Induction of mRNA coding for phenobarbital-inducible form of microsomal cytochrome P-450 in rat liver by administration of 1,1-Di(p-chlorophenyl)-2,2-dichloroethylene and phenobarbital

In the preceding paper (Yoshioka, H., et al. (1984) J. Biochem. 95, 937-947), we reported that 1,1-di(p-chlorophenyl)-2,2-dichloro-ethylene (DDE) induced the phenobarbital (PB)-inducible form of microsomal cytochrome P-450 (P-450(PB-1) in rat liver. In order to study more precisely the molecular events responsible for the induction of this particular form of cytochrome P-450 by the two chemical compounds, we determined the amounts of the mRNA coding for P-450(PB-1) in the liver of rats given a single dose of PB or DDE. RNA was extracted from the livers of the treated rats and the determination of the specific mRNA was carried out by using the rabbit reticulocyte lysate translation system and by a dot hybridization method using cloned P-450(PB-1) cDNA (Fujii-Kuriyama, Y., et al. (1982) Proc. Natl. Acad. Sci. U.S. 79, 2793-2797) as the probe. The amounts of P-450(PB-1) mRNA determined by these two methods at various time points of the induction process showed good agreement. These observations further confirmed the induction of an identical form of cytochrome P-450 by DDE and PB. The maximum level of P-450(PB-1) mRNA, which was about 8-fold higher than the control level, was attained at 20-30 h and at 48-72 h after the administration of PB and DDE, respectively. The mRNA level showed a rapid decrease after the peak in the liver of PB-treated rats, but the decrease was much slower with DDE-treated rats. We conclude that DDE had a more persistent inducing effect on the mRNA level than PB, although these two compounds induced an identical form of cytochrome P-450 in the liver microsomes of the animals.     

Detection of phenobarbital-induced cytochrome P-450 in rat hepatic microsomes using an enzyme-linked immunosorbent assay

The major phenobarbital-inducible form of cytochrome P-450 (cytochrome P-450 PB) was purified to homogeneity from rat liver microsomes and rabbit antibodies prepared against the purified enzyme. Using these antibodies, an enzyme-linked immunosorbent assay (ELISA) was developed for the detection of cytochrome P-450 PB in microsomes which was sensitive at the nanogram level. The content of cytochrome P-450 PB was determined in hepatic microsomes from rats treated with various xenobiotics. Phenobarbital and Aroclor 1254 pretreatments resulted in several-fold increases in immunoreactive cytochrome P-450 PB over control levels. ELISA measurements of cytochrome P-450 PB were also carried out over a 48-h time course of phenobarbital induction in liver microsomes. Significant increases over control levels were seen at 16 h and beyond. Measurements of ELISA-detectable cytochrome P-450 PB were made in microsomes following the administration of CCl4 to phenobarbital-pretreated rats. Immunoreactive cytochrome P-450 PB was observed to decrease less rapidly than the spectrally detectable enzyme in the microsomal membranes. Inhibition of heme synthesis was carried out by the administration of 3-amino-1,2,4-triazole (AT) to rats. Concomitant pretreatment with phenobarbital and AT resulted in levels of ELISA-detectable cytochrome P-450 PB which were significantly increased over control levels, while spectrally detectable levels of total holoenzyme remained unchanged. These results support the idea that this cytochrome P-450 may exist, at least partly, in the microsomal membrane in an inactive or apoprotein form.     

Characterization of ethanol-inducible human liver N-nitrosodimethylamine demethylase

Through the use of monospecific antibodies directed against hepatic cytochrome P-450j, an enzyme induced in rats treated with ethanol or isoniazid, we have purified from human liver the related cytochrome P-450 termed HLj. HLj resembles rat P-450j and P-450 LM3a, the homologous cytochrome in rabbit liver, in its NH2-terminal amino acid sequence, in being in highest concentration in liver microsome samples prepared from two patients intoxicated by ethanol and one patient given isoniazid, and in catalyzing the metabolic activation of the procarcinogen N-nitrosodimethylamine. Furthermore, each of nine human liver RNA samples contained a species of mRNA hybridizable to a cloned HLj cDNA. We conclude that HLj is related by structure, function, and some regulatory characteristics to rat P-450j and rabbit P-450 LM3a, cytochromes critical for metabolism of several clinically relevant cytotoxic and carcinogenic agents.     

