Intrinsic signals in the sexually dimorphic circulating growth hormone profiles of the rat.

Male rats secrete growth hormone (GH) in episodic bursts every 3.5-4 h. Between the peaks, GH levels are undetectable. In females, GH secretory profiles are characterized as continuous because hormone concentrations are always measurable in the circulation. These gender differences in the circulating GH profiles are responsible, to varying degrees, for observed sexual dimorphisms ranging from body growth to the expression of hepatic cytochrome P450 (P450, CYP) isoforms. Using hypophysectomized rats in which restored gender-dependent plasma GH profiles were manipulated, we have investigated the importance of the interpulse period in the masculine episodic plasma GH profile in regulating expression (mRNA, protein and/or specific catalytic activity) of male-specific CYP2A2, 2C11, 2C13 and 3A2, female-specific CYP2C12 and female-predominant CYP2A1, 2C6 and 2C7. We observed that some isoforms were induced or suppressed by discerning the length of the GH-devoid interpulse period, others responded to the pulse amplitudes, still others recognized the mean circulating concentrations of GH and some were regulated by a combination of these signals. We conclude that concealed in the gender-dependent circulating GH profiles are numerous intrinsic signals, both inductive and repressive, individually "tailored" to be recognized by each isoform of P450. There would appear to be no one signal in each gender-dependent GH profile responsible, in toto, for the characteristic sexually dimorphic expression of some dozen hepatic P450s in male and female rats.

Epidermal growth factor regulation of female-dependent CYP2A1 and CYP2C12 in primary rat hepatocyte culture.

In the present study, we describe the effects of medium composition in primary cultures of rat hepatocytes on the expression of two major constituent female-dependent CYP isoforms, CYP2C12 and CYP2A1. When female rat hepatocytes were cultured with the serum-free medium HepatoZYME, currently used to attain long-term maintenance of hepatocyte phenotypic expression, CYP2C12 mRNA and protein levels were markedly suppressed, despite the constant presence of growth hormone, the essential regulator of liver CYP2C12. Conversely, rat hepatocytes cultured in the serum-free medium Dulbecco's modified Eagle's medium-F12K, also supplemented with growth hormone, sustained near normal expression levels of CYP2C12 mRNA and protein for the 7 days of observations. Although media composition had no significant effect on mRNA expression of CYP2A1, protein content decreased dramatically in hepatocytes cultured with HepatoZYME medium. We were able to demonstrate the plasticity of the cells by restoring/suppressing the expression of CYP2C12 and CYP2A1 mRNA by reverting the culture conditions. Addition of the mitogen epidermal growth factor present in the HepatoZYME formulation to the Dulbecco's modified Eagle's medium-F12K culture media appreciably decreased expression of both CYP2C12 and CYP2A1 in female hepatocytes, while briefly sustaining levels of the cyclin inhibitor p21. Lastly, reduced CYP protein content observed in hepatocytes cultured with epidermal growth factor was not the result of an absence or reduction in the CCAAT/enhancer-binding protein alpha, a requisite transcription factor for CYP2C12 expression.

Infusion of gender-dependent plasma growth hormone profiles into intact rats: effects of subcutaneous, intraperitoneal, and intravenous routes of rat and human growth hormone on endogenous circulating growth hormone profiles and expression of sexually dimorphic hepatic cyp isoforms.

