T H E .JOURNAL OP RIOLOCICAI. CHEMISTRY

Printed in I1 S.A. Vol. 2.58, No. 16, Issue of August 26, pp. 101R2-10186, 19R3

Cloning of DNA Complementary to Cytochrome P-450 Induced by Pregnenolone- 16a-carbonitrile CHARACTERIZATION OF ITS mRNA, GENE, AND INDUCTION RESPONSE*

(Received for publicstion, February 24, 1983)

James P. Hardwick$, Frank J. Gonzalezg, and Charles B. Kasper From the McArdle Laboratory for Cancer Research, University of Wisconsin, Madison, Wisconsin 53706

The major form of cytochrome P-450 (P-45opCN) was isolated from rats administered pregnenolone- 16a- carbonitrile (PCN). Messenger RNA coding for P- 450PCN was enriched by polysome immunoadsorption and utilized to construct a library of cDNA clones in pBR322. P-45opCN clones were isolated from this library by differential colony hybridization using [32P]cDNA probes transcribed from PCN-induced and PCN-induced P-45opCN immunoenriched poly(A) RNA. The P-45opCN clone with the largest cDNA insert (pP45opCN-10) was verified to contain sequences com- plementary to P-45opCN mRNA by hybrid selection- translation. pP450pCN-10 was composed of approxi- mately 1900 base pairs and had a restriction map that overlapped at least 3 other cDNA clones selected by differential colony hybridization. Denaturing-agarose gel electrophoresis and nitrocellulose blot-hybridiza- tion using nick-translated 32P-labeled pP450pCN- 10 in- dicated that pP45opcN mRNA is 2500 f 150 nucleo- tides in length; pP45opCN- 10, therefore, represents ap- proximately 76% of its corresponding mRNA sequence. Southern blot analysis of rat DNA using pP450PCN revealed that approximately 50 to 60 kilobases of DNA reacted with the PCN probe, suggesting the P-45opCN gene is either a very large gene or other genomic segments exist that react with the probe, such as pseu- dogenes or related P-450 genes that share homology.

The mechanism of P-45opCN induction was examined by isolating poly(A) RNA at various times after steroid administration and quantitating for P-45OpcN mRNA using pP450pCN-10 as a hybridization probe. PCN administration produced a rapid elevation of P-45opCN mRNA which reached maximal levels (7-fold above control) 12 h after administration. In contrast, cyto- chrome P-450b mRNA, which is readily induced by phenobarbital, was only slightly elevated (approxi- mately 2-fold) after PCN administration.

The cytochrome P-450s are a group of enzymes embedded in the endoplasmic reticulum and nuclear envelope that func-

*This work was supported by Grants CA-23076, CA-17300, and CA-07175 from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduer- tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Present address, National Institute of Environmental Health Sciences, P.O. Box 12233, Research Triangle Park, NC 27709.

Present address, Laboratory of Developmental Pharmacology, National Institute of Child Health and Human Development, Build- ing 10, Room 8C-416, National Institutes of Health, Bethesda, MD 20205.

tion in the metabolism of a variety of endogenous compounds, drugs, and chemical carcinogens. In the latter case, the P-450 catalyzed mixed function oxidase system can either activate or inactivate certain carcinogenic compounds (1-6). The cel- lular cytochrome P-450 population at the time of carcinogen exposure, therefore, determines whether the chemical will be metabolized to its proximate carcinogenic form or be inacti- vated and subsequently eliminated (1). Since many exogenous compounds can induce the mixed function oxidase system, it has been suggested that the induction process may be an activation mechanism whereby a chemical can stimulate its own metabolism (7).

