Bernard H. Shapiro

Modification of gonadectomy-induced increases in brain monoamine metabolism by steroid hormones in male and female rats

Concentrations of monoamines (dopamine, DA; serotonin, 5-HT) and their major metabolites (homovanillic acid--HVA; dihydroxyphenylacetic acid--DOPAC; 5-hydroxyindolacetic acid--5-HIAA) were measured in selected brain areas of chronically gonadectomized, steroid- or oil-treated male and female rats. Concentrations of DOPAC and HVA were markedly increased in the hypothalamus (male, female), striatum (male, female) and brainstem (male) following gonadectomy, whereas the levels of DA remained unaltered in most of the brain areas examined. Most of the changes were reversed or attenuated by chronic estradiol (EB) substitution. In contrast, chronic treatment with physiological concentrations of testosterone (TP) reduced indexes of DA turnover only in the striatum of ovariectomized (OVX) and brainstem of orchidectomized (ORDX) rats. ORDX-related increases in striatal levels of DOPAC and HVA were not reversed by either EB or TP. ORDX increased the levels of 5-HIAA (hypothalamus, striatum) and decreased those of 5-HT (hypothalamus, hippocampus). These changes were reversed by chronic treatment with either TP or EB. Brain metabolism of 5-HT remained unaltered following OVX. Gonadectomy and chronic steroid replacement therapy appear to alter brain monoamine metabolism in a brain region and sex-dependent manner. Our data demonstrate that gonadectomy-related increases in the activity of brain monoaminergic neurons in both male and female rats was attenuated more effectively with physiological concentrations of estradiol than with testosterone. Insensitivity of monoaminergic neurons in a number of brain areas (e.g., hypothalamus, striatum) to the action of testosterone was evident in both sexes.

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. We have studied sex differences in the expression levels of seven different commonly used housekeeping genes.

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 ≈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. Whereas previous studies identified profound reductions in the pulse amplitudes of the masculine growth hormone profile as the cause for the diminished hormone secretion during aging, pulse heights are not recognized by the cytochromes as regulatory signals. Instead, we have shown that just a nominal secretion of growth hormone during the usual growth hormone-devoid interpulse period in the masculine episodic profile can explain the complete repression of male-specific CYP2C11, CYP3A2, and CYP2A2 and induction of female-dependent CYP2C12, CYP2C6, and CYP2A1 observed in senescent male rats.

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 b5, 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 (4 mg), 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.

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. We observed that female-specific CYP2C12 as well as female-predominant CYP2A1, 3A1, and 2C6 could be induced by growth hormone concentrations equal to as little as 6, 0.6, and 0.06% of the mean circulating hormone profile found in normal females. Irrespective of sex, all four female-dependent isoforms were far more responsive to the continuous growth hormone profile than the episodic pattern. Lastly, female-derived hepatocytes in general responded with strikingly greater induction levels of P450s than male hepatocytes exposed to the same growth hormone profiles. The present findings demonstrate intrinsic, irreversible sex differences in growth hormone-regulated female-dependent P450s.

Androgenic repression of hexobarbitone metabolism and action in Crl:CD‐1 (ICR)BR mice

1 Mice of the Crl:CD‐1 (ICR)BR strain exhibit a sexual dimorphism in hexobarbitone metabolism and action. Compared to females, males have a lower Vmax and a higher Km for hepatic microsomal hexobarbitone hydroxylase. In agreement with the enzyme studies, hexobarbitone‐induced sleeping times were greater for males than for females. 2 Results from experiments measuring hexobarbitone metabolism and action in castrate, testosterone and gonadotropin‐treated mice indicate that the sexual differences in drug metabolism and action found in Crl:CD‐1 (ICR)BR mice are due to the normally repressive effects of testicular androgens on the activities of the hepatic mono‐oxygenases. These findings are in dramatic contrast to studies with rats where it has been shown that androgens induce mono‐oxygenases. Furthermore, in the case of the mouse, changes in the activity of hexobarbitone hydroxylase in response to alterations in androgen levels require weeks, while in the rat, androgenic‐induced changes are apparent within a matter of days.

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. In agreement with in vivo findings, we observed that induction levels of the isoforms were always greatest when the male hepatocytes were exposed to the masculine-like episodic growth hormone profile and least stimulated by the continuous feminine-like hormone profile. When administered alone, dexamethasone consistently increased isoform levels. However, when administered with growth hormone, the glucocorticoid was always antagonistic, suppressing growth hormone induction of CYP2C11, CYP2C13, and CYP2A2. Finally, the P450 isoforms were completely unresponsive to all treatments when the hepatocytes were derived from female rats, supporting earlier findings that expression levels of sexually dimorphic P450 isoforms are inherently irreversible between sexes.

Feminine saccharin preference in the genetically androgen insensitive male rat pseudohermaphrodite

Abstract Saccharin preference in the Stanley-Gumbreck, male rat pseudohermaphrodite is characteristically feminine. The pseudohermaphrodite consumes significantly greater amounts of 0.25,.0.5, and 1.0% saccharin solutions than male littermates. The enhanced saccharin preference of pseudohermaphrodites suggest incomplete or absent masculine imprinting of brain centers regulating fluid consumption. Saccharin preference was unaffected by gonadectomy in pseudohermaphrodites with previous experience in selecting saccharin.

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., postpuberal) above normal activity levels of hepatic multicytochrome P450‐dependent drug metabolizing enzymes. We report that in spite of apparent normal health for the better part of their lives, daily administration of therapeutic‐like doses of phenobarbital to male and female rat pups during the first postpartum week reduced life expectancy by ∼20%. Necropsy at the time of natural death revealed an associated two‐ to threefold increase in the incidence of tumors in barbiturate‐exposed rats of both sexes and a three‐ to fourfold increase in urinary tract pathologies in male rats. At 2 yr of age, in agreement with an overexpression of hepatic CYP2C6 and CYP2C7, both in vitro and in vivo drug metabolism was more rapid in the phenobarbital‐imprinted male and female animals. Moreover, when the senescent rats were rechallenged with a nominal dose of the barbiturate, males and females neonatally exposed to phenobarbital exhibited a dramatic overinduction of multicytochrome P450‐dependent drug metabolizing enzymes as well as an overexpression of individual isoforms of cytochrome P450 implicated in enhanced susceptibility to tumorigenesis. Our findings support the growing realization that many adult diseases have their origins in early life by emphasizing that unlike adults, the new born is “plastic”, and even therapeutic drugs may produce “silent” programming defects that subtly, but irrevocably, jeopardize life‐long well‐being.

Sexual dimorphism in avian hepatic monooxygenases

Adult white Leghorn chickens exhibited a sexual dimorphism in hepatic microsomal monooxygenases determined from the concentrations of total cytochromes P450 and b5, and the metabolism of drug (hexobarbital, coumarin and ethoxyresorufin) and steroid (androstenedione and testosterone) substrates that were 2- to 4-fold greater in roosters than in hens. Caponizing at 6 weeks of age reduced the activities of the monooxygenases to levels comparable to those found in intact hens. In spite of the fact that testosterone replacement maximally stimulated comb growth in the capons and elevated (i.e. masculinized) hepatic monooxygenase activities in the hens to male-like levels, androgen replacement was ineffective in increasing the subnormal enzyme levels in the capons. While the failure of testosterone administration to restore monooxygenase levels in the capons may be explained by the immaturity of the birds at orchiectomy, the present results demonstrate, that like some mammals, birds may display gender differences in hepatic monooxygenases that are regulated by the testes.