Early expression of requisite developmental growth hormone imprinted cytochromes P450 and dependent transcription factors.

The sexually dimorphic expression of cytochromes P450 (CYP) drug metabolizing enzymes has been reported in all species examined. These sex differences are initially expressed during puberty and are solely regulated by sex differences in the circulating growth hormone (GH) profiles. Once established, however, the different male- and female-dependent CYP isoforms are permanent and immutable, suggesting that adult CYP expression requires imprinting.

Feminization imprinted by developmental growth hormone.

Previously, we identified early developmental exposure to growth hormone (GH) as the requisite organizer responsible for programming the masculinization of the hepatic cytochromes P450 (CYP)-dependent drug metabolizing enzymes (Das et al., 2014, 2017). In spite of the generally held dogma that mammalian feminization requires no hormonal imprinting, numerous reports that the sex-dependent regulation and expression of hepatic CYPs in females are permanent and irreversible would suggest otherwise.

Growth hormone: a newly identified developmental organizer.

The sexually dimorphic expression of cytochromes P450 (CYP) drug-metabolizing enzymes has been reported in all species examined. These sex differences are only expressed during adulthood and are solely regulated by sex differences in circulating growth hormone (GH) profiles. Once established, however, the different male- and female-dependent CYP isoform profiles are permanent and immutable, suggesting that adult CYP expression requires imprinting.

Permanent uncoupling of male-specific CYP2C11 transcription/translation by perinatal glutamate.

Perinatal exposure of rats and mice to the typically reported 4mg/g bd wt dose of monosodium glutamate (MSG) results in a complete block in GH secretion as well as obesity, growth retardation and a profound suppression of several cytochrome P450s, including CYP2C11, the predominant male-specific isoform--all irreversible effects. In contrast, we have found that a lower dose of the food additive, 2mg/g bd wt on alternate days for the first 9days of life results in a transient neonatal depletion of plasma GH, a subsequent permanent overexpression of CYP2C11 as well as subnormal (mini) GH pulse amplitudes in an otherwise normal adult masculine episodic GH profile. The overexpressed CYP2C11 was characterized by a 250% increase in mRNA, but only a 40 to 50% increase in CYP2C11 protein and its catalytic activity.

Irreversible perinatal imprinting of adult expression of the principal sex-dependent drug-metabolizing enzyme CYP2C11.

We proposed to determine whether, like other sexual dimorphisms, drug metabolism is permanently imprinted by perinatal hormones, resulting in its irreversible sex-dependent expression. We treated newborn male rats with monosodium glutamate (MSG), a total growth hormone (GH) blocker, and, using cultured hepatocytes, examined expression of adult CYP2C11, the predominant cytochrome-P450 expressed only in males, as well as the signal transduction pathway by which episodic GH solely regulates the isoform's expression. In addition, adolescent hypophysectomized (hypox) male rats served as controls in which GH was eliminated after the critical imprinting period.

Growth hormone-independent suppression of growth hormone-dependent female isoforms of cytochrome P450 by the somatostatin analog octreotide.

Octreotide is a potent somatostatin analog therapeutically used to treat several conditions including hyper growth hormone secretion in patients with acromegaly. We infused octreotide into female Sprague Dawley rats every 12h for 6 days at levels considerably greater than typical human therapeutic doses. Resulting circulating growth hormone profiles were characterized by ∼25% reduction in plasma levels, including both pulse and interpulse components, but still contained in an otherwise female-like "continuous" secretory profile.

Noncanonical suppression of GH-dependent isoforms of cytochrome P450 by the somatostatin analog octreotide.

Octreotide is a potent somatostatin analog therapeutically used to treat several conditions including hyper GH secretion in patients with acromegaly. We infused, over 30 s, octreotide into male rats every 12 h for 6 days at levels considerably greater than typical human therapeutic doses. Unexpectedly, resulting circulating GH profile was characterized by pulses of higher amplitudes, longer durations, and greater total content than normal, but still contained an otherwise male-like episodic secretory profiles.

Inherent sex-dependent regulation of human hepatic CYP3A5.

Background And Purpose: Expression of hepatic cytochromes P450 (CYP) in all species examined, including humans, is generally sexually dimorphic. We examined the sex-dependent expression of CYP3A5 and the hormone-regulated molecular mechanism(s) responsible for any dimorphism.

Experimental Approach: CYP3A5 levels as well as nuclear translocation and promoter binding of transcription factors regulating CYP3A5 expression were measured in primary hepatocyte cultures derived from men and women exposed to physiological-like levels of growth hormone alone, dexamethasone alone and the combined regimen.

Extensive sex- and/or hormone-dependent expression of rat housekeeping genes.

Aim: Identify sex- and hormone-independent housekeeping genes in rat liver by using a commercially available quantitative reverse transcription-polymerase chain reaction array designed to measure the expression of 32 rat housekeeping genes.

Results: We found that the levels of five of the genes were sexually dimorphic, 22 genes were overexpressed, and one was underexpressed in multi-hormone-deficient hypophysectomized rats of both sexes. Only three genes fulfilled the stability criteria determined by geNorm and NormFinder as suitable housekeeping genes.