The dramatic sexual dimorphism in rat hepatic CYPs is determined by gender differences in the circulating GH profiles. Accordingly, each responsive isoform of CYP is induced or suppressed by different components, i.e., signaling elements, in the GH profiles. It was the purpose of this study to determine whether the signaling elements in the sexually dimorphic plasma GH profiles identified in GH-depleted rats are recognized by the hepatic CYPs in intact rats exposed to a multiplicity of signals contained in the normal gender-dependent GH profiles. To accomplish this goal, we imposed (via osmotic minipumps) the continuous feminine GH profile upon normal male rats and superimposed (via intra-atrial catheters) the episodic masculine profile upon normal females. Monitored circulating GH profiles indicated that the administered GH had little or no effect on the normally secreted gender-dependent endogenous profiles. Basically, we observed that the degree of constancy of GH in the circulation (continuous in females and episodic in males) is the primary determinant establishing sexually dimorphic expression of eight hepatic CYPs in intact rats. However, the characteristic expression levels of each isoform observed in male and female rat liver are determined by an interaction of more subtle signals in the GH profiles reflected in the concentration and persistence of the feminine continuous profile as well as the frequency, duration, and amplitude of pulse and interpulse periods in the masculine episodic profile. In the course of the study, unexpected findings led us to compare the effectiveness of s.c.- and i.p.-infused GH and rGH with hGH. Briefly, male- and female-dependent hepatic CYPs were undoubtedly most responsive to rGH infused by i.p.-implanted osmotic pumps.

Nominal growth hormone pulses in otherwise normal masculine plasma profiles induce intron retention of overexpressed hepatic CYP2C11 with associated nuclear splicing deficiency.

Restoration of circulating masculine GH profiles at minipulse amplitudes (i.e. approximately 10% of normal) to hypophysectomized male rats and neonatal administration of monosodium glutamate (MSG), producing a similar plasma GH profile, both result in an overexpression (approximately 200-300%) of CYP2C11 messenger RNA (mRNA), the predominant hepatic cytochrome P450 (CYP) drug-metabolizing enzyme in adult male rats. Coincident with the severalfold elevation in transcript level is a modest 10-30% overexpression of CYP2C11 protein and its catalytic activities. Using hepatic tissue from adult, neonatally MSG-treated rats, we have cloned a variant species of CYP2C11 mRNA containing all of the essential elements of a full-length complementary DNA, including initiating codon, termination codon, and polyadenylase tail. In addition, the transcript contains a 742-bp intervening sequence (identical to the complete terminal intron) between the last and penultimate exons, and an intron-specific oligo probe for Northern blotting demonstrates the presence of the variant transcript in liver of MSG-treated rats. Associated with the overexpression and intron retention of the transcript is a 50% reduction in the nuclear splicing capacity of the liver for model precursor CYP2C11 mRNA. It is proposed that this splicing defect may be a consequence of the mini-GH pulses (secreted in otherwise normal masculine plasma profiles) signaling abnormal processing of precursor CYP2C11 mRNA to produce a substantial portion of intron retained, nontranslatable transcript.

Latent overexpression of hepatic CYP2C7 in adult male and female rats neonatally exposed to phenobarbital: a developmental profile of gender-dependent P450s.

For more than 20 years it has been known that neonatal exposure to phenobarbital results in a delayed, but permanent overexpression of drug-metabolizing enzymes in adult male and female rats. Accordingly, to identify the specific isoform(s) of P450 responsible for the imprinted overexpression of hepatic monooxygenases, we have monitored the developmental profile of some dozen hepatic P450 isoforms in 4- to 150-day-old male and female rats neonatally treated with the barbiturate. Some of the cytochrome P450s (CYP), i. e., CYP2A1, 2A2, 2C6, 3A1, and 3A2, exhibit the typical transient response in which isoform levels (mRNA, protein, and/or specific catalytic activity) rise precipitously at the time of phenobarbital administration and rapidly decline to preinduction levels after withdrawal of the barbiturate. Other isoforms, i.e., CYP1A1, 1A2, 2C7, 2C11, 2C12, and 2C13, were neither constitutively expressed nor phenobarbital inducible in the neonate. Only one of these isoforms, female predominant (M:F, approximately 1:2) CYP2C7, exhibited a barbiturate-induced delayed, but persistent approximately 30 to 50% overexpression from puberty through adulthood. We propose that at the time of exposure, neonatally administered phenobarbital produces a "silent" programming defect resulting in a delayed, but persistent overexpression of the isoform, contributing, at least in part, to a permanent elevation of hepatic drug-metabolizing enzyme activities.

Differential expression of gender-dependent hepatic isoforms of cytochrome P-450 by pulse signals in the circulating masculine episodic growth hormone profile of the rat.

