P450 2D Research Articles

Purification and Characterization of [<sup>3</sup>H] Mepyramine (Histamine H<sub>1</sub> Antagonist)-Binding Protein from Rat Liver: A Highly Homologous Protein with Cytochrome P450 2D

A protein having a high-affinity binding site for [3H]mepyramine (MBP) was purified to homogeneity from rat liver membranes. The purified MBP has a single type of binding site for [3H]mepyramine with Kd value of 18.5 nM, and its molecular weight was determined to be 56,000 by SDS polyacrylamide gel electrophoresis. Amino acid sequences of twelve tryptic peptides derived from MBP are highly homologous with those of rat debrisoquine 4-hydroxylase (cytochrome P450 2D1) and other rat P450 2D subfamily members. In immunoblotting analysis, an antibody against rat P450 2D1 stained a band corresponding to MBP with Mr of 56,000; its migration position was clearly different from that of rat P450 2D1. Substrates and inhibitors of debrisoquine 4-hydroxylase potently displace [3H]-mepyramine binding to MBP. Quinine and quinidine showed 400 and 80 times, respectively, higher affinity for MBP than for debrisoquine 4-hydroxylase. These results suggest that MBP is a novel P450 2D family member.

CDNA Cloning of a Novel Rat Brain Cytochrome P450 Belonging to the CYP2D Subfamily

Two cDNA clones designated 2d-29 and 2d-35 encoding a novel P450 belonging to the P450 2D subfamily were isolated from control and imipramine-treated rat train Libraries,respectively. cDNA 2d-29 and 2d-35 contained 1786 and 2754 bp nucleotides each. Although their 5′- and 3′-untranslated sequences were different from each other, their open reading frames were identical, encoding a protein which had 5 substitutions of amino acids in the C-terminal region compared with P450 2D4. Northern blot analysis showed that the expression levels of 2d-29 and 2d-35 in the brain were lower than that of P450 2D4 in the liver. However, it is noteworthy that the expression of 2d-35 was not detected in the fiver but in the brain. These results suggest that a novel P450 is expressed in the brain under different regulation.

Purification and Characterization of Constituent Testosterone 2α-Hydroxylase (Cytochrome P4502α) from Mouse Liver

Hepatic microsomal testosterone/androstenedione 2α-hydroxylase (i.e., cytochrome P4502α was purified from female CD-1 mice. Protein purification was monitored in eluates from Fractogel, DEAE-sephacel, and hydroxylapatite columns at heme absorbing 417 nm and by cytochrome P450 content, reactivity to a monoclonal antibody against female-specific rat cytochrome P450 2C12, and testosterone 2α-hydroxylase activity. The catalytic activity of the purified cytochrome P4502α, exhibiting a high degree of regioselectivity and stereospecificity, was basically restricted to the 2α-hydroxylation of testosterone and androstenedione; representing >96% and >92% of these respective metabolites. Polyclonal antibodies against cytochrome P4502α exhibited a dose-dependent and very selective inhibition of testosterone 2α-hydroxylation. The specific cytochrome P450 content of the purified cytochrome P4502α fraction was 12.06 nmol/mg protein. The specific testosterone 2α-hydroxylase activity of the purified protein was 14 nmol/min/nmol cytochrome P450, which was about 60-fold higher than the respective microsomes. The apparent subunit molecular weight of cytochrome P4502α was 51,000 and the protein appeared as a single band on sodium dodecyl sulfate polyacrylamide gels. The amino-terminal sequence analysis indicates that cytochrome P4502α is a member of the murine cytochrome P450 2d family.

Effect of ethanol on cytochrome P450 in the rat brain.

After a single dose of ethanol (0.8 ml/kg) administered intraperitoneally, the P450 content of the rat brain increased from 62 +/- 19 to 230 +/- 97 pmol/g (wet weight) of tissue (mean +/- SD, n = 5). Most of this increase could be accounted for by a 10- to 20-fold increase in the olfactory lobes and hypothalamic preoptic area. The P450s were identified by Western blot analysis and by microsequencing of the N-terminal ends after resolution of the proteins on SDS gels. They were identified as P450 2C7, 2C11, 2E1, 4A3, 4A8, and a member of the P450 2D family. In P450 extracted from the brains of control rats, P450 2C and 4A were also detectable but at a much lower concentration. P450 1A1, 2A1, 2B1, or 3A was not detected in the brains of either control or ethanol-treated rats. Oral administration of the same dose of ethanol resulted in a similar increase in the whole brain but smaller effects in the olfactory lobes. This effect of ethanol on the P450 in the brain has implications for the mechanism of toxicity and the development of tolerance to ethanol and for the effects of other drugs and environmental pollutants that act on the central nervous system.