Agents that induce drug metabolizing enzymes fall into several classes, some of which are represented by (a ) pheno- barbital, (b ) 3-methylcholanthrene, (c) isosafrole, (d ) 2-ace- tylaminofluorene and N-hydroxy-2-acetylaminofluorene, ( e ) PCN.’ Efforts have recently been expanding in attempting to elucidate the molecular details of the 3-methylcholanthrene (8-13), phenobarbital (14-21), and 2-acetylaminofluorene (22) induction responses. The largest body of information deals with the 3-methylcholanthrene-like class of inducers. This induction process is controlled by the Ah locus in the mouse (reviewed in Ref. 8) and is mediated by a receptor which enters the nucleus as a receptor-ligand complex (9, 10) and causes an accumulation of cytochrome P1-450 mRNA (11). Furthermore, cytoplasmic mRNA accumulation may result from an increase in the rate of transcription of the cytochrome Pl-450 gene (12). Likewise, 3-methylcholanthrene administration to rats also results in increased cytochrome P- 448 mRNA (13).

The class of agents represented by phenobarbital elevates cellular levels of epoxide hydratase (14-16), NADPH-cyto- chrome P-450 oxidoreductase (15-17), and cytochrome P- 450b (16, 18-21) mRNAs. In vitro transcription using isolated nuclei has demonstrated that mRNA accumulation results from the augmentation of transcriptional rates for the respec- tive genes (40). Finally, 2-acetylaminofluorene and N-hy- droxy-2-acetylaminofluorene specifically elevate epoxide hy- dratase mRNA (22), and this induction process is accom- panied by an elevation of epoxide hydratase pre-mRNA, sug- gesting a transcriptional activation (22). An intriguing aspect of these studies is the finding that 2-acetylaminofluorene, but not its N-hydroxy derivative, transiently elevates levels of NADPH-cytochrome P-450 oxidoreductase and cytochrome P-450b nuclear pre-mRNAs and cytoplasmic mRNAs (22). These enzymes, however, are not elevated after either acute or chronic exposure to 2-acetylaminofluorene (22;.

Much less is known about the inducing agent pregnenolone- 16a-carbonitrile. Administration of this compound produces

-~

’ The abbreviation used is: PCN, pregnenolone-16n-carbonitrile.

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Cloning of Pregnenolone-16a-carbonitrile-induced Cytochrome P-450 cDNA 10183

an increased microsomal ethylmorphine N-demethylase activ- ity (23), and treated animals are found to have an increased resistance to carbon tetrachloride (24) and dimethylnitrosa- mine toxicity (25, 26). Recently, a PCN inducible cytochrome P-450 (P-45opCN) was isolated from rats, the reconstituted form of which exhibits an enhanced ethylmorphine N-de- methylase activity (27). Of considerable interest is the finding that dexamethasone, a physiologically active glucocorticoid, also elevates levels of P-45opCN (28), suggesting that this cytochrome may have a role in the metabolism of endogenous compounds. Another aspect of the PCN induction response that is noteworthy is the ability of the steroid to elevate P- 450PCN in primary hepatocyte cultures (29). This increase was accompanied by an elevation of translatable mRNA coding for the cytochrome, suggesting that PCN may have a mecha- nism of action similar to the glucocorticoid hormones (29). Dose response curves, however, indicate that substantially higher amounts of the steroid dexamethasone are required to achieve an induction response for P - 4 5 0 ~ ~ ~ equivalent to other glucocorticoid induced enzymes (29).

In order to determine the process by which the steroids PCN and dexamethasone increase levels of P-450pCNj cDNA clones containing sequences complementary to P - 4 5 0 ~ ~ ~ mRNA were constructed and characterized. One of these clones was then utilized to determine the approximate size of P-450pvN mRNA and to analyze its gene by Southern blot analysis. In addition, quantitative hybridization studies were performed to establish the effects of PCN on P-45opCN and P-450b mRNAs.

MATERIALS AND METHODS

Purification of PCN-induced Cytochrome P-450-Male Sprague- Dawley rats (150-170 g body weight) received an intraperitoneal 1- ml injection of 100 mg/ml of PCN (Searle and Company, Chicago, IL) dissolved in a solution of 0.9% NaCl and 0.005% Tween 80, 24 h prior to killing. Cytochrome p - 4 5 0 ~ ~ ~ was purified from liver micro- somes as described by Elshourbagy and Guzelian (27). Rabbit IgG produced with purified P-45opCN was isolated as previously described (30). Double immunodiffusion analysis revealed a single precipitin line when anti-P-450pCN IgG was reacted with different concentra- tions of solubilized liver microsomes from PCN-induced animals as well as purified P-450pcN.2 No cross-reactivity was observed with cytochromes P-450b or P-450~. as previously demonstrated (27).