The masculine profile of growth hormone (GH) secretion characterized by episodic bursts ( approximately 200-300 ng/ml plasma) every 3.5 to 4 h, separated by interpulse periods devoid of detectable hormone, was restored at various peak heights to hypophysectomized, thyroxine-supplemented male rats to determine the minimum signaling amplitudes of the hormone pulse required to maintain male-like expression levels of gender-dependent hepatic cytochrome P-450s (CYP P-450s). Restoration of the pulse to as little as 2.5% of normal elevated CYP2C11 (the predominant isoform in male liver) protein and dependent catalytic activities to approximately 50% of normal, whereas transcript concentrations increased to 150% of physiologic. Renaturalizing the masculine plasma GH profile to 5% of normal was sufficient to increase CYP2C11 protein and catalytic activity to intact levels while further elevating mRNA to approximately 200% of normal (subsequently declining to intact concentrations with physiologic pulses). In dramatic contrast, CYP2C7 (mRNA and protein) declined to barely detectable levels following hypophysectomy and remained completely unresponsive to GH until replaced with the physiologic masculine profile. The repressive effects of the episodic GH profile on CYP2A2 and CYP3A2 expression similarly required replacement of near physiologic pulse amplitudes. Exhibiting an intermediate response to the masculine profile, restoration of 25% of the normal pulse amplitude was sufficient to significantly elevate CYP2A1 and CYP2C6 expression levels in hypophysectomized rats. These findings illustrate the importance of the pulse amplitudes (in addition to the interpulse periods) in the circulating masculine GH profile as differential signals regulating the expression and/or repression of each sex-dependent hepatic P-450 isoform in the rat.

Gender differences in the responsiveness of the sex-dependent isoforms of hepatic P450 to the feminine plasma growth hormone profile.

Most of the constitutive hepatic P450 isoforms expressed in the rat exhibit dramatic gender differences. Whereas only male hepatocytes contain CYP2A2, 2C11, and 3A2, only female hepatocytes express CYP2C12 and 3- to 4-fold greater levels of CYP2C7. This sexually dimorphic expression of hepatic P450 isoforms is regulated by the gender-dependent secretory GH profiles, i.e. episodic in males and continuous in females. In the case of the feminine GH profile, the continuous presence of the hormone in the circulation completely suppresses male-specific CYP2A2, 2C11, and 3A2, while stimulating full expression of female-dependent CYP2A1, 2C7, 2C12, and non-P450 testosterone 5alpha-reductase (type 1). The gender-dependent expression of the P450s can be reversed by exposing male rats to the continuous feminine plasma GH profile and females to the episodic masculine GH profile. Under these conditions, females will now express the male-specific isoforms and suppress the female-dependent forms, whereas the opposite will occur in the males. Nevertheless, it is not clear whether the levels of expression or suppression are comparable in male and female rats exposed to the same sex-dependent GH profiles. In the present study, we have renaturalized the circulating feminine GH profile in euthyroid-maintained, hypophysectomized female and male rats at six concentrations ranging from 3-100% of normal. Continuous monitoring of GH levels revealed indistinguishable plasma profiles in females and males at each dosage administered. In the case of females, restoration of the feminine-like plasma GH profile at a concentration that was 3% of the normal level restored expression levels (i.e. mRNA, protein, and/or catalytic activity) of female-dependent CYP2C12, 2A1, and 5alpha-reductase to 50% or greater of normal and fully suppressed expression of male-specific CYP2A2, 2C11, and 3A2. Twice the dosage of the hormone (6% of normal) was required to restore female-predominant CYP2C7 to 50% of normal in hypophysectomized female rats. In contrast, we found that all of the measured isoforms were significantly less responsive to the inductive and suppressive effects of the feminine-like GH profile when administered to male rats. While suppression of the male-specific isoforms (i.e. CYP2A2, 2C11, and 3A2) in male rats required concentrations of GH in the feminine profile 2-3 times greater than were effective in female rats, no dosage of the hormone was as effective in inducing female-dependent P450s (i.e. CYP2A1, 2C7, and 2C12) in males as in females. Clearly, the continuous feminine GH profile was more effective at inducing and suppressing gender-dependent isoforms of hepatic P450 when restored to female rats, where it is normally secreted, than in males. As GH profiles appear to be the sole factor responsible for regulating the sexually dimorphic expression of hepatic P450 isoforms in adult rats, the differential responsiveness of male and female rats to the feminine GH profile are likely to be inherently induced by irreversible imprinting during a critical developmental period.

