Complementary DNA cloning of cytochrome P-450s related to P-450(M-1) from the complementary DNA library of female rat livers. Predicted primary structures for P-450f, PB-1, and PB-1-related protein with a bizarre replacement block and their mode of transcriptional expression

Three kinds of cDNA clones were isolated from a cDNA library synthesized from female rat liver mRNA by cross-hybridization with the P-450(M-1) cDNA as a probe and sequenced. One clone appears to be the previously isolated P-450f cDNA clone with an additional 5'-untranslated and coding sequence which are lacking in the previously reported clone (Gonzalez, F. J., Kimura, S., Song, B.-J., Pastewka, J., Gelboin, H. V., and Hardwick, J. P. (1986) J. Biol. Chem. 261, 10667-10672), though several nucleotide differences were seen. Another one is for P-450PB-1 mRNA previously isolated, and the last has an almost identical nucleotide sequence to P-450PB-1 (the same report cited above) except for one region of 159 base pairs where the sequence homology between the two is abruptly broken down. This nonhomologous region appears to correspond exactly to the entire eighth exon, estimated by comparison with the gene structure of the related P-450 (P-450(M-1)). This replacement in P-450PB-1 (ps) causes a frameshift in the open reading frame, resulting in the generation of a truncated form of P-450 with a strange replacement block and lacking the heme-binding region. This observation suggests that the mRNA whose cDNA was cloned here was produced from a recombinant gene generated by gene conversion or from alternative splicing of a cryptic exon. Sex- and age-dependent expression of the mRNAs investigated by dot blot analysis revealed that normal- and pseudo-type PB-1 mRNA were expressed in both male and female rat livers, though their age-dependent expression was different in male and female animals. In addition, both the mRNAs were specifically expressed in the female brain of 8 weeks, whereas practically no expression was observed in kidney and lung of both sexes.     

Pregnenolone 16 alpha-carbonitrile-inducible P-450 gene family: gene conversion and differential regulation.

A cytochrome P-450 cDNA clone, designated pP450PCN2, homologous to the previously characterized pregnenolone 16 alpha-carbonitrile (PCN)-induced P-450 cDNA (pP450PCN1; F. J. Gonzalez, D. W. Nebert, J. P. Hardwick, and C. B. Kasper, J. Biol. Chem. 260:7435-7441), was isolated from a rat liver cDNA expression library by use of a polyclonal anti-P450PCN1 antibody. This P-450 cDNA contains 2,014 base pairs and yields an open reading frame of a protein consisting of 504 amino acids (Mr = 57,760). P450PCN2 cDNA and protein shared 90% nucleotide and 89% amino acid similarity with P450PCN1 cDNA and protein, respectively. The 5' untranslated, coding, and 3' untranslated regions between the two cDNAs share 94, 93, and 79% similarities, respectively. Nucleotide differences in the coding regions, however, are not evenly distributed. Complete homology exists between the two mRNAs for 425 nucleotides (positions 346 through 771). Other regions of 93 nucleotides containing only one difference and 147 nucleotides containing two differences exist toward the 3' end of the coding regions. These data suggest the possibility that a gene conversion event(s) have occurred subsequent to duplication of the ancestral P450PCN gene. Oligonucleotide probes unique for P450PCN1 and P450PCN2 cDNAs were used to examine the levels of their respective mRNAs in noninduced and PCN-induced liver cells and in male and female rats of various ages. P450PCN1 mRNA was not detectable in either male or female rats at any ages. In contrast, P450PCN2 mRNA was present at a low level in newborn rats and became elevated in both males and females at 1 week of age. Levels of p450PCN2 mRNA continued to increase in males until 12 weeks, whereas the mRNA in females reached peak levels at 2 weeks of age but declined continuously at the onset of puberty (between 4 and 12 weeks). These levels of P45PCN2 mRNA closely parallel the increases in testosterone 6 beta-hydroxylase activity and P450PCN2 protein level, as analyzed by Western blots. P450PCN1 mRNA was induced by PCN, dexamethasone, and phenobarbital in both male and female rats. P450PCN2 mRNA was not significantly induced by PCN or dexamethasone but was readily induced by phenobarbital. Testosterone 6 beta-hydroxylase activity was also induced severalfold by PCN, dexamethasone, and phenobarbital. These data demonstrate that P450PCN1 and P450PCN2 genes are differentially regulated during development and after administration of inducing compounds and furthermore suggest that both enzymes possess testosterone 6 beta-hydroxylase activity.     