Intrasplenic transplantation of isolated adult rat hepatocytes: sex-reversal and/or suppression of the major constituent isoforms of cytochrome P450.

Adult male and female rat hepatocytes were individually transplanted into the spleens of adult male and female rats. The recipients were euthanized at either eight, sixteen, thirty, or forty-five weeks following transplantation, at which time hepatic and splenic levels of liver-specific rat albumin mRNA as well as sex-dependent transcript levels of CYP2C11, -2C12, -2C7, -2A1, and -3A2-which accounts for > 60% of the total concentration of hepatic constituent cytochrome P450-were determined. Whereas the pre-infused hepatocytes expressed their expected cytochrome P450 sexual dimorphisms (female-specific CYP2C12, male-specific CYP3A2, and female-predominant CYP2A1), their post-transplantational competence now reflected the sexual dimorphisms of the recipient (as observed in the host's liver), which supports the concept that the sex-dependent growth hormone circulating profiles are the determinants regulating the expression levels of hepatic cytochrome P450.

Intrinsic sexually dimorphic expression of the principal human CYP3A4 correlated with suboptimal activation of GH/glucocorticoid-dependent transcriptional pathways in men.

Cytochrome P450 (CYP)3A4 is the principal and most abundant human isoform of CYP responsible for the metabolism of more than 50% of all consumed drugs and innumerable endogenous compounds. Expression of CYP3A4 is sexually dimorphic and regulated by the combined actions of GH and glucocorticoids. In the case of the rat, nearly all of the CYPs are "intrinsically" or "inherently" sexually dimorphic, meaning that the expressed sex differences are permanent and irreversible.

Inherent sexually dimorphic expression of hepatic CYP2C12 correlated with repressed activation of growth hormone-regulated signal transduction in male rats.

Because of its myriad physiologic functions, it is not surprising that the actions of growth hormone (GH) are mediated by recruiting/activating dozens of signaling molecules involved in numerous transduction pathways. The particular signal transduction pathway activated by the hormone is determined by the affected target cell, the sexually dimorphic secretory GH profile (masculine episodic or feminine continuous) to which the cell is exposed, and the individual's sex. In this regard, expression of female-specific CYP2C12, the most abundant cytochrome P450 in female rat liver, is solely regulated by the feminine GH profile.

Sex-dependent expression of CYP2C11 in spleen, thymus and bone marrow regulated by growth hormone.

CYP2C11, the most commonly expressed isoform of cytochrome P450 in male rat liver, was measured in spleen, thymus and bone marrow by quantitative real-time PCR and enhanced Western blotting. CYP2C11 concentrations in the lymphoid tissues were a fraction of that observed in liver, but like the liver, were sexually dimorphic (M>F) with mRNA and protein levels in agreement. Although the response to hypophysectomy varied according to tissue and sex, expression levels of CYP2C11 in all measured tissues remained greater in males.

Attenuated expression of episodic growth hormone-induced CYP2C11 in female rats associated with suboptimal activation of the Jak2/Stat5B and other modulating signaling pathways.

Inherent sex differences in various parameters of growth, musculoskeletal function, metabolism, and cytochrome P450 (P450)-dependent drug metabolism have been reported in rats and humans administered typical intermittent/episodic growth hormone (GH) replacement therapy. Having infused and monitored the identical physiologic masculine (episodic) growth hormone profile to both hypophysectomized male and female rats, we observed that induction levels of hepatic CYP2C11 were 35 to 40% lower in females. Associated with the reduced expression of the P450 isoform in the episodic GH-treated females were dramatically lower activation levels of Janus kinase (Jak2), signal transducers and activators of transcription (Stat5A and 5B) as well as 50% less binding of Stat5B to the CYP2C11 promoter.

Effect of perinatal low protein diets on the ontogeny of select hepatic cytochrome p450 enzymes and cytochrome p450 reductase in the rat.

In the present study, we administered two low protein diets (LPDs) to rats during pregnancy and lactation and determined their effect on the ontogeny of select hepatic cytochrome P450 (P450) isoforms in their offspring. The L93 and LM76 LPDs were derived from the American Society of Nutrition recommended AIN93G and a modified version of the AIN76A purified control diets, respectively. The LPDs contained 8% crude protein in the form of casein, whereas the purified control diets contained 19% casein.

A molecular basis for the sexually dimorphic response to growth hormone.

Once reserved solely for the treatment of short stature, the now readily available recombinant GH has expanded the use of the hormone to include the treatment of cardiovascular, renal, muscular, skeletal, immunological, psychosocial, and metabolic abnormalities associated with GH deficiency. There are also proposals for the widespread use of the hormone to ameliorate or reverse aging. However, this extensive use of GH has revealed intrinsic sexual dimorphisms in which females are considerably less responsive to the therapeutic regimen than are males.

Two low protein diets differentially affect food consumption and reproductive performance in pregnant and lactating rats and long-term growth in their offspring.