Interactions of gender, growth hormone, and phenobarbital induction on murine Cyp2b expression.

The interactions of gender, growth hormone, and phenobarbital induction on Cyp2b expression were examined in phenotypically normal (lit/+) and growth-hormone deficient "little" (lit/lit) mice. Using an immunocrossreactive monoclonal antibody designed to identify rat CYP2B1 and 2B2 proteins, we observed three hepatic Cyp2b proteins in control (lit/+) females, but only two proteins, one at trace levels, in control males. Phenobarbital administration to lit/+ mice increased the expression of the two Cyp2b isoforms in the males by 3- to 4-fold, but produced an approximately 75% reduction in the female-expressed proteins. Whereas growth hormone depletion (lit/lit) had no effect on the expression profile of Cyp2b proteins in females, it had a de-repressive effect in males, resulting in the expression of three proteins at concentrations now comparable to those observed in female liver. Generally, phenobarbital had no inductive effects in the lit/lit mice of both sexes. In all groups, transcript levels measured by a CYP2B1 probe were in agreement with the protein findings. In contrast, Cyp2b mRNA identified by an oligonucleotide probe for CYP2B2 were repressed completely by growth hormone in both sexes, and was expressed as a female-predominant transcript in the lit/lit mice. In spite of an apparent high degree of sequence homology between the rat CYP2B and murine Cyp2b gene families, the present findings highlight fundamental differences in their constitutive and gender-dependent expression, growth hormone regulation, and phenobarbital inducibility.

A comparison of cytochrome P450-dependent testosterone 2alpha-hydroxylase in rat (P4502C11) and mouse (P4502alpha).

Hepatic P450 2C11 in the rat and P450(2alpha) in the mouse are unique in being the only isoforms in their respective species with testosterone 2alpha-hydroxylase activity. Comparing gender differences, tissue distribution and physicochemical properties, we investigated whether this uncommon catalytic activity shared by the two isoforms is dependent upon a high degree of homology. Using additional substrates (e.g. androstenedione, hexobarbital), we observed that P450(2alpha) and P450 2C11 produced no metabolites in common. Moreover, concentrations of antisera prepared against purified P450(2alpha) that inhibited 95% of P450(2alpha)-dependent testosterone 2alpha-hydroxylase activity had only a minimal inhibitory effect (< 20%) on P450 2C11-dependent testosterone 2alpha-hydroxylase and were similarly unreactive to the rat isoform isolated on Western blots. Comparison of the isoforms' N-terminal amino acid residues and two internal peptide fragments indicated almost no sequence homology (< 4%). Gender-dependent tissue expression levels of P450(2alpha) and P450 2C11 revealed additional dichotomies. Whereas hepatic P450(2alpha) was moderately female-predominant (M/F; 0.62), hepatic P450 2C11 was clearly male-specific (M/F; 32.9). Murine P450(2alpha) mRNA was equally and substantially expressed in liver, kidney and brain; by contrast earlier studies reported that rat P450 2C11 was exclusively expressed in liver. The present results indicate that the unique testosterone 2alpha-hydroxylase activities of P450(2alpha) and P450 2C11 are expressed by two very different proteins exhibiting minimal homology.

Gender, age and dose effects of neonatally administered aspartate on the sexually dimorphic plasma growth hormone profiles regulating expression of the rat sex-dependent hepatic CYP isoforms.