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.     

Reciprocal regulation of sex-dependent expression of testosterone 15 alpha-hydroxylase (P-450(15 alpha)) in liver and kidney of male mice by androgen. Evidence for a single gene

Testosterone 15 alpha-hydroxylase activities and its mRNA levels are higher in kidneys than in livers from male 129/J mice. Castration of 129/J male mice resulted in repression of P-450(15 alpha) in kidney, but increased it in liver. Two types of cDNA (p15 alpha-29 (Type I) and -15 (Type II)) encoding P-450(15 alpha) were previously cloned from 129/J female livers (Burkhart, B.A., Harada, N., and Negishi, M. (1985) J. Biol. Chem. 260, 15357-15361). With the use of p15 alpha-29 as a probe, Type I and II P-450(15 alpha) cDNAs were isolated from libraries of 129/J kidney poly(A)+ RNA. The nucleotide sequences of the cDNAs showed that Type I and II cDNAs from liver and kidney were identical and shared 98.3% similarity. The deduced amino acid sequence from a full-length Type I cDNA indicated that Type I P-450(15 alpha) consists of 494 amino acids with a molecular weight of 56,594. Nine amino acid substitutions were found in the Type II clone in 432 amino acids overlapping Type I. Type I cDNA clones accounted for approximately 90% P-450(15 alpha) clones isolated from a male kidney library, whereas approximately 90% of cDNA clones in a female kidney library were Type II. Liver cDNA libraries from males and females contained similar ratios of Type I and II. Effects of castration on Type I and II mRNAs were determined by Southern hybridization of a 32P-labeled ClaI-ClaI fragment from p15 alpha-29 to cDNAs synthesized from kidney and liver poly(A)+ RNAs prepared from sham-operated, castrated 129/J mice. The double-stranded cDNAs were digested with ClaI and PstI prior to gel electrophoresis to create the diagnostic restriction fragments specific for Type I or II. Castration resulted in decreased levels of Type I mRNA in male kidney. In male liver, only Type I mRNA rose significantly in response to castration. Testosterone administration returned the Type I mRNA to normal levels in castrated mice. It therefore appears that the high levels of P-450(15 alpha) in male kidney were due to androgen-dependent induction of Type I mRNA. Both Types I and II were repressed in male liver, which results in decreased levels of P-450(15 alpha). Androgen was responsible for the repression and expression of Type I in liver and kidney, but not Type II.     

Pituitary regulation of the male-specific steroid 6 beta-hydroxylase P-450 2a (gene product IIIA2) in adult rat liver. Suppressive influence of growth hormone and thyroxine acting at a pretranslational leve;

Oligonucleotide probes that distinguish between two closely related mRNAs encoding steroid 6 beta-hydroxylases of rat P-450 gene family CYP3A were used to individually assess their responsiveness to pituitary hormone regulation. Northern blot analysis revealed that the elevation of immunoreactive P-450 IIIA2 in livers of hypophysectomized rats reflects an elevation of the constitutive, male-specific P-450 IIIA2 (P-450 2a) and not an induction of the drug-inducible P-450 IIIA1 (P-450p). P-450 IIIA2 mRNA levels in intact adult male rats were found to be markedly reduced by GH administered as a continuous infusion at levels as low as 1 mU/h, indicating that GH acts at a pretranslational step to suppress expression of this P-450 enzyme. In hypophysectomized male rats, however, this same hormone treatment was only partially effective at suppressing P-450 IIIA2 mRNA and protein, suggesting that other pituitary-dependent factors contribute to the suppression observed in the intact rats. Further analysis revealed that T4, but not ACTH or human CG, can act in concert with GH to effect a more complete suppression of hepatic P-450 IIIA2 mRNA and protein in hypophysectomized rats. T4 also suppressed the expression of another GH-regulated, male-specific hepatic enzyme, designated P-450 IIA2 (P-450 RLM2), particularly in hypophysectomized female rats. In contrast, the GH-responsive P-450 IIA1 (P-450 3) was much less affected by T4 treatment. Thus, while T4 can modulate P-450 IIIA2 expression, it does not serve as a universal regulator for hepatic expression of GH-responsive P-450s.     