Cloned cytochrome P-450 cDNA. Nucleotide sequence and homology to multiple phenobarbital-induced mRNA species

Phenobarbital (PB) treatment of rats of various strains leads to the accumulation of liver mRNAs which encode two or three immunochemically related but electrophoretically separable cytochrome P-450 polypeptides. These mRNAs hybridize efficiently to a single cloned cDNA derived from mRNA of PB-treated rats and, therefore, must have extensive sequence homology. The nucleotide sequence of this cloned cDNA was determined and shown to encode the COOH-terminal 211 amino acids of one of the major cytochrome P-450 isozymes induced in rat liver by PB. Together with the recently reported sequence data of Fujii-Kuriyama et al. (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sagawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797) for cloned rat cytochrome P-450 cDNA, our data suggest that differences between two closely related P-450 isozymes are restricted to the COOH-terminal half of the polypeptides, with highly divergent regions flanking a tridecapeptide which has been previously shown to be highly conserved in two dissimilar forms of rabbit liver cytochrome P-450. The significance of other interesting features of the cDNA sequence such as a second long (409 residues) open frame, an unusual poly(A) addition signal, and the absence of long hydrophobic stretches in the encoded polypeptide is discussed.     

The phenobarbital-type induction of rat liver microsomal monooxygenases by perfluorodecalin

Induction of perfluorodecalin (PFD) of the liver microsomal system of metabolism of xenobiotics has been studied and compared with the inductions by phenobarbital (PB) and 3-methylcholanthrene (MC). It has been shown that PFD increases the content of cytochrome P-450, NADPH-cytochrome c reductase activity. Like PB, PFD induces the activities of benzphetamine-N-demethylase, aldrine-epoxidase, 16 beta-androstendion-hydroxylase. Using specific antibodies against cytochromes P-450b and P-450c (which are the main isoenzymes of cytochrome P-450 in the PB- and MC-microsomes respectively), an immunological identity of the cytochrome P-450 isoforms during PFD and PB induction has been found. According to the rocket immunoelectrophoresis the content of cytochrome P-450 in PFD-microsomes, which is immunologically indistinguishable from P-450b, was approximately 70% of the total cytochrome P-450. Two forms of cytochrome P-450 were isolated from the liver microsomes of PFD-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from the PB-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromotographic behavior on DEAE-Sephacel column, molecular weight determined by sodium dodecyl sulphate (SDS) electrophoresis in polyacrylamide gel, immunoreactivity, peptide mapping, catalytic activity. The data presented demonstrate that PFD induced in rat liver microsomes the cytochrome P-450 forms whose immunological properties and substrate specificity correspond to those of the PB-type cytochrome P-450. These findings suggest that PFD and PB, which differ in their chemical structure, induce in the rat liver microsomes identical forms of cytochrome P-450.     

Characterization of complementary DNA clones coding for two forms of 3-methylcholanthrene-inducible rat liver cytochrome P-450

Double-stranded DNA complementary to the partially purified mRNA prepared from 3-methylcholanthrene (MC)-treated rat liver was constructed and cloned in Escherichia coli. Twenty clones were verified to carry a complementary DNA (cDNA) insert coding for MC-inducible cytochrome P-450 by positive hybridization translation assay and immunochemical assay with anti-cytochrome P-450 antibody. The identified cDNA clones were divided into at least two groups on the basis of comparison of restriction maps of the cDNA inserts. A clone pAU157 whose cDNA insert was approximately 2.7 kb in length contained nearly full-length mRNA information for cytochrome P-450MC or P-450c, which is the major form of MC-inducible cytochrome P-450. Other cDNA clones pTZ286-pTZ330 contained the 1.2 kb sequence complementary to cytochrome P-450d mRNA. RNA blot analysis revealed that pAU157 and pTZ286-pTZ330 cDNA clones were derived from 22S and 18S mRNAs, respectively, both of which were induced in rat liver by MC treatment. Sequence analysis revealed that there were closely homologous sequence regions in pAU157 and pTZ286-pTZ330 cDNA inserts and most of the homologous sequences were localized in two limited coding regions of the two cytochrome P-450 species. pAU157 encoded the total amino acid sequence of cytochrome P-450MC or P-450c and pTZ286-pTZ330 coded for the C-terminal 368 amino acid residues of cytochrome P-450d. Two highly homologous regions were found in the amino acid sequences of these cytochrome P-450 species.     