We fed 2 low protein diets (LPD) to rats during pregnancy and lactation, and compared food intake and reproductive performance in the dams, and long-term growth in their offspring. The L93 and LM76 LPDs were derived from the American Society of Nutrition's recommended AIN93G and a modified version of the AIN76A purified control diets, respectively. The LPDs contained 8% crude protein in the form of casein and differed in their fat and carbohydrate sources.

Sex-dependent expression of seven housekeeping genes in rat liver.

Background And Aim: Sexual differences in the transcript levels of various genes including the hepatic isoforms of cytochrome P450 have been extensively studied. Expression of these sexual dimorphic genes have been quantified by Northern blotting, nuclear run on assays and reverse transcriptase-polymerase chain reaction (RT-PCR) methods using numerous housekeeping genes to normalize results. Earlier reports apparently assumed that these internal controls were sex-independent.

Inducibility of male-specific isoforms of cytochrome p450 by sex-dependent growth hormone profiles in hepatocyte cultures from male but not female rats.

Although in vivo expression levels of the male-specific hepatic isoforms of cytochrome P450 (P450) (CYP2C11, CYP2C13, CYP2A2, and CYP3A2) are determined by the episodic growth hormone profile secreted by male rats, these isoforms have been completely refractory to growth hormone regulation in hepatocyte culture. By using species-specific rat growth hormone, at subphysiologic in vivo concentrations administered in two daily episodic pulses, we successfully induced CYP2C11 and CYP2A2 to near normal concentrations. Whereas inductive levels of CYP2C13 were subnormal, CYP3A2 was unresponsive to all hormonal treatments, quickly declining to undetectable concentrations.

Sexually dimorphic regulation of hepatic isoforms of human cytochrome p450 by growth hormone.

Sex differences in drug metabolism have been reported in numerous species, including humans. In rats and mice, sex-dependent differences in circulating growth hormone profiles are responsible for the differential expression of multiple sex-dependent isoforms of cytochrome P450, which is the basis for the sexual dimorphism in drug metabolism. In contrast, very little is known about sex differences in human isoforms of cytochrome P450 and their regulation by growth hormone.

Neonatal phenobarbital imprints overexpression of cytochromes P450 with associated increase in tumorigenesis and reduced life span.

Perinatal exposure to phenobarbital produces a range of permanent reproductive, growth, locomoter, and learning dysfunctions in animals as well as humans. In addition, the affected individuals exhibit latently expressed (i.e.

Inadequacy of the Janus kinase 2/signal transducer and activator of transcription signal transduction pathway to mediate episodic growth hormone-dependent regulation of hepatic CYP2C11.

CYP2C11, the most commonly expressed hepatic cytochrome P450 isoform in male rats, is induced by the masculine "episodic" secretory growth hormone profile. A considerable number of reports have indicated that episodic growth hormone effects are mediated by the activation of the Janus kinase 2 (Jak2)/signal transducer and activator of transcription (Stat)5B signal transduction pathway. We observed that restoration of the normal masculine plasma growth hormone pulse in hypophysectomized male rats did indeed rapidly activate (phosphorylate) Jak2, shortly followed by activation and nuclear translocation of Stat5B.

Intrinsic sex differences determine expression of growth hormone-regulated female cytochrome P450s.

The masculine profile of cytochrome P450s found in male liver is determined by the episodic secretion of growth hormone characteristic of males. In turn, the female pattern of P450s observed in female rat liver is regulated by the continuous secretion of growth hormone characteristic of the female. In order to determine if intrinsic and possibly permanent sex differences exist in the response of hepatic P450s to growth hormone regulation, we compared the effects of the episodic and continuous growth hormone profiles on the expression of female-dependent isoforms in cultured hepatocytes isolated from both sexes.

Interpulse growth hormone secretion in the episodic plasma profile causes the sex reversal of cytochrome P450s in senescent male rats.

Humans as well as other mammals experience an aging-related decline in drug metabolism as well as a diminution in growth hormone secretion. In the case of rats, these events are more pronounced in senescent males, whose expression of male-specific isoforms of cytochrome P450, the major drug-metabolizing enzymes and constituting approximately 60-70% of the total cytochrome P450 in male rat liver, is completely suppressed, whereas female-dependent isoforms are remarkably induced to female-like levels. Overlooked in these independently reported studies is the fact that "signals" inherent in the masculine episodic and female continuous growth hormone profiles regulate expression and/or suppression of the dozen or so sex-dependent cytochrome P450 isoforms in rat liver.

Spurious observation of splenic cyp2b1 expression.

Phenobarbital (PB) induction of the CYP2B subfamily was studied in the livers and spleens of male and female rats. Animals were treated with either PB (10 mg/kg) or vehicle for 4 consecutive days. A reverse transcriptase-polymerase chain reaction (RT-PCR), quantitative Northern blotting, Western blotting, and a radioenzymatic assay were used to observe differential levels of CYP2B1 and CYP2B2 mRNAs, proteins, and catalytic activities.