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.     

Gene structure and nucleotide sequence for rat cytochrome P-450c

Two clones from rat genomic libraries that contain the entire gene for rat cytochrome P-450c have been isolated. lambda MC4, the first clone isolated from an EcoR1 library, contained a 14-kb insert. A single 5.5-kb EcoR1 fragment from lambda MC4, the EcoR1 A fragment, hybridized to a partial cDNA clone for the 3' end of the cytochrome P-450c mRNA. This fragment was sequenced using the dideoxynucleotide chain termination methodology with recombinant M13 bacteriophage templates. Comparison of this sequence with the complete cDNA sequence of cytochrome P-450MC [Yabusaki et al. (1984) Nucleic. Acids Res. 12, 2929-2938] revealed that the EcoR1 A fragment contained the entire cytochrome P-450c gene with the exception of a 90-bp leader sequence. The gene sequence is in perfect agreement with the cDNA sequence except for two bases in exon 2. A second genomic clone, lambda MC10, which was isolated from a HaeIII library, contains the missing leading sequence as well as 5' regulatory sequences. The entire gene is about 6.1 kb in length with seven exons separated by six introns, all of the intron/exon junctions being defined by GT/AG. Amino- and carboxy-terminal information are contained in exons 2 and 7, respectively. These exons contain the highly conserved DNA sequences that have been observed in other cytochrome P-450 species. Potential regulatory sequences have been located both 5' to the gene as well as within intron I. A comparison of the coding information for cytochrome P-450c with the sequence of murine cytochrome P3-450 and rat cytochrome P-450d revealed a 70% homology in both the DNA and amino acid sequence, suggesting a common ancestral gene. Genomic blot analyses of rat DNA indicated that the 3-methylcholanthrene-inducible family of cytochrome P-450 isozymes is more limited in number compared to the phenobarbital-inducible isozymes. Cross-hybridization studies with human DNA suggest a high degree of conservation between rat cytochrome P-450c and its human homolog although gross structural differences do exist between the two genes.     

Molecular induction by phenobarbital of a rat hepatic form of cytochrome P-450: expression of a 4-kilobase messenger RNA

A differential screening procedure was employed to isolate a cDNA clone corresponding to a major phenobarbital (PB)-inducible form of rat hepatic cytochrome P-450. The G-C homopolymer-tailing technique was utilized to construct a cDNA library in the PstI site of plasmid pBR322. The library represented PB-induced poly(A+)RNA sequences from hepatic polysomes of 150-g male Sprague-Dawley rats. Hybrid-selection experiments against total PB-inducible RNA were performed with plasmid DNA derived from clones enriched in PB-inducible information. The mRNA molecules that specifically hybridized were subjected to in vitro translation, were immunoprecipitated with antibody raised in rabbits against purified cytochrome P-450b (P. E. Thomas, D. Korzeniowski, D. Ryan, and W. Levin (1979) Arch. Biochem. Biophys. 192, 524-532), and were electrophoresed under sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoretic conditions. One cDNA clone, designated PB-8, contained a 600-bp insert partially coding for a PB-inducible cytochrome P-450 species that comigrated on SDS-gel electrophoresis with highly purified P-450b. A single injection of PB, 15-18 h before sacrifice, increased the level of polysomal poly(A+)RNA complementary to the isolated cDNA clone by approximately 16-fold. Northern blot hybridizations of polysome-derived poly (A+)RNA, electrophoresed in denaturing agarose gels, demonstrated that the size of the mRNA corresponding to the isolated clone was 4 kb. Isolated heteronuclear RNA species demonstrated a time-dependent increase in the synthesis of a similar 4-kb RNA molecule. By genomic blot hybridization to EcoRI-restricted DNA, at least three complementary DNA fragments migrating at 5.1, 3.2, and 2.9 kb were observed with 32P-labeled PB-8 as a probe. These data, together with restriction endonuclease mapping and partial cDNA sequence information of the PB-8 cDNA, suggest that the PB-8 clone represents a previously unreported cDNA clone for a form of cytochrome P-450 inducible by PB.     