Phenobarbital induction of rat liver cytochromes P-450b and P-450e. Quantitation of specific RNAs by hybridization to synthetic oligodeoxyribonucleotide probes

Cytochromes P-450b and P-450e are extremely homologous and immunochemically indistinguishable proteins that are coordinately induced by phenobarbital in rat liver. To assess the effect of phenobarbital on mRNA levels for each of these hemoproteins we performed solution hybridization and Northern blot experiments with synthetic oligodeoxynucleotide probes of defined sequence. Our data demonstrate that phenobarbital administration to rats resulted in marked increases in levels of hepatic mRNA for both cytochrome P-450b and cytochrome P-450e, with a 4- to 5-fold greater accumulation of P-450b mRNA vis à vis P-450e mRNA. The level of hepatic mRNA increased from less than 3 molecules/cell of each mRNA in untreated rats, to 630 and 130 molecules/cell for P-450b and P-450e, respectively, in phenobarbital-treated rats. Data obtained in Northern blot hybridization experiments demonstrated that the size of the mRNAs for each protein were identical, being approximately 1800 bases in length.     

Tissue-specific expression of rat mRNAs homologous to cytochromes P-450b and P-450e.

The tissue-specific expression of cytochrome P-450b and P-450e mRNAs was examined with synthetic 18-mer oligomer probes in the liver, lung, kidney, and testis of control and inducer pretreated adult rats. RNAs homologous to the P-450e probe were detected in trace amounts in control and 3-methylcholanthrene (MC) induced livers and at high levels in livers from phenobarbital (PB) induced animals. P-450e mRNA levels were below detection limits in the other tissues examined, regardless of pretreatment. In contrast, mRNAs homologous to the P-450b oligomer were detected at low levels in control and inducer pretreated lung and testis, and at high levels in PB induced liver. No P-450b mRNAs were detected in these assays in RNA isolates from the kidney or from control or MC pretreated liver. Solution hybridization data indicated that the rat lung contained 9-12%, and the testis, 6-9%, respectively, of the levels of P-450b mRNA measured in the PB induced liver. Results from oligo(dT)-cellulose and poly(U)-affinity experiments indicated that the hepatic mRNAs for P-450b and P-450e were present predominantly in the bound, polyadenylated fraction, whereas the homologous lung and testes P-450b mRNAs predominated in the flow-thru fractions.     

Cytochrome P-450 isoforms in the liver of rats treated with phenobarbital, 3-methylcholanthrene and Aroclor 1254 studied by monoclonal antibodies

Seven types of monoclonal antibodies to cytochrome P-450 were obtained from the rat liver. Liver microsomal samples from intact rats and those pretreated with phenobarbital, 3-methylcholanthrene, Aroclor 1254, pregnenalone carbonitrile, beta-naphthoflavone and imidazole were stained with these antibodies using immunoblotting technique. The study made it possible to draw the following conclusions. Firstly, two types of these antibodies react with two cytochrome P-450 isoforms, P-450b and P-450 PB/PCN-E. Secondly, two types of antibodies react with three cytochrome P-450 isoforms: P-450a, P-450b and P-450PB/PCN-E. Antibodies of the latter three clones react with two cytochrome P-450 isoforms: P-450c and P-450d. Antibodies of all seven clones can be used for immunomorphological identification of cytochrome P-450 on rat liver paraffin sections.     