Rat testosterone 7 alpha-hydroxylase. Isolation, sequence, and expression of cDNA and its developmental regulation and induction by 3-methylcholanthrene

A P-450, designated P-450a, with high testosterone 7 alpha-hydroxylase activity was purified from rat liver microsomes. Specific polyclonal antibody against this P-450 was used to screen a lambda gt11 expression cDNA library and a 1687-base pair cDNA was isolated and sequenced. The deduced protein had 492 amino acids, a calculated Mr of 56,016, and it shared 51 and 45% amino acid similarities to P-450e and P-450f, respectively. Regions of similarity were distributed in distinct areas of high and low similarity along the P-450a primary sequence. P-450a cDNA was introduced into yeast cells using the expression vector pAAH5, and the resultant yeast microsomes contained both a protein of identical electrophoretic mobility to that of rat P-450a and testosterone 7 alpha-hydroxylase activity. These results confirm enzyme reconstitution data and antibody inhibition data that P-450a possesses testosterone 7 alpha-hydroxylase activity. The antibody and cDNA probes were used to examine the mechanism of regulation of P-450a by inducers and during development. P-450a and its mRNA were present at low level in newborn rats and increased to maximal level at 1 week of age in both males and females. At age 12 weeks, however, the P-450a level decreased in males but remained elevated in females. Concomitant with the decrease in P-450a in adult males was an increase in level of another immunologically related P-450. In adult male rats, P-450a was induced almost 5-fold by administration of 3-methylcholanthrene and this induction was the result of an increase in its mRNA. These results establish testosterone 7 alpha-hydroxylase as a member of the P-450e gene family that is developmentally regulated, sex-dependent, and markedly inducible by 3-methylcholanthrene.     

Adult male-specific and neonatally programmed rat hepatic P-450 forms RLM2 and 2a are not dependent on pulsatile plasma growth hormone for expression

Rat hepatic cytochrome P-450 form RLM2 is a testosterone 15 alpha-hydroxylase reported to be male-specific on the basis of purification studies (Jansson, I., Mole, J., and Schenkman, J. B. (1985) J. Biol. Chem. 260, 7084-7093). The sex dependence, developmental regulation, xenobiotic induction, and hormonal control of P-450 RLM2 expression were studied using P-450 form-specific immunochemical and catalytic assays. Polyclonal antibodies raised to rat hepatic P-450 3 (P-450 gene IIA1) were found to cross-react strongly with P-450 RLM2, but not with 10 other rat P-450 forms, suggesting that P-450 3 and P-450 RLM2 are highly conserved in primary structure. Western blotting of liver microsomes under conditions where P-450s 3 and RLM2 are resolved electrophoretically revealed that P-450 RLM2 is markedly induced at puberty in male rats, with no protein detected (less than or equal to 5% of adult male levels) in adult females or immature animals of either sex. A similar developmental dependence was observed for hepatic microsomal testosterone 15 alpha-hydroxylase activity, which was found to be catalyzed primarily by P-450 RLM2. P-450 RLM2 was resistant to induction by several xenobiotics and in the case of phenobarbital and beta-naphthoflavone, was suppressed by 50-60%. Studies on the steroid hormonal regulation of P-450 RLM2 revealed that its adult male-specific expression is imprinted (programmed) in response to neonatal testosterone exposure. Ovariectomy studies demonstrated that suppression by estrogen does not contribute significantly to the absence of P-450 RLM2 in adult female rats. Although the male-specific developmental induction of P-450 RLM2 in response to neonatal testosterone is strikingly similar to that of P-450 2c (testosterone 2 alpha/16 alpha-hydroxylase; gene IIC11), P-450 RLM2 expression is not dependent on the pulsatile pituitary growth hormone secretion required for P-450 2c synthesis. Rather, hypophysectomy of adult male rats increased P-450 RLM2 and its associated testosterone 15 alpha-hydroxylase activity by 50-100%.(ABSTRACT TRUNCATED AT 400 WORDS)     

Hormonal regulation of levels of the messenger RNA encoding hepatic P450 2c (IIC11), a constitutive male-specific form of cytochrome P450.

