaroff, 1992) was
assayed using [4-14C]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.
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Results and Discussion |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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), 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
animals2 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 CYP2E13, 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.4 We can only
conclude that the spurious results using the 18-mer Northern probe for CYP2B1
mRNA is due to the presence of a cross-reacting, PB-inducible transcript not
currently identifiable in existing databases.
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Meena R. Sharma
Parameswaran Periandythevar
Bernard H. Shapiro
Laboratories of Biochemistry, University of Pennsylvania School of Veterinary Medicine, Philadelphia, Pennsylvania
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Footnotes |
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1 Abbreviations used are: P450, cytochrome P450; PB, phenobarbital; RT-PCR,
reverse transcriptase-polymerase chain reaction; AMV, avian myeloblastosis
virus. 
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).
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: prasunsh{at}vet.upenn.edu
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References |
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TopAbstractMaterials and MethodsResults and DiscussionReferences |
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