The accumulation of distinct mRNAs for the immunochemically related cytochromes P-450c and P-450d in rat liver following 3-methylcholanthrene treatment

Treatment of rats with 3-methylcholanthrene leads not only to a marked accumulation in the liver of translatable mRNA coding for a 56-kilodalton polypeptide representing cytochrome P-450c, the major 3-methylcholanthrene-induced cytochrome P-450 of rat liver, but also to the accumulation of comparable amounts of mRNA encoding a 52-kilodalton polypeptide which is immunoprecipitated with antibodies prepared against rat liver cytochrome P-450c. Further electrophoretic and immunochemical characterization of the latter translation product demonstrates that it corresponds to cytochrome P-450d, the major isosafrole-induced form of rat liver cytochrome P-450. The mRNAs for cytochromes P-450c and P-450d can be completely separated by electrophoresis in denaturing agarose gels and have chain lengths of approximately 4000 and 2000 nucleotides, respectively. These two mRNAs do not show detectable sequence homology to the mRNAs coding for the major phenobarbital-induced forms of cytochrome P-450 (P-450b and P-450e) since in Northern blotting experiments they fail to hybridize under conditions of low to moderate stringency to cloned probes for the latter mRNAs.     

Regulation of cytochrome P-450p by phenobarbital and phenobarbital-like inducers in adult rat hepatocytes in primary monolayer culture and in vivo

Treatment of rats with phenobarbital increases the hepatic concentration of P-450p, a form of cytochrome P-450 believed to be controlled primarily by a mechanism that stereospecifically recognizes glucocorticoids like dexamethasone and anti-glucocorticoids like pregnenolone-16 alpha-carbonitrile [Schuetz, E.G., & Guzelian, P.S. (1984) J. Biol. Chem. 259, 2007]. To test the possibility that phenobarbital induces P-450p indirectly by increasing the availability of endogenous glucocorticoids in the liver, we added phenobarbital and phenobarbital-like inducers to primary monolayer cultures of adult rat hepatocytes incubated in serum-free medium without glucocorticoids and found stimulated de novo synthesis of P-450p measured as increased incorporation of [3H]leucine into immunoprecipitable P-450p protein. With some of the inducers, notably the organochlorine pesticides chlordane and trans-nonachlor, there was a greater accumulation of P-450p measured on quantitative immunoblots than could be accounted for by the increase in P-450p synthesis. "Pulse-chase" experiments confirmed that these compounds significantly lengthen the half-life of P-450p up to 60 h as compared to the values in control (11 h) or dexamethasone-treated (10 h) cultures. Treatment of rats with chlordane, trans-nonachlor, or other cyclodiene organochlorine pesticides confirmed that these agents increase the concentration of P-450p in liver microsomes analyzed on immunoblots of two-dimensional electrophoretic gels. The time courses of induction in trans-nonachlor-treated rats of P-450p protein and of P-450PB proteins induced by phenobarbital were similar as were the amounts of P-450PB mRNA and P-450p mRNA measured by hybridization to cloned cDNA probes. However, analysis of structure-activity relationships among polychlorinated biphenyls revealed that isomers with two ortho chlorinated positions maximally induced P-450PB whereas isomers with three and four ortho chlorines maximally induced P-450p in rats and in hepatocyte culture, respectively. We conclude that P-450p is induced by the phenobarbital class of inducers through direct contact with the hepatocytes involving decreased degradation of the protein and stimulation of its synthesis in a manner similar but not identical with that of P-450PB.     

NADPH-dependent reduction of amaranch in liver microsomes characterized the quantity of low spin forms of cytochrome P-450.

We confirmed that NADPH-dependent anaerobic amaranch reduction in rat liver microsomes is compatible with the interaction of the dye with Fe(III) heme of cytochrome P-450 as the type II substrate. This process is rate-limiting in the whole reaction. High positive correlation (r = 0.949) between the values of Vmax for reaction of NADPH-dependent anaerobic amaranch reduction and the relative content low spin forms of cytochrome P-450 determined by ESR in microsomes from liver of control and induced by PB, BP, IS and 4-MP rats was observed. Relative content of low spin forms of cytochrome P-450 determined by ESR was increased according to BP less than PB less than control less than IS approximately 4-MP; Vmax values increased according to BP less than PB less than control less than IS less than 4-MP. Thus, reaction of NADPH-dependent anaerobic amaranch reduction may be used for determination of low spin forms of cytochrome P-450 at physiological conditions.     