A cDNA clone for rat hepatic cytochrome P450 2c (gene product IIC11) was isolated and used to study the sex specificity, expression during development, and hormonal regulation of the mRNA encoding this protein in rat liver. P450 2c mRNA levels were about 16-fold higher in males than in females and were only slightly increased in male rats after administration of phenobarbital, a drug that dramatically raises the levels of mRNAs encoding several other members of the P450 II family. In contrast to the mRNA encoding P450 f (gene product IIC7), which increases gradually over the first 6 weeks of life, P450 2c mRNA showed a dramatic increase at puberty, between 4.5-5.5 weeks of life. The roles of sex steroids and GH in controlling this male-specific, developmentally regulated mRNA were then examined. A dependence on adult androgen was demonstrated by the 2- to 4-fold decrease in P-450 2c mRNA levels after castration of adult male rats and their restoration to normal by administration of the synthetic androgen methyltrienolone. Prolonged treatment (15 days) of ovariectomized female rats with this androgen also increased the levels of P450 2c mRNA and its encoded testosterone 16 alpha-hydroxylase to those of intact males. In male rats treated with estradiol valerate, mRNAs for P450 2c and alpha 2u-globulin, a major male-specific hepatic secretory protein that is under complex hormonal control, fell to negligible levels. None of these hormonal perturbations had a detectable effect on the levels of PB-1 (gene product IIC6) mRNA, which is not expressed in a sex-dependent manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural and regulatory analysis of the male-specific rat liver cytochrome P-450 g: repression by continuous growth hormone administration

Complementary DNA clones encoding the male-specific rat liver cytochrome P-450 g have been isolated by cross-hybridization with sequences from the female-specific rat liver cytochrome P-450 15 beta. Tissue distribution analysis indicates the liver as the organ with major expression of this cytochrome P-450 gene. Minimal P-450 g expression was also detected in prostate, kidney, heart, and brain. A developmental analysis reveals liver expression in the 8-week-old male and to a lesser extent in the 4-week-old male, but no detectable expression is seen in females of these ages or in 1- and 2-week-old rats from both sexes. Hypophysectomy of female rats dramatically increases hepatic expression of P-450 g, whereas continuous GH administration represses hepatic expression in male or female hypophysectomized rats. In similarity to P-450 15 beta and P-450 16 alpha, therefore, the cytochrome P-450 g gene in liver is GH regulated.     

cDNA cloning and inducible expression during pregnancy of the mRNA for rabbit pulmonary prostaglandin omega-hydroxylase (cytochrome P-450p-2)

We have isolated cDNA clones of the mRNA for prostaglandin omega-hydroxylase (cytochrome P-450p-2) (Yamamoto, S., Kusunose, E., Ogita, K., Kaku, M., Ichihara, K., and Kusunose, M. (1984) J. Biochem. (Tokyo) 96, 593-603) in rabbit lung by using synthetic oligonucleotides as probes. The cDNA sequence contains an open reading frame of 1,470 nucleotides, the first 9 amino acids of which correspond to the residues 17-25 of cytochrome P-450p-2 determined from protein analysis. The predicted primary structure contains amino acid sequences of 23 tryptic fragments of cytochrome P-450p-2 and the deduced amino acid composition is in agreement with that determined from the purified protein. The complete polypeptide, including residues 1-16, contains 506 amino acids with a calculated molecular weight of 58,515. Cytochrome P-450p-2 shared 74% amino acid similarity with rat hepatic lauric acid omega-hydroxylase (cytochrome P-450LA omega) (Hardwick, J.P., Song, B.-J., Huberman, E., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 801-810), whereas it showed less than 25% similarity to other forms of cytochrome P-450, indicating that the two cytochrome P-450s constitute a unique cytochrome P-450 gene family. DNA blot analysis of the total genomic DNA of rabbits suggest the presence of several genes or gene-like DNA sequences which cross-hybridized with the cloned cDNA. RNA blot analysis showed that progesterone treatment increased the amount of mRNA hybridizable to the cDNA by about 100-fold in the lung of rabbits as compared with the basal level without the treatment. This high level of the mRNA was also observed in the lung of pregnant rabbits.     

Identification of a novel P450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene

A novel P450 cDNA was isolated from a rat lung lambda gt11 library by hybridization with the rat P450 IIB1 cDNA probe. The cDNA-deduced amino acid sequence of this clone was 71% and 73% similar to rat IIA1 and IIA2 P450s; it was, therefore, designated IIA3 as the third member of the rat IIA subfamily. IIA3 demonstrates only 55% amino acid similarity with IIB1. Interestingly, this P450 also shared 85% and 94% amino acid similarities with human IIA3 and a mouse testosterone 15 alpha-hydroxylase P450, respectively, indicating that these P450s are orthologous counterparts to rat IIA3. Chromosome mapping, using mouse-hamster somatic cell hybrids, revealed that the IIA3 gene is localized on mouse chromosome 7. The IIA3 mRNA was detected in rat lung, and its level was induced 3-fold by treatment of rats with 3-methylcholanthrene. No IIA3 mRNA was seen in the liver, kidney, or intestine, even after long exposure of Northern blot filters to X-ray film. In contrast, the orthologous mouse and human IIA3 genes are expressed in liver.     