Zmmunoenrichment of P - 4 5 0 ~ ~ ~ Messenger RNA and Construction of cDNA Clones-Tightly membrane-bound polysomes were isolated from rats administered PCN 16 h prior to killing essentially as described (la), except the initial homogenization buffer contained 20 mM MgCl,, 10 mM vanadyl-ribonucleoside complex, and 100 pg/ml of heparin. Polysome immunoadsorption was performed as outlined previously (30). Briefly, a r1 t i -P -450~~~ IgG was incubated with PCN- induced polysomes (0.5 pg of IgG/unit of A260) for 3 h at 0 "C. Staphylococcus aureus ghost (Calbiochem-Behring, LaJolla, Ca) were added and the incubation was continued for 10 min. Polysome-bound ghosts were sedimented and washed before the RNA was released as described (30). Subsequent to an extraction with phenol and chloro- form ( l : l ) , poly(A) was purified by two rounds of oligo(dT)-cellulose chromatography (15).

Poly(A) RNA enriched in P-45OPcN mRNA sequences was used to construct a cDNA clone bank in pBR322. This was accomplished using reverse transcriptase, S1 nuclease, and oligo(dG)-oligo(dC) tailing as previously described in detail (30). Double-stranded cDNA (200 ng) from 10 pg of poly(A) RNA yielded approximately 1400 ampicillin-sensitive and tetracycline-resistant colonies. These were screened by differential colony hybridization with [32P]cDNA synthe- sized from PCN-induced poly(A) RNA and P-45OPcN immunoen- riched poly(A) RNA (30). Eighty-five colonies were selected that specifically annealed with the immunoenriched probe and their plas- mid DNA purified (31). DNA was analyzed by PstI digestion and agarose gel electrophoresis. Hybrid selection-translation was carried out by established procedures (32). RNA electrophoresis in formal-

, J. P. Hardwick, F. J. Gonzalez, and C. B. Kasper, unpublished results.

dehyde gels and blot-hybridization was performed as described (16). Quantitation of specific mRNAs was performed by hybridizing in uitro 32P-labeled poly(A) RNA to DNA bound to nitrocellulose filters as outlined (16). Rat DNA was isolated from purified nuclei (15) according to the method of Blin and Stafford (33). Southern blots were carried out a s described (34) with the inclusion of a depurination step to facilitate transfer of large DNA fragments (35). Nick-trans- lation of plasmid DNAs was performed as described (36).

RESULTS

Preparation and Characterization of Cytochrome P-45OPcN cDNA Clones-Membrane-bound polysomes were isolated from rats administered PCN 17 h prior to killing. These polysomes were then used for enriching cytochrome p - 4 5 0 ~ ~ ~ mRNA by polysome immunoadsorption. Immunoenriched poly(A) RNA was isolated and double-stranded cDNA was synthesized with reverse transcriptase and inserted into the PstI site (8-lactamase gene) of pBR322 by the dC-dG tailing method. Transformation with 200 Fg of double-stranded cDNA was carried out with Escherichia coli HblOl and ap- proximately 1400 ampicillin-sensitive and tetracycline-resist- ant colonies were obtained. These were then screened by differential colony hybridization using [32P]cDNA synthe- sized from PCN-induced and PCN-induced immunoenriched P-450PCN mRNA poly(A) RNA. Ninety colonies that reacted with the immunoenriched probe were selected and their DNA was isolated, digested with PstI, and analyzed by acrylamide gel electrophoresis. The clone pP450pCN-10 was found to have an insert of approximately 1900 base pairs and an internal cleavage site for PstI (Fig. 1). This clone was mapped with several restriction enzymes and compared with other plasmids containing smaller inserts. As expected, the restriction map of pP450pcN-10 overlapped with pP45opCN-3, pP45opCN-7, and pP45opCN-9 (Fig. 1). In addition, all clones contained common termini, which, by DNA sequence analysis, were found to contain the poly(A) tract of the mRNA and the concensus poly(A) addition site.'