Complementary DNA and amino acid sequence of rat liver microsomal, xenobiotic epoxide hydrolase

The coding nucleotide sequence for rat liver microsomal, xenobiotic epoxide hydrolase was determined from two overlapping cDNA clones, which together contain 1750 nucleotides complementary to epoxide hydrolase mRNA. The single open reading frame of 1365 nucleotides codes for a 455 amino acid polypeptide with a molecular weight of 52,581. The deduced amino acid composition agrees well with those determined by direct amino acid analysis of the rat protein, and the amino acid sequence is 81% identical to that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and that of rabbit epoxide hydrolase. Analysis of codon usage for epoxide hydrolase, and comparison to codon usage for NADPH-cytochrome P-450 oxidoreductase and cytochromes P-450b, P-450d, and P-450PCN, suggest that epoxide hydrolase is more conserved than cytochromes P-450b and P-450PCN; comparison of the extent of sequence conservation for 12 homologous proteins between the rat and rabbit, including cytochrome P-450b, supports this hypothesis, and indicates that much of epoxide hydrolase is constrained to maintain its hydrophobic character, consistent with its intramembranous location. The predicted membrane topology of epoxide hydrolase delineates 6 membrane-spanning segments, less than the 8 or 10 predicted for two cytochrome P-450 isozymes; the lower number of membrane-spanning segments predicted for epoxide hydrolase correlates with its lesser dependence on the membrane for maintenance of its tertiary structure and catalytic activity.     

Cloning and sequence analysis of a rat liver cDNA coding for a phenobarbital-inducible microheterogenous cytochrome P-450 variant: regulation of its messenger level by xenobiotics

A rat liver cDNA library was prepared from total polyribosomal poly(A)+ RNA extracted from phenobarbital-treated animals. A cDNA clone coding for a phenobarbital-inducible cytochrome P-450 (PB P-450) was identified by differential colony hybridization to cDNAs synthesized from liver poly(A)+RNAs isolated from phenobarbital-treated rats for positive selection and cDNAs from either untreated rats or beta-naphthoflavone-treated rats as negative controls, followed by hybrid-selected translation and analysis of the translation products by immunoprecipitation. As the cloning and screening strategies involve no prior enrichment for specific mRNAs, they also permit the identification of sequences coding for phenobarbital-induced proteins other than cytochromes P-450. This relatively straightforward approach is generally applicable to the molecular cloning of sequences coding for other inducible cytochromes P-450. Nucleic acid sequencing data indicated that the cloned PB P-450 cDNA codes for a cytochrome P-450 variant [designated P-450e(U.C.)] that is very similar, but not identical, to P-450e. Sequence analysis of the section of cDNA specifying the 3'-non-coding region of the mRNA revealed that it lacked the usual poly(A) addition site signal sequence but contained three inverted repeat structures. Solution hybridization analysis demonstrated that PB P-450 mRNA is increased 20-fold by phenobarbital treatment and decreased 3-fold by beta-naphthoflavone treatment.     

Induction of cytochrome P-450 in cultured rat hepatocytes. The heterogeneous localization of specific isoenzymes using immunocytochemistry.

Primary cultures of rat hepatocytes were exposed to phenobarbitone, clofibric acid, beta-naphthoflavone, isosafrole or dexamethasone for 3 days, and the induction of several cytochrome P-450 isoenzymes was demonstrated by increased catalytic activity, by Western blotting and by immunocytochemistry. The profiles of isoenzymes induced in vitro were compared with those induced in liver microsomes of rats dosed with the same agents. Clofibric acid, an agent which has not been thoroughly investigated previously, was shown to induce both in vivo and in vitro several P-450 isoenzymes normally inducible by phenobarbitone (PB1a, PB3a and PB3b) or steroids (PB2c). Immunocytochemical studies demonstrated that the inducible isoenzymes of cytochrome P-450 are not distributed evenly throughout the hepatocyte population, and increasing concentrations of phenobarbitone or beta-naphthoflavone in the medium results in an increasing proportion of 'induced' cells. However, whereas maximal concentrations of beta-naphthoflavone resulted in virtually all cells containing induced levels of MC1b, a maximal concentration of phenobarbitone resulted in only 30% of the cells containing induced levels of PB3a/PB3b. These results are discussed in relation to the heterogeneous distribution and induction of cytochrome P-450 in the intact liver.     

Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P-450MC

We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.