Isolation and characterization of genomic clones for two chicken phenobarbital-inducible cytochrome P-450 genes

A cDNA clone specific for a chicken phenobarbital-inducible cytochrome P-450 was used to screen a chicken genomic library. Twenty-nine clones were isolated, restriction mapped, and divided into two non-overlapping groups. The cDNA clone hybridized to 12 kilobases of DNA from both groups. Both groups contained restriction fragments which hybridized to both 5' and 3' fragments of the cDNA clone, and it was concluded that the two groups were derived from two separate genes. Southern transfer analysis of individual chicken DNAs and quantitative hybridization analysis indicated that these two genes are independent and are present as single copies/haploid genome. Comparison of restriction digests of the cloned DNAs and total genomic DNA discounted the possibility that other closely related P-450 genes are present in the chicken genome.     

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

The major form of cytochrome P-450 (P-450PCN) was isolated from rats administered pregnenolone-16 alpha-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-450PCN clones were isolated from this library by differential colony hybridization using [32P]cDNA probes transcribed from PCN-induced and PCN-induced P-450PCN immunoenriched poly(A) RNA. The P-450PCN clone with the largest cDNA insert (pP450PCN-10) was verified to contain sequences complementary to P-450PCN mRNA by hybrid selection-translation. pP450PCN-10 was composed of approximately 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-hybridization using nick-translated 32P-labeled pP450PCN-10 indicated that pP450PCN mRNA is 2500 +/- 150 nucleotides in length; pP450PCN-10, therefore, represents approximately 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-450PCN gene is either a very large gene or other genomic segments exist that react with the probe, such as pseudogenes or related P-450 genes that share homology. The mechanism of P-450PCN induction was examined by isolating poly(A) RNA at various times after steroid administration and quantitating for P-450PCN mRNA using pP450PCN-10 as a hybridization probe. PCN administration produced a rapid elevation of P-450PCN mRNA which reached maximal levels (7-fold above control) 12 h after administration. In contrast, cytochrome P-450b mRNA, which is readily induced by phenobarbital, was only slightly elevated (approximately 2-fold) after PCN administration.     

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.     

cDNA cloning and functional expression in yeast Saccharomyces cerevisiae of beta-naphthoflavone-induced rabbit liver P-450 LM4 and LM6

A cDNA library was constructed from liver mRNA of a beta-naphthoflavone-induced rabbit. Two clones pLM4-1 and pLM6-1 containing 2.2-kbp inserts that hybridized at low stringincy with a mouse P1 P-450 probe were selected. The clone pLM4-1 was fully sequenced and found to contain a full-length cDNA coding for cytochrome P-450 LM4. Partial sequence and restriction mapping made it possible to identify pLM6-1 as coding for the major part of cytochrome P-450 LM6. Cloned LM4-1 cDNA was reformed by deletion of the 5' and 3' non-coding regions before insertion into yeast expression vectors PYe DP1/10. A similar operation was performed on pLM6-1 cDNA after replacement of the missing N-terminus-coding sequences by homologous sequences form the pLM4-1 clone resulting in a chimeric cytochrome P-450 coding sequence. Expression of cloned rabbit cytochrome P-450 into transformed yeast was optimized by studying the effect of the nature of the DNA sequence just preceding the initiation codon on the level of cytochrome P-450 production. Yeast synthesized cytochromes P-450 were characterized by immunoblotting, spectra and catalytic activity determinations. Cloned cytochrome P-450 LM4 was found by all criteria to be identical to the authentic rabbit one. The chimeric cytochrome P-450 that contains the 143 N-terminal amino acids of cytochrome P-450 LM4 and the remaining 375 amino acids of cytochrome P-450 LM6 was found to exhibit most of the authentic cytochrome P-450 LM6 catalytic properties. Enzymatic and evolutionary implications of these results are discussed.     

cDNA cloning of cytochrome P-450 related to P-450p-2 from the cDNA library of human placenta. Gene structure and expression