Newborn male and female rat pups were injected with either 2 mg or 4 mg monosodium aspartate (MSA)/g body weight or diluent on alternate days for the first 9 days of life. Both doses of the amino acid had profound effects on the sexually dimorphic growth hormone secretory profiles in adulthood. There were no measurable levels of growth hormone in any of the plasma samples obtained during 8 continuous hr of serial blood collections from the adult males and females treated neonatally with 4 mg of MSA. Male rats treated with half the dose of the amino acid (i.e., 2 mg MSA/g) exhibited typical masculine profiles of growth hormone release, except that the amplitudes of the ultradian pulses were reduced to 10-20% of normal male levels. Otherwise, like normal males, the peaks occurred about every 3-4 hr and the intervening 2.5-hr troughs had undetectable levels of growth hormone. In a similar sense, females treated with 2 mg of MSA maintained their sexually dimorphic pattern of plasma growth hormone, i.e., frequent pulses of hormone followed by short-lived troughs. However, the peaks rarely exceeded 20 ng/ml and the troughs usually fell to a measurable 8 to 10 ng/ml resulting in an approximate 75% reduction in the mean plasma concentration. Growth hormone- and gender-dependent expression of CYP2C7, 2C11, 2C12, 2C13, 2A1, 2A2, and 3A2 (mRNAs, proteins, and catalytic activities) were generally unaffected by neonatal exposure to 2 mg of MSA. In contrast, the higher 4-mg dose of the amino acid completely or near completely suppressed male-specific CYP2C11, 2C13, 2A2, and 3A2 expression while inducing small increases in female-specific CYP2C12 and female-predominant CYP2A1 in the treated males. Females exposed to the 4 mg MSA dose exhibited less severe isoform changes characterized by small reductions in CYP2C12 and 2C7 levels. Whereas expression levels of most of the CYP isoforms in both sexes were lowest in the pubertal (47-day-old) rats, and occasionally higher in the adults (207-day-old) as compared with the early postpubertal (70-day-old) rats, the effects of neonatal MSA were the same at all ages studied. Since each of the CYP isoforms are regulated by different "signaling elements" in the sexually dimorphic plasma growth hormone profiles, it is possible to correlate MSA-induced alterations in CYP expression levels to specific changes in the gender-dependent growth hormone profiles.

Feminization of hepatic cytochrome P450s by nominal levels of growth hormone in the feminine plasma profile.

The feminine profile of continuous growth hormone secretion was restored at various concentrations to hypophysectomized, thyroxine-supplemented female rats to determine the minimum signaling concentrations of the hormone required to maintain female-like expression levels of gender-dependent hepatic cytochrome P450s (P450s). Rat growth hormone was infused by intraperitoneally implanted osmotic minipumps, and the resulting circulating concentrations and profiles were determined by radioimmunoassay of serially collected plasma samples. Restoration of feminine growth hormone profiles at 3% of physiological concentration completely suppressed male-specific CYP2C11, CYP2C13, CYP2A2, and CYP3A2. Although significant levels of female-dependent isoforms were expressed at this growth hormone concentration, their full expression required, somewhat higher plasma concentrations of the hormone; CYP2A1 and 5 alpha-reductase were increased to normal female levels with only 6-12% of physiological concentrations of the hormone, normal expression levels of CYP2C12 required approximately 12-25% physiological hormone levels, and CYP2C7 required approximately 25-50% of the normal growth hormone profile to attain female-like expression levels. When determined, protein and specific catalytic activities were in agreement with mRNA levels, supporting the conclusion that growth hormone regulates gender-dependent expression of P450 isoforms by transcription initiation. There was little effect of gender, hypophysectomy, or growth hormone replacement on CYP2C6, growth hormone receptor, and growth hormone-binding protein mRNAs. In contrast, insulin-like growth factor-1 mRNA was sexually dimorphic (male > female), virtually disappeared after hypophysectomy, and was restored to female-like levels with plasma growth hormone concentrations equaling 12-25% of normal. These findings demonstrate the effectiveness of nominal growth hormone concentrations (undetectable by available radioimmunoassay) in an otherwise feminine plasma profile to maintain female-like expression levels of gender-dependent P450s.

Imprinted overinduction of hepatic CYP2B1 and 2B2 in adult rats neonatally exposed to phenobarbital.