Urinary excretion of amphetamine and 4′-hydroxyamphetamine by sprague dawley and dark agouti rats

Urinary excretion of amphetamine and 4′-hydroxyamphetamine has been studied in male and female Sprague Dawley (SD) and Dark Agouti (DA) rats. The DA rat is an animal model for the cytochrome P450 (P450) 2D poor metabolizer. Rats were given d-ampethamine sulfate (5 mg/ kg, i. p.) and urines were collected at 12 hour intervals for extraction and analysis of the amphetamines by HPLC. There was no significant difference between the sexes of either SD and DA rats in urinary 4′-hydroxyamphetamine and amphetamine excretion, but significant differeces were seen between the two strains. The percentage of dose per ml urine recovered as 4′-hydroxyamphetamine from the urine over 24 hours was 11.1 and 9.1 in the SD male and female rats, and 2.3 and 2.5 in DA male and female rats, respectively. The percentage of dose per ml urine recovered as amphetamine was correspondingly lower in the SD male and female rats, 1.1 and 1.0, than that of the DA male and female rats, 5.9 and 5.0. These results support our hypothesis that P450 2D is involved in hepatic 4−hydroxydroxylation of amphetamine in rats.

Evidence of partial involvement of P-450 2D in mutagenic activation of benzo(a)pyrene in liver S-9 fraction from untreated rats.

In order to clarify which species of cytochrome P-450 is involved in activation of benzo(a)pyrene (BP) in untreated rat liver, strain and sex differences in the ability of rat liver 9000 g supernatant (S-9) to mutagenically activate BP was investigated using Ames test. The numbers of histidine revertants in Ames test after pre-incubation of TA 98 strain of Salmonella typhimurium and BP with liver S-9 from male rats were markedly higher than those obtained using female rats. In addition, a marked strain difference (Wistar > DA) in the ability of liver S-9 from Wistar and DA rats to activate BP was observed. Antibody against cytochrome P-450 2D inhibited up to 50% of the revertant formation by the activation of BP with liver S-9 from male Wistar rats. These results indicate the partial involvement of cytochrome P-450 2D subfamily as well as cytochrome P-450 species specific to male rats in activation of BP to ultimate mutagen in untreated rat liver.

A Novel P450 Expressed at the High Level in Rat Brain

Only P450 2D mRNA could be detected in Northern blot analysis of rat brain poly(A) + RNA using probes from eight different P450s involved in drug metabolism. Four cerebral P450 cDNAs were isolated from a rat brain cDNA library using rat P450 2D2 cDNA as a probe, three of which were identified to rat P450 2D4. The expression of 2D4 mRNA was investigated by the reverse transcriptase-polymerase chain reaction and southern blot analysis using an oligonucleotide probe specific for P450 2D4. P450 2D4 mRNA was expressed at the high level in brain. Furthermore, it was detected in all other tissues examined, although to a lesser extent except in the liver and small intestine. However, total mRNAs of other P450 2D members (2D1, 2D2, 2D3, and 2D5) were mainly expressed in liver. The total level of expression of these mRNAs varied markedly among tissues. These results suggest that P450 2D4 may have a specific physiological function in the brain.

Differential effects of neonatally administered glutamate on the ultradian pattern of circulating growth hormone regulating expression of sex-dependent forms of cytochrome P450