We have isolated and analyzed cDNA (designated P-450HP cDNA) clones from a human placenta cDNA library, using the cDNA for rabbit pulmonary cytochrome P-450p-2, a prostaglandin omega-hydroxylase, as a hybridization probe. The cDNA obtained encoded a polypeptide comprising 511 amino acids with a calculated molecular mass of 58987 Da, and the amino acid sequence similarity with P-450p-2 and rat liver laurate omega-hydroxylase (P-450LA omega) was only about 50%. RNA blot analysis showed that the mRNA hybridizable with the human P-450HP cDNA was inducibly expressed 3-5-fold in rabbit small intestine and lung by gestation, but the expression remained constant in rabbit liver and kidney. This mode of expression was quite different from that of P-450p-2 and P-450LA omega. Interestingly, the mRNA hybridized with the cDNA of P-450HP was found to be expressed in all the human tumor tissues so far examined, in sharp contrast with the facts that almost all the other species of P-450s are known to disappear in the tumor tissues. Taken together, the deduced hemoprotein termed P-450HP dose not seem to be the human counterpart of rabbit P-450p-2 or rat P-450LA omega, and is presumably a new member of the P-450 family including P-450p-2 and P-450LA omega. Furthermore, the corresponding genomic DNA was also cloned and analyzed. The gene of P-450HP spanned 18.8 kb and was separated into 11 exons by 10 introns whose locations were completely different from those of P-450 genes so far determined.     

Multiple constitutive but phenobarbital-inducible rat hepatic nuclear RNA species homologous to a novel cytochrome P-450 cDNA

A cDNA clone, pPB8, representing partial information for a phenobarbital-inducible rat hepatic cytochrome P-450, immunochemically related to cytochrome P-450b and/or P-450e, hybridized to multiple hepatic nuclear RNA species. In addition to the 3.7 +/- 0.2 kb mRNA encoding this novel cytochrome P-450 isozyme, pPB8 hybridized to nuclear RNAs of 4.9 +/- 0.3, 5.4, 5.7 +/- 0.2, and 6.3 +/- 0.1 kb. These nuclear RNAs were constitutively expressed and were inducible to various extents by phenobarbital administration. The time course of induction of these nuclear RNA components suggested product-precursor relationships. A "full-length" cDNA clone, pPB8/7, synthesized from poly(A)+ RNA homologous to pPB8, detected two mRNA species of 4.6 and 1.8 kb. The 4.6 kb nuclear RNA was inducible by 3-methylcholanthrene, Aroclor 1254, and phenobarbital, while the 1.8 kb nuclear RNA was not appreciably affected. It is suggested that pPB8 and pPB8/7 were synthesized from distinct mRNAs that share homology in their 3' regions.     

Isolation and characterization of full-length mouse cDNA and genomic clones of 3-methylcholanthrene-inducible cytochrome P1-450 and P3-450

Polysome immuno-adsorption, with immunoglobulin G directed against two 3-methylcholanthrene-induced mouse liver cytochrome P-450 proteins, was used to enrich mRNA from 3-methylcholanthrene-treated C57BL/6N mouse liver. cDNA transcribed from the P-450-enriched mRNA was then cloned into the Okayama-Berg vector. Two cDNA classes were detected upon differential screening of the clone bank with [32P]cDNA derived from 3-methylcholanthrene-induced immuno-enriched versus control mRNA. Several representatives of these two classes were judged to be near full length by comparison with their corresponding mRNA mobilities on denaturing agarose gels. A continuous reading-frame near the 5' end of one cDNA class (P1-450) corresponds to a protein having 15 of 17 residues the same as the published N-terminal sequence of rat P-450c. A continuous reading frame near the 5' end of the other class (P3-450) corresponds exactly to the first 25 amino acids of the published N-terminal sequence of rat P-450d. The P1-450 cDNA is at least 700 bp longer than the P3-450 cDNA. Heteroduplex analysis and Southern blot hybridization demonstrate that these mRNAs share approx. 1100 bp of sequence homology. Genomic P1-450 and P3-450 clones were isolated from a gene library constructed from C57BL/6N mouse liver DNA. By heteroduplex analysis with the corresponding cDNA, the P1-450 gene spans about 6 kb and the P3-450 gene about 7 kb. The intron-exon patterns are very similar, with the second and seventh exons being much larger than the other five. The 3' terminal exon of P1-450 is about 500 bp longer than that of P3-450. These data suggest that both P1-450 and P3-450 have diverged from a common ancestral gene.     