Neonatally administered phenobarbital has been shown to cause a permanent, but delayed overexpression of hepatic drug-metabolizing enzymes, occurring at the time of sexual maturity. The present studies indicate that these above-normal levels of hepatic monooxygenases are not a result of a persistent overexpression of CYP2B1 and 2B2 proteins, the major phenobarbital-inducible isoforms of cytochrome P450. However, early exposure to the barbiturate permanently alters (i.e., imprints) the inductive responsiveness of CYP2B1 and 2B2 to subsequent phenobarbital challenge in adulthood. That is, neonatal administration of therapeutic-like levels of phenobarbital causes an overinduction (approximately 30-40%) of CYP2B1 and 2B2 mRNAs, proteins and specific catalytic activity (androstenedione 16 beta-hydroxylase) levels when the rats are rechallenged as adults with as little as 1 mg or 10 mg/kg b.wt. of the barbiturate. This "latent" defect in the inductive mechanism is associated in both sexes with an abnormality in the circulating growth hormone profiles characterized by subnormal secretory pulses. Because endogenous growth hormone normally inhibits phenobarbital induction of CYP2B1 and 2B2, and the level of inhibition is directly related to the height of the secretory pulse, we have proposed that the overinduction of CYP2B1 and 2B2 in adult rats neonatally exposed to phenobarbital results, at least in part, from a "deinhibition" by the subnormal pulse amplitudes in the plasma growth hormone profiles.

Purification and characterization of constituent androstenedione 15 alpha-hydroxylase (cytochrome P450(15 alpha AD)) from mouse liver. Sex- and tissue-dependent expression.

Hepatic microsomal androstenedione 15 alpha-hydroxylase (i.e.cytochrome P450(15)alpha AD 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, by cytochrome P450 content, reactivity to monoclonal antibody against female-specific rat cytochrome P450 2C12, and androstenedione 15 alpha-hydroxylase activity. The catalytic activity for androgens of the purified cytochrome P450(15)alpha AD, exhibiting a high degree of regioselectivity and stereospecificity, was restricted to the 7 alpha- and 15 alpha-hydroxylation of androstenedione, representing, respectively, > 5% and > 93% of the total metabolites. Polyclonal antibodies against cytochrome P450(15)alpha AD exhibited a concentration-dependent and very selective inhibition of hepatic microsomal androstenedione 7 alpha- and 15 alpha-hydroxylation and a 60% inhibition of benzphetamine demethylation, the latter drug appearing to be a much more effective substrate than androgens. Cytochrome P450(15)alpha AD accounted for about 3% of the total P450 in female mouse liver microsomes. The apparent subunit molecular weight of P450(15)alpha AD was 53,000, and the protein appeared as a single band or sodium dodecyl sulfate-polyacrylamide gels. The isoform was intensely expressed in both liver and lung of CD-1 female mice and was female-predominant in the livers of five or eight strains examined; it was sex-independent in the remaining three strains. Amino-terminal sequence analysis indicates that cytochrome P450(15)alpha AD is a member of the murine cytochrome P450 2c subfamily.

Neonatal phenobarbital-induced persistent alterations in plasma testosterone profiles and testicular function.

Daily sc injections of phenobarbital at anticonvulsant therapeutic doses for the rat (40 mg/kg) for the first 7 days of life resulted in below normal levels of serum testosterone from around birth to before puberty, normal levels during puberty and above normal levels of the androgen after puberty and in adulthood. Cluster analysis of the plasma testosterone secretory profiles obtained at 15-min intervals from phenobarbital-treated rats at 65 and 165 days of age revealed a significant increase in both the peak amplitudes and their durations resulting in a 100% increase in the amount of hormone secreted during the peak periods. In general, most of the rats (control and experimental) secreted testosterone as two large peaks, each 3 to 4 hr in duration, during the 10-hr lights-on collection period. In addition to permanently disrupting the ultradian profiles of plasma testosterone, neonatal exposure to the barbiturate altered testicular responsiveness to steroidogenic regulatory agents. That is, neonatal exposure to phenobarbital enhanced the responsiveness to exogenous hCG as measured by an above-normal increase in testosterone concentration. Moreover, phenobarbital-induced reductions in serum testosterone levels were delayed in adult rats neonatally exposed to the barbiturate. Whereas a single challenge dose of phenobarbital (1 or 10 mg/kg) reduced serum testosterone concentrations in control animals by almost 80% within 3 hr, a decline in serum androgen levels in the neonatally phenobarbital exposed males was not observed until 12 hr after the challenge dose. These results indicate that postpartum exposure to therapeutic levels of phenobarbital can permanently disrupt testosterone secretory profiles and alter pathways regulating testicular steroidogenesis.