Isolation and characterization of cDNA clones for cytochromes P-450 immunochemically related to rat hepatic P-450 form PB-1

Rat hepatic cytochrome P-450 PB-1 is a prominent constitutive P-450 form whose levels increase approximately 2-3 fold upon phenobarbital administration. Antibodies raised against this protein recognized two major proteins in immunoblots of rat liver microsomal proteins and precipitated comparable amounts of two electrophoretically separable hepatic mRNA translation products. The levels of the two mRNAs encoding these polypeptides were increased substantially upon phenobarbital administration. The anti-PB-1 antibodies were used to screen a cDNA library, and two distinct cDNA clones, pTF-1 and pTF-2, were isolated. These clones contain inserts of 1227 and 410 base pairs, respectively, and show 80% nucleic acid sequence homology in their region of overlap. The DNA sequences of these clones show 54% sequence homology to the corresponding portions of the mRNA encoding P-450 PB-4, a major phenobarbital-inducible form of rat liver P-450, and can be optimally aligned with the PB-4 sequence without introducing insertions or deletions. The level of hepatic mRNA which hybridizes to clone pTF-2 increases approximately 2-4-fold after phenobarbital treatment, whereas mRNA which hybridizes to pTF-1 does not change in concentration after this treatment. mRNA, which hybridizes to pTF-1, is, however, 4-fold more abundant in livers of female rats than in livers of male rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue-specific regulation of cytochrome P-450 dependent testosterone 15 alpha-hydroxylase

Testosterone 15 alpha-hydroxylase activity in kidney microsomes is higher in male mice than in female mice, while in the liver the activity is higher in females than in males. Cytochrome P-450 15 alpha, a specific form of cytochrome P-450 having testosterone 15 alpha-hydroxylase activity, accounts for virtually all of the testosterone 15 alpha-hydroxylase activity in female kidney microsomes, while other isozymes of testosterone 15 alpha-hydroxylase are present in male kidney microsomes. In female kidney, P-450 15 alpha expression is regulated by a single sex-dependent locus, called Rsh for "regulation of steroid hydroxylase." The higher level of P-450 15 alpha expression in male kidneys is dependent on androgens. Of all mice strains, 129/J seems to be the least dependent on androgens to maintain a high expression of P-450 15 alpha in male kidneys. Castration of male mice lowers kidney levels of P-450 15 alpha but in the liver, P-450 15 alpha levels rise after castration. This reciprocal regulation of P-450 15 alpha genes in liver and kidney was investigated by isolating cDNA clones encoding P-450 15 alpha from liver and kidney cDNA libraries. Two highly homologous cDNA clones encoding P-450 15 alpha designated type I and type II were identified, and levels of type I and type II mRNA in liver and kidney were determined by differential restriction mapping of double-stranded cDNA prepared from mRNA from these tissues.(ABSTRACT TRUNCATED AT 250 WORDS)     

Rip locus: regulation of female-specific isozyme (I-P-450(16 alpha) of testosterone 16 alpha-hydroxylase in mouse liver, chromosome localization, and cloning of P-450 cDNA