In order to definitively demonstrate that pP450pCN-10 con- tained sequences complementary to cytochrome P-450PCN mRNA, hybrid selection-translation was performed. pP45OpcN-10 and pBR322 DNAs were bound to separate nitrocellulose filters and each incubated with 17-h PCN- induced poly(A) RNA under hybridization conditions. The filters were extensively washed and bound poly(A) RNA was eluted and subjected to translation in the nuclease-treated reticulocyte lysate system. An equivalent number of dpms of "%labeled polypeptides synthesized from RNA, adsorbed to either pP450pcN-10 or pBR322 filters, were then examined for immunoprecipitable cytochrome P-45op,". An autoradi- ograph of sodium dodecyl sulfate-polyacrylamide gel contain- ing the immunoprecipitable material revealed that the cyto- chrome was synthesized to a much greater extent from p P 4 5 0 p < ~ 1 0 adsorbed mRNA (Fig. 2, lane 2) as compared to pBR322 filter-bound mRNA (Fig. 2, lane I ) . The 51,000-

I ,P450PcN-3

I 1 ,P450~cri 7

I I I ,P450PCN.9

I I .P450PCN 10 Earn HI P.: I Eca R I

2W 400 600 800 1 w O 12W 1400 1600 1SWbp I I 4 I I I I I I I 1

FIG. 1. Restriction endonuclease maps of selected P - 4 5 0 ~ ~ ~ cDNA clones. Plasmid DNA was digested with restriction enzymes and electrophoresed on polyacrylamide gels in parallel with AluI, and HinfI digests of pBR322. Restriction maps were constructed by ana- lyzing single and double enzyme digestions. The 3' sequence corre- sponding to the poly(A) tract of the p - 4 5 0 ~ ~ ~ mRNA is at the right of the figure.

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10184 Cloning of Pregnenolone- I6a-carbonitrile-induced Cytochrome P-450 cDNA

dalton polypeptide furthermore co-migrated with in uiuo syn- thesized cytochrome P-450prN. These results demonstrate that pP450p(.N-10 contains sequences complementary to cy- tochrome P-4501.~~ mRNA.

Determination of the Size of Cytochrome P"f5@p( 'N mRNA- Poly(A) RNA was isolated from control and 17-h PCN- induced rats, denatured, and electrophoresed on a formalde- hyde containing agarose gel. After blotting to nitrocellulose paper, hybridization with nick-translated [:"P]pP4501>CN-10, and autoradiography, a single RNA species was detected that migrated a t 2500 f 150 nucleotides (Fig. 3, lane I ). This species was significantly increased in a poly(A) RNA popu- lation from 17-h PCN-induced animals (Fig. 3, lane 2). Cy- tochrome P-450p(.N mRNA, therefore, is slightly larger than the 2150-nucleotide mRNA coding for cytochrome P-450b, the major phenobarbital induced form of cytochrome P-450 (16). The size differences between the P"i501>(.N and P-450b

1 2

Frc. 2. Hybrid translation-selection of P - 4 5 0 ~ ~ mRNA us- ing pP45OpcN-10. Poly(A) RNA isolated from 17-h PCN-induced animals was hybridized with nitrocellulose filters containing either pBR322 or p P 4 5 0 ~ ~ ~ - 1 0 DNA. RNA adsorbed to pBR322 (lane I ) and p P 4 5 0 ~ ~ - 1 0 (lane 2 ) was translated and analyzed for p - 4 5 0 ~ ~ ~ by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In each case the cytochrome was immunoprecip- itated from 40,000 dpm of translation products. The arrow designates the mobility of cytochrome P - 4 5 0 ~ ~ isolated from rat microsomes.