Phenobarbital induction of hepatic CYP2B1 and CYP2B2: pretranscriptional and post-transcriptional effects of gender, adult age, and phenobarbital dose.

Chemical induction of hepatic monooxygenases should not be viewed as an extracorporal process but rather as one that is liable to be influenced by numerous endogenous factors. In this regard, we examined the interactions of gender, adult age, and barbiturate dose on the course of phenobarbital induction of hepatic CYP2B1 and CYP2B2. We observed that femaleness and youth were associated with the greatest inhibition, so that both the rate and initiation of CYP2B1 and CYP2B2 induction were suppressed most in the young adult (65 days of age) females, followed by the mature adult (150 days of age) females and then by the young adult males, with the mature adult males exhibiting the least suppression of phenobarbital induction. The differential expression rates of hepatic CYP2B1 mRNA in the young and mature male and female rats, similarly reflected at the protein level, suggest that gender- and age-dependent suppression of CYP2B1 occurs at a pretranscriptional or transcriptional level. In contrast, transcript levels of CYP2B2 were unaffected by gender or age. However, accumulation of cytochrome P450 (P450) 2B2 protein was affected by the animal's age and gender, suggesting regulation of a post-transcriptional event. Highly selective (androstenedione 16beta-hydroxylase) as well as nonspecific (total P450 and hexobarbital hydroxylase) P450 2B1- and 2B2-dependent catalytic activities were in agreement with protein levels. Determination of gender- and age-dependent circulating growth hormone profiles indicates that the continuous secretion of the hormone characteristic of the female is more suppressive of CYP2B induction than the episodic pattern growth hormone secretion found in males. The considerably elevated growth hormone pulse amplitudes observed in the young rats of both genders seem to be an additional inhibitory signal antagonizing phenobarbital induction of CYP2B1 and CYP2B2. Phenobarbital administration did not interfere with the normal gender- and age-dependent growth hormone secretory profiles. Last, although as little as 1 mg/kg phenobarbital increased CYP2B1 mRNA concentrations by 100%, there was no translation into detectable levels of protein. In contrast, the same low dose of barbiturate inducing an equal percent increase in CYP2B2 mRNA did result in an expression of protein. Unlike use of the 10 mg/kg dose, CYP2B1 and CYP2B2 induction by phenobarbital at 1 mg/kg was unaffected by age or gender.

Growth hormone regulation of male-specific rat liver P450s 2A2 and 3A2: induction by intermittent growth hormone pulses in male but not female rats rendered growth hormone deficient by neonatal monosodium glutamate.