Characterization of a cDNA for the unexpressed form of cytochrome P-450g from the (-g) rat and differentiation of its mRNA from that of the (+g) phenotype using specific oligoprobes

Our laboratory recently isolated a cDNA for cytochrome P-450g (IIC13), a male-specific, highly polymorphic P-450 isozyme, from livers of the high phenotype (+g) of Sprague-Dawley rats [McClellan-Green et al. (1989) Biochemistry 28, 5832-5839]. Hybridization studies using a specific oligonucleotide probe for P-450 (+g) indicated that equivalent amounts of P-450g mRNA were present in livers of both the high and low phenotypes (+g and -g) of male Sprague-Dawley, Fischer (inbred -g), or ACI (inbred +g) rats. In the present study, we isolated one full-length and one nearly full-length cDNA clone coding for the unexpressed form of cytochrome P-450g from a cDNA library constructed from mRNA from a (-g) male Sprague-Dawley rat. The longest cDNA had an open reading frame of 1473 nucleotides which coded for a 490 amino acid polypeptide of Mr 55,839. Although the 5'-noncoding leader sequence and the 3'-noncoding region were unchanged, the coding sequence of the (-g) phenotype differed from that of the cDNA isolated from the (+g) phenotype by nine bases changes. These base changes would result in seven amino acid differences between the protein sequences for the two phenotypes. Two specific oligonucleotide probes for (+) P-450g and (-) P-450g containing three base differences between the (+g) and (-g) sequences hybridized differentially to mRNA from the (+g) and (-g) phenotypes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hamster cytochrome P-450 IA gene family, P-450IA1 and P-450IA2 in lung and liver: cDNA cloning and sequence analysis.

Two cDNA clones, 2C19 and 4C1, were isolated from a lung cDNA library of 3-methylcholanthrene (MC)-treated hamster by using rat P-450c cDNA as a probe. The cDNA determined from 2C19 and 4C1 was 2,916 bp long and contained an entire coding region for 524 amino acids with a molecular weight of 59,408. The deduced amino acid sequence showed a 85% identity with that of rat P-450c indicating 2C19 and 4C1 encode the hamster P-450IA1 protein. Another cDNA clone, designated H28, was isolated from a MC-induced hamster liver cDNA library by using the hamster lung 2C19 or 4C1 cDNA clone as a probe. H28 was 1,876 bp long and encoded a polypeptide of 513 amino acids with a molecular weight of 58,079. The N-terminal 20 residues deduced from nucleotide sequence of H28 were identical to those determined by sequence analysis of purified hamster hepatic P-450MCI. The high similarity of the nucleotide and deduced amino acid sequences between H28 and P-450IA2 of other species indicated that H28 encoded a P-450 protein which belongs to the P-450IA2 family. Northern blot analysis revealed that the mRNAs for hamster P-450IA1 and IA2 were about 2.9 and 1.9 kb long, respectively. Hamster P-450IA1 mRNA was induced to the same level in lungs as in livers by MC treatment, whereas hamster P-450IA2 mRNA was induced and expressed only in hamster liver.     

Titration of mRNAs for cytochrome P-450c and P-450d under drug-inductive conditions in rat livers by their specific probes of cloned DNAs

By sequence analysis of the cloned cDNA or genomic DNA, we have recently deduced the complete primary structures of two forms of 3-methylcholanthrene-inducible cytochromes P-450 (P-450c and P-450d). Comparing these sequences, we identified two highly conserved regions, amino acid numbers from 35 to 200 and from 340 to 470. The nucleotide sequences corresponding to these homologous regions are also well conserved, whereas other regions have undergone considerable sequence divergence. In RNA blot analysis with unfractionated mRNA isolated from 3-methylcholanthrene-treated rat livers, Probe A (specific to P-450c sequence) hybridized with mRNA around 23 S, while Probe B (specific to P-450d sequence) hybridized with mRNA around 18 S. When common sequence between P-450c and P-450d was used as the probes (Probe C or D), two bands were clearly observed around 23 and 18 S mRNAs. With the common DNA sequence between P-450c and P-450d as a probe (93.7% homology), we studied the induction of specific mRNA for P-450c and P-450d by a single dose of several chemical compounds to rats. 3-Methylcholanthrene increased both P-450c and P-450d mRNA levels by 50 and 10 times above the control at 17 h after the administration, respectively. Despite the lower induction rates, the P-450d mRNA level was constantly higher than or at least similar to that of P-450c mRNA. beta-Naphthoflavone and Kaneclor KC 500 showed similar induction ability to 3-methylcholanthrene. On the other hand, isosafrole induced P-450d mRNA to a much greater extent than P-450c mRNA.     