Neonatal male rats were treated with monosodium glutamate (MSG) at either 0.5, 1.0, 2.0, 3.0 or 4.0 mg/g body weight on alternate days during the first 9 days of life. As adults, rats were catheterized to obtain unstressed, serial blood samples for the determination of ultradian patterns of circulating growth hormone. In addition, the levels of drug-metabolizing enzymes (i.e. hexobarbital hydroxylase, cytochromes P450 and b 5, NADPH-cytochrome P450 reductase and ethoxyresorufin O-deethylase as well as sex-dependent forms of cytochrome P450 [i.e. male-dependent cytochromes P450 2c (IIC11), 2a (IIIA2) and RLM2 (IIA2) and female-dependent cytochromes P450 2d (IIC12) and 3 (IIA1)] and/or their catalytic activities were measured in the hepatic microsomes of the treated rats. The results demonstrated a dose-dependent, graded response to MSG treatment. As the dose of MSG increased from 0.5 to 4.0 mg, there was a concurrent decline in the amplitudes of the characteristically masculine, episodic bursts of growth hormone, until at the highest dose (4mg), the pulses were no longer detectable. Associated with this dose-dependent alteration in the ultradian pattern of growth hormone secretion was a measurable change in the activities of the sex-dependent hepatic enzymes. As the pulse heights of the hormone declined to 10–20% of their normal amplitudes, the levels of the male-dependent enzymes (i.e. the drug-metabolizing enzymes, as well as the male forms of cytochrome P450 and their specific steroid hydroxylases) were maintained, and in some cases, exceeded the levels normally found in males. However, as the hormone pulse heights declined, there appeared an accompanying increase in the activities of some of the female-dependent enzymes. Finally, with the loss of all detectable levels of circulating growth hormone, the normal masculine profile of hepatic enzymes was reversed to an apparently normal (with the exception of cytochrome P450 2d) feminine profile. Summarizing, the results indicate that (1) neonatal administration of MSG can produce dose-dependent, graded, long-term developmental defects in the ultradian rhythm of circulating growth hormone and associated sex-dependent hepatic enzymes, and (2) while the male-dependent hepatic enzymes can be maintained at normal or even higher levels in the face of an up to 90% reduction in the pulse heights of plasma growth hormone, the activities of the female-dependent enzymes may begin to increase.

Pretranslational control by thyroid hormone of rat liver steroid 5 alpha-reductase and comparison to the thyroid dependence of two growth hormone-regulated CYP2C mRNAs.

The sexually differentiated microsomal enzyme steroid 5 alpha-reductase (NADPH: delta 4-3-oxosteroid 5 alpha-oxido-reductase, EC 1.3.99.5) catalyzes the NADPH-dependent conversion of testosterone to 5 alpha-dihydrotestosterone, a more potent androgen. In rat liver, this enzyme is expressed at a 10-fold higher level in adult females as compared to adult males. The pituitary regulation of this enzyme and its mRNA was studied in untreated and hypophysectomized rats and in rats rendered hypothyroid by treatment with the antithyroid drug methimazole. Hepatic 5 alpha-reductase activity was elevated 8-fold, to 85% of adult female levels, in adult male rats given growth hormone by continuous infusion. This same treatment was only partially effective in restoring 5 alpha-reductase in rats depleted of endogenous growth hormone by hypophysectomy, indicating that other pituitary-dependent factors contribute to the elevation observed in the inact animals. Further analysis revealed that thyroxine, but not adrenocorticotropic hormone (ACTH) or chorionic gonadotropin, could elevate 5 alpha-reductase activity and mRNA when given to the hypophysectomized rats and that this effect was enhanced by the presence of growth hormone. This thyroid hormone dependence was confirmed by the decrease in hepatic 5 alpha-reductase expression in hypothyroid rats and by its substantial restoration following thyroxine replacement. Thyroxine also stimulated expression of another female-predominant hepatic mRNA, encoding the steroid 16 alpha-hydroxylase cytochrome P-450f (IIC7), in a manner that was independent of the stimulatory effect of growth hormone on this transcript. In contrast, thyroid hormone did not significantly affect protein or mRNA levels of the growth hormone-stimulated, female-specific steroid sulfate 15 beta-hydroxylase P-450 2d (IIC12). These findings establish that thyroid hormones act at a pretranslational level to modulate the expression of some, but not all, growth hormone-stimulated hepatic mRNAs and demonstrate that both thyroxine and growth hormone can independently contribute to the sex-dependent expression of hepatic enzymes of steroid metabolism.

Depletion of Serum Growth Hormone in Adult Female Rats by Neonatal Monosodium Glutamate Treatment without Loss of Female-Specific Hepatic Enzymes P450 2d (IIC12) and Steroid 5α-Reductase*

The sexually dimorphic profiles of pituitary GH secretion play a key role in regulating the expression of several sex-dependent and developmentally controlled P-450 enzymes in rat liver. Current models for P-450 regulation by GH, however, are primarily based on hypophysectomy and GH replacement experiments. The present study examines the effects on hepatic P-450 expression of neonatal injections of monosodium glutamate (MSG), which allows for the nonsurgical suppression of adult GH levels. Furthermore, the levels of other pituitary-dependent hormones, such as testosterone and estradiol, are largely unchanged in the MSG-treated rats. Although hypophysectomy and GH replacement experiments have previously demonstrated that expression of the female-specific hepatic enzymes P-450 2d (gene product IIC12) and steroid 5 alpha-reductase is strikingly dependent on continuous GH exposure, neither enzyme was decreased in adult female rat liver after the elimination of plasma GH (less than 2 ng/ml) by neonatal MSG treatment. Moreover, although the loss of circulating GH appears to be largely responsible for the more than 10- to 20-fold elevation of the male-specific hepatic P-450 forms 2a (gene product IIIA2) and RLM2 (gene product IIA2) in hypophysectomized female rats, no such elevation of the male-specific P-450s was observed in the GH-deficient MSG-treated female rats. In contrast, the female-predominant forms P-450j (gene product IIE1) and 3 (gene product IIA1) were both elevated in adult rat liver after neonatal MSG treatment, in agreement with earlier hypophysectomy studies and demonstrating the suppressive effects that GH can exert on expression of these P-450 forms. Thus, although MSG and hypophysectomy both produce GH depletion, the responsiveness of the hepatic P-450s to these endocrine manipulations differs, allowing for an expanded understanding of the role of GH in P-450 expression.