1 2

4.3- 3.6- 2.8-

1.6-

0.7-

FIG. 3. Determination of the size of mRNA codiing for P- 4 5 0 ~ ~ . Poly(A) RNA (5 pg) isolated from control (lane I ) and 17-h PCN-induced (lane 2) polysomes was denatured and electrophoresed on a 2.2 M formaldehyde, 1% agarose gel, blotted to nitrocellulose, and hybridized with nick-translated ["zP]pP450p~N-10. Restriction fragments of pBR322 (16) were denatured and electrophoresed in parallel. The length of these fragments in kilobase pairs and their positions of migration is indicated at the left of the figure. Autora- diography was carried out with the aid of a Dupont Quanta I1 intensifying screen.

1 2 3 4 5 6

23.3- 9.5- 1

6.4-

4.2- I

2.2- 1.8-

b

* ..

FIG. 4. Blot-hybridization analysis of restriction enzyme digested rat liver nuclear DNA. High molecular weight nuclear DNA was digested in duplicate with EcoRI (lanes I and 4 ) , HindIII (lanes 2 and 5 ) . and PuuII (lanes 3 and 6 ) . electrophoresed on 0.8% agarose gels, depurinated, and blotted to nitrocellulose. After hybrid- ization with pP450b-5 (lanes 1-3) and pP450pCN-10 (fanes 4-6), fragments were visualized by autoradiography using a Dupont Quanta I1 intensifying screen. HindIII digested X phage was electrophoresed in parallel and the mobility and length (in kilobase pairs) of the resultant fragments is indicated at the left of the figure.

mRNAs and the presence of only a single species of mRNA in phenobarbital induced poly(A) RNA with either cDNA probe (Fig. 3 and Ref. 16) illustrated that each mRNA reacts only with its complementary cDNA probe. These mRNAs, therefore, do not share extensive sequence homology.

Analysis of Genomic DNA Complementary to Cytochrome P-45@,*.,~ mRNA--In order to determine if cytochrome P- 450r.rN mRNA is coded by a single copy gene, Southern blots were carried on rat liver nuclear DNA. DNA was digested with EcoRI, HindIII, and PuuII, subjected to agarose gel electrophoresis, partially depurinated, and blotted to nitro- cellulose. Blot hybridization was then performed with either nick-translated [:''P]pP450pcN-10 or pP450b-5 (16). The latter clone contains sequences complementary to cytochrome P- 450b mRNA. Interestingly, pP45opCN-10 and pP450b-5 an- nealed with multiple but distinctly different DNA fragments (Fig. 4). Summation of fragments that reacted with p P 4 5 0 p ~ ~ - 10 revealed a total of approximately 50 to 60 kilobases of DNA. Comparison of this result with that obtained by South- ern blot analysis of the P-450b gene (Fig. 4, lanes 1-3) revealed that many genomic fragments were of different size than the P"kjOp(.N gene fragments (Fig. 4, kznes 4-6). These results suggest that the genes for the two cytochromes are distinct, as expected, since the P-450pcx-10 cDNA clone does not react with P-450b mRNA.

Quantitation of Cytochrome P-45@1>(7.! and Cytochrome P - 4506 mRNAs after PCN Administration-Membrane-bound polysomes were isolated from rats at various times after a single dose of either PCN or phenobarbital. Poly(A) RNA was purified by oligo(dT)-cellulose chromatography, labeled with :IZP, and hybridized with nitrocellulose filters containing either pP450r>(.N or pP450b. The per cent of total RNA rep- resented by each mRNA was then quantitated. In control animals, P-450r>(.N mRNA is present a t approximately 200 copies/cell (calculated using control value of 0.1% dpm hy- bridized, see Ref. 16), which falls in the range of the mida- bundance mRNA class similar to cytochrome P-450b and oxidoreductase mRNAs (approximately 50 and 66 copies/cell,

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Cloning of Pregnenolone-16a-carbonitrile-induced Cytochrome P-450 cDNA 10185

respectively; Ref. 16). After administration of PCN, P-450PcN mRNA is rapidly elevated to 6-fold above control at 12 h and remained elevated up to 24 h (Fig. 5). In addition, cytochrome P-450b mRNA was also slightly elevated; however, it reached a level of only 2-fold above control values at 17 to 24 h after PCN administration (Fig. 5). These results demonstrate that a major effect of PCN is to cause an accumulation of p - 4 5 0 ~ ~ ~ mRNA and that this increase results in enhanced synthesis of cytochrome P-45OPCS.