Monooxygenase activity of Saccharomyces cerevisiae cells transformed with expression plasmids carrying rat cytochrome P-450MC cDNA

The recombinant plasmids pAMC1 and pJMC1 were constructed; the former contained the cytochrome P-450MC (P-450MC) cDNA expression unit consisting of yeast alcohol dehydrogenase I (ADH) promoter, rat P-450MC cDNA and ADH terminator, and the Leu 2 marker gene, and the latter contained the same expression unit and the leu 2-d gene. Saccharomyces cerevisiae AH22 cells transformed with each of the recombinant plasmids were examined for plasmid copy number, P-450MC mRNA level, P-450MC content, and monooxygenase activity. The S. cerevisiae AH22/pJMC1 cells contained about 2-fold higher levels of the plasmid, P-450MC mRNA, and P-450MC than the AH22/pAMC1 cells. Monooxygenase activity towards 7-ethoxycoumarin and acetanilide of the AH22/pJMC1 cells was 1.7-fold and 1.5-fold higher than that of the AH22/pAMC1 cells, respectively, whereas the activity of the AH22/pAMC1 cells towards 7-ethoxycoumarin and acetanilide was more than 1,000-fold 10-fold higher than that of the control AH22/pAAH5 cells which contain no P-450MC cDNA, respectively. Therefore, it is likely that monooxygenase activity of the AH22 cells carrying rat P-450MC cDNA was approximately proportional to the expression level of P-450MC cDNA.     

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.     

Characterization of rat cytochrome P-450MC synthesized in Saccharomyces cerevisiae

Rat cytochrome P-450MC cDNA was expressed in Saccharomyces cerevisiae AH22, SHY3 and NA87-11A cells under the control of the yeast ADH1 promoter and terminator. Although the three yeast strains transformed with the constructed expression plasmid, pAMC1, contained approximately three copies of the plasmid, the levels of both P-450MC mRNA and the corresponding protein in the AH22 cells carrying plasmid pAMC1 were 1.4- to 1.7-fold and 2-fold higher than in the other two strains, respectively. The P-450MC protein was purified from the microsomal fraction of AH22 cells carrying pAMC1 by a rapid purification method. The apparent molecular weight, chromatographic behavior, spectral properties, substrate specificity and immunochemical properties of the purified P-450MC protein were indistinguishable from those of rat liver P-450MC-I and P-450MC-II (Sasaki, T., et al. (1984) J. Biochem. 96, 117-126). The NH2-terminal amino acid sequence of the purified protein up to 10 residues was the same as those of P-450MC-I and P-450MC-II. In addition, HPLC analysis of the microsomal fraction of AH22 cells containing pAMC1 indicated that the synthesized P-450MC protein corresponds to P-450MC-II, but not P-450MC-I. With another purification method, we obtained the cleaved P-450MC protein which lacked the NH2-terminal 30 amino acids of intact P-450MC. The spectral properties and monooxygenase activities towards benzo(a)pyrene and 7-ethoxycoumarin of the cleaved P-450MC were nearly the same as those of intact P-450MC.     

Aflatoxin B1-4-hydroxylase is associated with cytochrome P3-450 in C57BL/6 mouse liver

Messenger RNA from the livers of Aroclor 1254 treated mice was used to produce a cDNA library. cDNA clones corresponding to cytochromes P1-450 and P3-450 were isolated from this library by screening with a probe for the rat cytochrome P-450c gene. Specific non-cross hybridizing probes for P1-450 and P3-450 were prepared from unique restriction fragments. The radiolabeled probes were hybridized to RNA from mice treated with a low (15 mg/kg) and high (150 mg/kg, 400 mg/kg) doses of beta-naphthoflavone. The low dose of beta-naphthoflavone was found to induce only P3-450 mRNA, whereas higher doses induced both P1-450 and P3-450 mRNA. Similarly, a low dose of beta-naphthoflavone induced aflatoxin B1-4-hydroxylase, whereas higher doses induced both aflatoxin B1-4-hydroxylase and aryl hydrocarbon hydroxylase activities. These results suggest that P3-450 mRNA codes for the cytochrome that is associated with aflatoxin B1-4-hydroxylase activity.