The constitutive expression of phenobarbital-inducible mouse cytochrome P-450 (I-P-450(16 alpha) at the mRNA level and its associated testosterone 16 alpha-hydroxylase activity in liver microsomes was a female characteristic in many inbred mice, including BALB/cJ, A/HeJ, and C57BL/6J. This sex-dependent constitutive expression of the mRNA and enzyme activity was severely reduced in females of mouse strain 129/J. The distribution patterns of the mRNA and activity levels in individual offspring of F1, F2, and F1 backcrosses to progenitors, generated from crosses between 129/J and BALB/cJ mice, indicated that the female-specific expression of I-P-450(16 alpha) is an autosomal dominant trait under the regulation of a sex-limited single locus. It was found that the genotypes of this locus exhibited concordance with that of the coumarin hydroxylase locus (Coh locus) in eight out of nine 9 X A recombinant inbred strains, suggesting the localization of this sex-limited locus on chromosome 7. We propose Rip (regulation of sex-dependent, constitutive expression of phenobarbital-inducible P-450) as the name of this sex-limited locus. With the use of the rat P-450e cDNA probe, a cDNA library from liver poly(A+) RNA of BALB/cJ was screened, and three distinct cDNAs (pf3, pf26, and pf46) were selected on the basis of their restriction patterns. Nucleotide sequences of the cDNAs revealed that pf3 and pf46 are clones overlapped, with the exception that the 27-bp DNA is inserted in the coding region of pf46. The nucleotide sequence (named pf3/46) obtained from the overlapping sequences of pf3 and pf46 contained 1473 or 1500 bp of open-reading frame, and the deduced amino acid sequence shared 93% similarity with those of rat P-450b. The 27-bp insertion resulted in nine extra amino acids just in front of the cysteine residue, the fifth ligand for heme binding. The mRNA with 27-bp insertion was ubiquitously present in other inbred mice such as A/HeJ and C57BL/6J, but not in 129/J. S-1 nuclease analysis estimated a ratio of p46 and pf3 to be 1:50. Nucleotide and deduced amino acid sequences of the 1473-bp open-reading frame in pf26 possessed 83% similarity to those of pf3/46. Hybridizations of oligonucleotide probes (pf26-cu and pf3/46-cu) specific to either pf26 or pf3/46 with liver poly(A+) RNA from males and females of BALB/cJ, 129/F, and F1 offspring demonstrated that the expression of pf26, but not pf3/46, mRNA was associated with the autosomal dominant inheritance of I-P-450(16 alpha).(ABSTRACT TRUNCATED AT 400 WORDS)     

Complete cDNA sequence of a major 3-methylcholanthrene-inducible cytochrome P-450 isozyme (P-450AFB) of Syrian hamsters with high activity toward aflatoxin B1

Cytochrome P-450AFB is major isozyme inducible by 3-methylcholanthrene in Syrian golden hamsters and shows high potency toward aflatoxin B1 activation. We have isolated and sequenced cDNA clones to P-450AFB by immunoscreening a hamster liver cDNA library in lambda gt11. The longest clone contains an open reading frame of 1482 nucleotides and encodes a protein of 494 amino acids with a molecular weight of 57,420. The sequence of P-450AFB shares a 73% and 65% homology with that of mouse P-450 15 alpha (IIA3) and rat P-450a (IIA1), respectively, indicating that P-450AFB is a unique gene of the P-450IIA subfamily. The apparent concentration of a mRNA species hybridizable to the clone as well as the concentration of a protein immunoreactive to P-450AFB was increased significantly by the treatment with 3-methyl-cholanthrene, which indicates that the increase in P-450AFB protein is due mainly to an elevation of the mRNA.     

Regulation of cholesterol 7 alpha-hydroxylase in the liver. Cloning, sequencing, and regulation of cholesterol 7 alpha-hydroxylase mRNA.

Monospecific antibody against purified rat liver cholesterol 7 alpha-hydroxylase cytochrome P-450 was used to screen a lambda gt11 cDNA library constructed from immuno-enriched polysomal RNA of cholestyramine-treated female rat liver. Two types of cDNA clones differing in the length of the 3'-untranslated region were identified, and DNA sequences were determined. The full length clone contains 3561 base pairs plus a long poly(A) tail. The amino acid sequence deduced from the open reading frame revealed a unique P-450 protein containing 503 amino acid residues which belonged to a new gene family designated family VII or CYP7. Southern blot hybridization experiments indicated that the minimal size of P-450 VII gene was 11 kilobase pairs (kb), and there was probably only one gene in this new family. Northern blot hybridization using specific cDNA probes revealed at least two major mRNA species of about 4.0 kb and 2.1 kb, respectively. These two mRNA species may be derived from the use of different polyadenylation signals and reverse-transcribed to two types of cDNA clones. Cholesterol 7 alpha-hydroxylase mRNAs were induced 2- to 3-fold in rat liver by cholestyramine treatment. The mRNA level was rapidly reduced upon the removal of the inducer. Similarly, cholesterol feeding induced enzyme activity, protein, and mRNA levels in the rat by 2-fold, suggesting that cholesterol is an important regulator of cholesterol 7 alpha-hydroxylase in the liver. On the other hand, dexamethasone and pregnenolone-16 alpha-carbonitrile drastically reduced the activity, protein, and mRNA levels. These experiments suggest that the induction of cholesterol 7 alpha-hydroxylase activity by cholestyramine or cholesterol and inhibition of cholesterol 7 alpha-hydroxylase activity by bile acid feedback are results of the rapid turnover of cholesterol 7 alpha-hydroxylase enzyme and mRNA levels.     