Signalling Elements in the Ultradian Rhythm of Circulating Growth Hormone Regulating Expression of Sex-Dependent Forms of Hepatic Cytochrome P450*

Neonatal male rats were treated with monosodium glutamate (MSG) at either 2 or 4 mg/g BW on alternate days during the first 10 days of life. As adults, the 4 mg MSG-treated rats displayed the obesity, growth retardation, and reduced pituitary weights that typify this syndrome. These animals had no detectable plasma GH as determined from serial blood samples taken every 20 min for 8 consecutive h. Associated with this loss of circulating GH was an induction of the female-specific hepatic cytochrome P450 2d (gene IIC12) and the disappearance of the male-specific form of cytochrome P450 2c (gene IIC11). The catalytic activities of cytochrome P450 2c (i.e. androgen 16 alpha- and 2 alpha-hydroxylase), sex-dependent hexobarbital hydroxylase and total cytochrome P450 were similarly feminized. Rats exposed to the 2-mg dose of MSG were also obese, but their growth rates and pituitary sizes were not as severely affected as in the 4 mg MSG-treated rats. Circulating GH in these lower dosed males was secreted in a pulsatile pattern similar to that in normal males, except that the pulse amplitude was reduced as much as 90%. In spite of this profound decline in GH peak heights in the 2 mg MSG-treated males, liver metabolism was characteristically masculine. That is, female-specific cytochrome P450 2d remained undetectable, while the male forms of cytochrome P450 2c, 2a (gene IIIA2), and RLM2 (gene IIA2), their respective catalytic steroid hydroxylase activities, and associated sex-dependent drug-metabolizing enzymes were expressed at the level of or greater than that in normal males. Thus, while an ultradian pulse of circulating GH is necessary for the characteristically masculine profile of sex-specific forms of hepatic cytochrome P450, neither the amplitude of the secretory peaks nor their total GH content is critical, and these can be greatly reduced from the normal male levels.

Female-predominant rat hepatic P-450 forms j (IIE1) and 3 (IIA1) are under hormonal regulatory controls distinct from those of the sex-specific P-450 forms.

Hepatic expression of cytochrome P-450j (alcohol-inducible nitrosamine demethylase; P-450 gene IIE1) and P-450 3 (testosterone 7 alpha-hydroxylase; P-450 gene IIA1) is female predominant in adult rats [female/male greater than or equal to 1.5-2 (P-450j) or 3-4 (P-450 3)]. This sex difference emerges during the postsuckling period, when P-450 3 declines significantly (60-70% decrease) in male, but not female, rats, and P-450j declines in both sexes, but to a lower constitutive level in males than in females. The biochemical factors and regulatory events that control these developmental changes were investigated and found to be distinct from those that regulate expression of the female-specific hepatic enzymes P-450 2d (P-450 gene IIC12) and steroid 5 alpha-reductase. Immunoquantitation of the changes in P-450j and P-450 3 levels in hormonally altered rats established that both P-450s are under gonadal control. However, while androgen and estrogen both suppress P-450j expression, estrogen stimulates and androgen suppresses the expression of P-450 3. Since many of the effects of gonadal hormones on hepatic enzyme levels are mediated by the pituitary, the contribution of pituitary hormones to P-450j and P-450 3 expression was evaluated. Hypophysectomy of adult rats of either sex elevated P-450j to the levels found in immature rats (3- to 5-fold increase). This elevation was largely reversed in both sexes by GH administered either intermittently or continuously, demonstrating that P-450j is under the suppressive control of this pituitary hormone. The regulation of P-450 3 was more complex. Hypophysectomy elevated P-450 3 to prepubertal levels in adult male rats, but had no effect on P-450 3 levels in adult females. In both sexes GH suppressed P-450 3 expression when administered to hypophysectomized rats intermittently, but stimulated P-450 3 expression when infused continuously. Corresponding changes in hepatic microsomal P-450 3-dependent testosterone 7 alpha-hydroxylase and P-450j-dependent aniline hydroxylase activities were observed in response to hypophysectomy and GH replacement, but only after supplementation of microsomal NADPH P-450 reductase [which was 63-77% suppressed by hypophysectomy] with exogenous purified NADPH P-450 reductase. These studies demonstrate that these female-predominant hepatic P-450 enzymes are regulated by different mechanisms, and that both are under hormonal regulatory controls distinct from those that govern expression of the female-specific hepatic enzymes.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Regulation of rat hepatic cytochrome P-450: age-dependent expression, hormonal imprinting, and xenobiotic inducibility of sex-specific isoenzymes.