Administration of phenobarbital also results in an elevation

L 1 I I I I I 0 4 8 12 16 20 24

HOURS AFTER PCN ADMINISTRATION

FIG. 5. Quantitation of mRNAs coding for P-450pcN and P- 450b, after administration of PCN. Tightly membrane-bound polysomes were isoalted from rats at various times after a single dose of PCN. Control animals received 0.9% saline and 0.005% Tween 80. Poly(A) RNA was purified, labelled with 32P, and hybridized to separate nitrocehlose filters containing either pP45opCN-10, pP450b- 5, or pBR322 DNA. The percentage of total dpms represented by each mRNA was calculated and the level of induction represented as a percentage of saline injected controls. Control values were 0.107 f 0.046 for P-450PcN mRNA (F". ) , and 0.027 t 0.008 for P-450b mRNA (o"-o). Eachpoint represents the mean of two experiments using pooled livers of three rats/experiment.

3200 I I I I I I I

2800 -

2400 - 2000 - 1 6 0 0 -

1200 - P =: 100

I I I I I I I

0 4 8 12 16 20 24

HOURS AFTER PHENOBARBITAL ADMINISTRATION

FIG. 6. Quantitation of mRNAs coding for P-45opCN and P- 450b after administration of phenobarbital. Tightly membrane- bound polysomes were isolated from rats at various times after administration of phenobarbital, and P-45OpCN (U) and P-450b (U) mRNAs were quantitated by filter hybridization. See Fig. 5 legend for details.

of P-450pCN mRNA (Fig. 6). However, in contrast to its rapid increase observed 3 to 6 h after PCN, P-45opCN mRNA levels were not detectably elevated until 8 to 12 h after phenobar- bital administration. The maximum level of 4-fold above control for P-45opCN mRNA is also markedly less than the 30-fold increase in P-450b mRNA seen 24 h after administra- tion of the drug (Fig. 6).

DISCUSSION

Polysome immunoadsorption was utilized to enrich mRNA coding for cytochrome p - 4 5 0 ~ ~ ~ . This mRNA population was used to construct a cDNA clone bank from which pP45opCN- 10 was selected by differential colony hybridization and hybrid selection-translation. These procedures have been used suc- cesssfully to isolate cDNA clones complementary to epoxide hydratase (30), cytochrome P-450b (16), NADPH-cytochrome P-450 oxidoreductase (16), and ornithine aminotransferase (37) mRNAs. Since immunoadsorption can enrich an mRNA by 20- to 50-fold, a higher fraction of specific cDNA clones are obtained from a midabundance mRNA population; hence, the possibility of obtaining a near full length cDNA clone is increased.

The pP450pcs-10 cDNA clone has an insert of approxi- mately 1900 base pairs which corresponded to 76% of the P- 450PCN mRNA. Elshourbagy and Guzelian (27) previously found that cytochrome P-45OpCS was distinctly different from phenobarbital inducible cytochrome P-450b, based on differ- ent catalytic activities in a reconstituted system and lack of immunological cross-reactivity. Our studies demonstrating that the mRNAs coding for the two cytochromes have differ- ent molecular weights and that the respective cDNAs do not cross-hybridize with the mRNAs further establish the unique- ness of these cytochrome P-450s. Cytochrome P-450b mRNA is approximately 2150 k 100 nucleotides in length (16), whereas cytochrome P-45OpCS mRNA contains 2500 k 150 nucleotides. Restriction mapping of pP450pCs-10 also re- vealed that it is distinctly different from the map of cDNA complementary to P-450b mRNA (16, 38).