Feminization of rat hepatic P-450 expression by cisplatin. Evidence for perturbations in the hormonal regulation of steroid-metabolizing enzymes

The biochemical basis for the complex effects of the anti-cancer drug cisplatin on hepatic cytochrome P-450 activity was studied in adult male rat liver using P-450 form-specific steroid hydroxylase assays and antibody probes. Cisplatin treatment of adult male rats resulted in a marked and prolonged feminization of the pattern of P-450 enzymes expressed in hepatic tissue. The adult male-specific cytochrome P-450 forms designated P-450 2c (P-450 gene IIC11), P-450 2a (gene IIIA2), and P-450 RLM2 were decreased by 70-90% after 7-14 days, with parallel decreases in their respectively associated microsomal steroid hydroxylase activities. Concomitantly, hepatic levels of the female-predominant enzymes P-450 3 (gene IIA1) and P-450j (gene IIE1) were elevated approximately 2-4-fold. The female-specific microsomal enzyme androstenedione 5 alpha-reductase was induced approximately 20-fold by cisplatin; however, no elevation of the female-specific P-450 2d was detected. The underlying hormonal basis for these effects of cisplatin was then examined. Serum testosterone levels were found to be depleted by cisplatin in a time- and dose-dependent manner which correlated with the observed changes in these hepatic enzymes. Furthermore, castration of adult rats altered the profile of these enzymes in a manner which resembled that observed with cisplatin treatment, suggesting that androgen depletion was the primary cause for the observed feminization of hepatic enzyme expression. Consistent with this possibility, the synthetic androgen methyltrienolone effectively blocked the changes in hepatic enzyme expression induced by cisplatin. Moreover, hepatic enzyme feminization was significantly reversed by chorionic gonadotropin, which fully restored serum testosterone levels in the cisplatin-treated rat. Luteinizing hormone-releasing hormone challenge experiments demonstrated that the responsiveness of the pituitary to this hypothalamic regulator of testicular androgen production was unimpaired by cisplatin treatment, indicating that hypothalamic production or secretion of luteinizing hormone-releasing hormone may be deficient in the cisplatin-treated animals. These studies establish that the effects of cisplatin on hepatic P-450 enzyme expression result from its interruption of the hypothalamic-pituitary stimulation of testicular androgen production and that this, in turn, leads to a depletion of circulating androgens required for maintenance of normal P-450 enzyme expression in adult male rats.     

Structural analysis and specific expression of microsomal cytochrome P-450(M-1) mRNA in male rat livers

cDNA clones for the P-450(M-1) mRNA, which exhibits a male-specific expression in rat livers, were isolated by using synthetic oligonucleotides as the probes. Sequence analysis of the cDNAs showed that P-450(M-1) mRNA contains 1,853 nucleotides in addition to a poly(A) chain, and a single open reading frame of 1,500 nucleotides encodes a polypeptide of 500 amino acids with a Mr = 57,187. The predicted NH2-terminal sequence of 30 amino acids agrees well with that of the purified protein determined by Edman degradation, and the predicted primary structure included all the partial sequences of six internal peptides of P-450(M-1) obtained by the proteolytic digestion and a conserved amino acid sequence containing a putative heme-binding cysteine, proximate to the COOH terminus of the molecules. P-450(M-1) showed relatively high sequence similarity with P-450b (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sogawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U.S.A. 79, 2793-2797) (52% similarity), P-450-3b (Ozols, J., Heinemann, F. S., and Johnson, E. F. (1985) J. Biol. Chem. 260, 5427-5434) (64%), P-450-1 (Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., and Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354) (74%), P-450PBc1 (Leighton, J. K., DeBrunner-Vossbrinck, B. A., and Kemper, B. (1984) Biochemistry 23, 4598-4603) (71%), while its sequence similarity with 3-methylcholanthrene-inducible P-450c and P-450d is rather low. Consequently, P-450(M-1) could be structurally classified into the phenobarbital-inducible type of P-450 gene family. RNA blot analysis using a synthetic oligonucleotide specific for P-450(M-1) revealed that P-450(M-1) mRNA was expressed exclusively in the livers of mature male rats in a sex-specific manner, but not in other tissues so far examined.