The influence of age, sex, and hormonal status on the expression of eight rat hepatic cytochrome P-450 (P-450) isoenzymes was evaluated by both catalytic and immunochemical methods. The male specificity of P-450 2c(male)/UT-A, the major microsomal steroid 16 alpha-hydroxylase of uninduced rat liver [Waxman, D.J. (1984) J. Biol. Chem. 259, 15481-15490], was shown to reflect its greater than or equal to 30-fold induction at puberty in male but not in female rats. The female specificity of P-450 2d(female)/UT-I was shown to reflect its developmental induction in females. P-450 PB-2a/PCN-E was shown to mediate greater than or equal to 85% of microsomal steroid 6 beta-hydroxylase activity; the male specificity of this P-450 largely reflects its developmental suppression in female rats. Neonatal gonadectomy and hormonal replacement experiments established that neonatal androgen "imprints" or programs the male rat for developmental induction of P-450 2c(male)/UT-A, for maintenance of P-450 PB-2a/PCN-E, and for suppression of P-450 2d(female)/UT-I, all of which occur in male rats at puberty. By contrast, the expressed levels of P-450 isoenzymes PB-1/PB-C, 3/UT-F, PB-4/PB-B, ISF-G, and beta NF-B were mostly unaffected by the rats' age, sex, and hormonal status. Studies on the sex specificity of P-450 induction established that the response of these latter five isoenzymes to the P-450 inducers phenobarbital, beta-naphthoflavone, pregnenolone-16 alpha-carbonitrile, and isosafrole is qualitatively and quantitatively equivalent in females as in males.

Rat hepatic cytochrome P-450 isoenzyme 2c. Identification as a male-specific, developmentally induced steroid 16 alpha-hydroxylase and comparison to a female-specific cytochrome P-450 isoenzyme.

Rat hepatic cytochrome P-450 isoenzyme 2c, purified to homogeneity from uninduced, adult rat liver (Waxman, D.J., Ko, A., and Walsh, C. (1983) J. Biol. Chem. 258, 11937-11947), was shown to exhibit a unique NH2-terminal amino acid sequence as well as distinctive peptide maps and immunochemical properties when compared to seven other purified rat liver P-450 isoenzymes. P-450 2c was an efficient monooxygenase catalyst with several xenobiotic substrates; P-450 2c also catalyzed 16 alpha- and 2 alpha-hydroxylations of testosterone, androst-4-ene-3,17-dione and progesterone (total turnover = 7-9 min-1 P-450(-1) at 25 microM steroid substrate) with the ratio of 2 alpha to 16 alpha hydroxylation varying from less than or equal to 0.02 to 1.6 depending on the steroid's C-17 substituent. Six different microsomal steroid hydroxylase activities characteristic of purified P-450 2c and sensitive to specific inhibition by anti-P-450 2c antibody were induced at puberty in male but not female rat liver. Microsomal steroid hydroxylations catalyzed by other P-450 isoenzymes exhibited age and sex dependencies distinct from those of the P-450 2c-mediated activities. Immunochemical analyses confirmed that this sex dependence and developmental induction reflected alterations in P-450 2c polypeptide levels. Attempts to chromatographically detect P-450 2c in either immature male or adult female microsomes were unsuccessful and led to purification of P-450 2d (female), a catalytically distinct and female-specific form. Peptide mapping and immunochemical analyses suggested significant structural homologies between the two sex-specific isoenzymes, P-450 2c and P-450 2d (female). A significant suppression of P-450 2c levels (up to 70-80%) was observed upon administration of several classical P-450 inducers. These studies establish that P-450 2c corresponds to the male-specific and developmentally-induced steroid 16 alpha-hydroxylase of rat liver and suggest that the expression of P-450 2c versus P-450 2d (female) may provide a biochemical basis for the sex differences characteristic of rat liver xenobiotic metabolism.