Southern blot analysis of rat nuclear DNA using pP45opCN- 10 revealed that 50 to 60 kilobases of DNA contained se- quences corresponding to P-450pCN mRNA. These results indicate that either (i) the P-45OpCS gene is very large and contains up to 25-fold more intron sequences than exon, (ii) there is present in the rat genome one or several pseudogenes of the P-45opCN gene, or (iii) the P-450pcN gene contains sequences present in other P-450 genes or closely related genes. These possibilities cannot be clarified until the seg- ments of DNA that react with pP450pCN-10 are isolated by genomic cloning and characterized. However, it is noteworthy that clone 46, a cDNA clone containing sequences comple- mentary to cytochrome PI-450 in mouse (39) reacts with a single EcoRI restriction fragment of mouse DNA (12). This result would appear to eliminate the possibility that all cyto- chrome P-450 genes contain DNA sequence homology. Fur- ther evidence for the lack of homology between cytochrome P-450 genes is the finding that single and distinct (electro- phoretically separated) mRNAs react with cDNA clones of P- 4 5 0 ~ ~ ~ and P-450b (16) (Fig. 3). There may exist, however, classes or subsets of P-450 genes that are structurally related. Characterization and chromosomal localization of the P- 4 5 0 ~ ~ ~ gene and related sequences should provide further insight into the organization of P-450 genes.

An intriguing aspect of these studies is the finding that PCN administration results in an elevation of P-45OPCN mRNA approximately 6-fold above control. This increase was detectable as early as 3 h after administration. Cytochrome P-450b mRNA was also elevated 2-fold at later times after

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10186 Cloning of Pregnenolone-16a-carbonitrile-induced Cytochrome P-450 cDNA PCN treatment; however, this induction does not seem ade- quate to account for the 14-fold increase in cytochrome P- 450b detected immunologically after PCN administration (28). Other factors may serve to elevate this enzyme, such as an increase in translational efficiency or stabilization of the protein. No detectable increase was noted for epoxide hydra- tase, NADPH-cytochrome P-450 oxidoreductase, or serum albumin mRNAs after PCN administration,' emphasizing the specificity of the induction response. Phenobarbital, however, readily increases epoxide hydratase and oxidoreductase mRNAs (14-17) in addition to cytochrome P-450b mRNA (Fig. 6; Refs. 16, 18-21). Our direct quantitative measure- ments of P-450rcN mRNA induction confirm and extend the studies of Elshourbagy et al. (29). Using in vitro translation and immunochemical quantitation, they demonstrated that PCN added to hepatocyte cultures elevated P-45opCN and its corresponding translatable mRNA (29). Interestingly, the lev- els of P-45OPcN were also elevated by dexamethasone admin- istration; however, the dose required for achieving a maximal increase in the cytochrome was 10-fold higher than that required for maximal induction of tyrosine aminotransferase (28). In addition, the latter enzyme actually decreased after PCN administration (28). These results led to the proposal that PCN induction of P-450rcN is dissociable from the clas- sical glucocorticoid receptor mediated induction response (28). Interestingly, phenobarbital administration also results in an increase in P-450PCN mRNA that reached 4-fold above base1 levels at 24 h (Fig. 6). In this case, the increase in P- 450r.(.N mRNA level is markedly slower than the rapid increase (&fold at 6 h) detected after PCN administration (Fig. 5). These data suggest that the mechanisms by which phenobar- bital and PCN induce P-45opCN mRNA may be distinctly different. For instance, phenobarbital may cause a stabiliza- tion of P-45OPcN mRNA, whereas PCN may specifically aug- ment transcription of the p - 4 5 0 ~ ~ ~ gene. These possibilities await further experimentation.

Acknowledgments-We thank Patricia McQuiddy for expert and valuable technical assistance. We are also grateful to Daniel Simmon of our laboratory for allowing us to present his comparative Southern blot analysis of the cytochrome P-450b and p - 4 5 0 ~ ~ ~ genes. We also thank Tony B. Martinez of Searle and Company, Chicago, IL for the gift of pregnenolone-16oc-carbonitrile. Finally, the efficient secretarial assistance of Ingrid E. <Jordan is greatly appreciated.

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J P Hardwick, F J Gonzalez and C B Kasperinduction response.

pregnenolone-16 alpha-carbonitrile. Characterization of its mRNA, gene, and Cloning of DNA complementary to cytochrome P-450 induced by

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