Growth hormone (GH) secretory patterns regulate the expression of several sex-dependent liver cytochrome P450 (CYP) genes. Studies using the hypophysectomized rat model have established that the intermittent plasma GH secretory pattern associated with adult male rats markedly stimulates liver expression of the male-specific CYP 2C11, a testosterone 2 alpha- and 16 alpha-hydroxylase, but is not required for expression of other male-specific liver enzymes, including CYP 2A2, a testosterone 15 alpha-hydroxylase, and CYP 3A2, a testosterone 6 beta-hydroxylase. In the present study, the effects of intermittent GH treatment on liver CYP expression were studied in adult rats rendered GH deficient by neonatal administration of monosodium glutamate (MSG), which depletes circulating adult GH without the global loss of other pituitary-dependent hormones that is associated with hypophysectomy. Restoration of the normal masculine circulating GH profile of six daily pulses (180-225 ng GH/ml/peak) in MSG-treated male rats by the use of an external pumping apparatus led to a substantial (30-50%) restoration of normal male levels of CYP 2A2 and CYP 3A2 activity, protein, and mRNA. GH pulsation at the nonphysiological frequencies of two or four times per day was less effective unless given at a dose that resulted in supraphysiological plasma GH levels. Although intermittent GH treatment can induce male-specific P450 expression in hypophysectomized female rats, the same hormone treatment did not stimulate CYP 2A2 or CYP 3A2 expression in MSG-treated female rats. Liver GH receptor mRNA levels at adulthood were not significantly altered by neonatal MSG treatment, suggesting that the unresponsiveness of MSG-treated females and the previously reported low responsiveness of MSG-treated males to GH-induced CYP 2C11 expression are not due to the absence of GH receptor. Moreover, normal liver IGF-1 mRNA levels were expressed in the MSG-treated female rats, suggesting that the liver GH receptor is functional in these animals. The present findings establish that the adult male-specific enzymes CYP 2A2 and CYP 3A2 can be positively regulated by intermittent GH pulsation despite their GH-independent expression in hypophysectomized rats. Moreover, neonatal MSG treatment, particularly in female rats, may lead to the loss of factors other than GH that are required for full expression of the pulsatile GH-stimulated CYP 2A2, 3A2, and 2C11 genes.

Simulataneous isolation of NADPH-cytochrome P-450 reductase and cytochrome P-450 using tentacle ion-exchange chromatography and interspecies comparison of the reductase activity.

Using the same initial Fractogel (tentacle) ion-exchange chromatography to isolate murine cytochrome P-450, mouse hepatic NADPH-cytochrome P-450 reductase (EC 1.6.2.4) was simultaneously isolated from solubilized liver microsomes and purified on a DE-52 column to a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme had a molecular mass of 77 kD, and its specific activity was 25.4 mumol.min-1.mg protein-1. Purified constitutive mouse liver NADPH-cytochrome P-450 reductase was successfully reconstituted in vitro with dilauroylphosphatidyl-choline and constitutive purified mouse testosterone 2 alpha-hydroxylase (cytochrome P-450(2)alpha) with an observed activity of 13.8 nmol.min-1.nmol P-450-1. Although the partially purified reductase obtained from the Fractogel column was contaminated by significant levels of two unidentified proteins, it was as equally effective in the reconstituted system as the DE-52-derived purified reductase. Lastly, we found that rat and mouse NADPH-cytochrome P-450 reductases were similarly effective in supporting the catalytic activity of rat cytochrome P-450 2B1, but the murine reductase was 50% more effective than the rat reductase in a reconstituted system containing mouse cytochrome P-450(2)alpha.

Neonatal phenobarbital-induced defects in age- and sex-specific growth hormone profiles regulating monooxygenases.

Growth hormone was secreted in sexually dimorphic patterns in both 65- and 150-day-old rats (i.e., "on-off" pulsatile for males and "continuous" pulsatile for females), but as a result of a 200-400% increase in pulse levels the mean concentration of hormone in the circulation was about two times as great in the younger animals. Neonatal exposure to phenobarbital at anticonvulsant therapeutic doses for the rat reduced the pulse amplitudes of circulating growth hormone in both the 65- and 150-day-old males but only in the 65-day-old females. As expected, neonatal administration of the barbiturate produced an almost immediate increase in the activities of the hepatic monooxygenases, as measured by hexobarbital metabolism, which declined to noninduction levels after treatment ceased. Contrary to the well-known transient effects of phenobarbital, at around the time of sexual maturity when gender-dependent differences in hepatic monooxygenases appear (males > females), we observed a second "round" of enzyme induction that persisted in both sexes for the remainder of the study (180 days). Because growth hormone is the primary regulator of sex-dependent hepatic monooxygenases, we have proposed that the abnormal plasma growth hormone profiles produced by neonatal phenobarbital are responsible for the permanent induction of hepatic monooxygenases.