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Biomedical subjects

B H Shapiro

Publications and source records attributed to B H Shapiro.

At least 37 records · Page 2Linked to original sources
PubMed1995

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.

Aging↗
PubMed1995

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.

Animals↗
PubMed1995

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.

Animals↗
PubMed1994

Subnormal concentrations in the feminine profile of circulating growth hormone enhance expression of female-specific CYP2C12.

When newborn female rats were treated with monosodium glutamate, 4 mg/g body weight, on days 1 and 3 of life, circulating growth hormone concentrations were permanently reduced 75-85% in adulthood, whereas the feminine secretory profile characterized by frequent growth hormone pulses, separated by short-lived, measurable troughs, persisted. Associated with this reduction in growth hormone secretion was a mild obesity and a slight depression in peripubertal body weight. In contrast, expression of growth hormone-dependent, female-specific CYP2C12 was increased by almost 100% when measured at both its protein and mRNA levels. In agreement, this supraphysiological expression of CYP2C12 was reflected at a pharmacologic level by a simultaneous elevation in in vitro and in vivo hexobarbital metabolism. When growth hormone secretion was pulsatile (i.e. masculine) or was eliminated from the circulation (i.e. hypophysectomy), hepatic CYP2C12 protein and mRNA were undetectable. The present findings suggest that the normal levels of plasma growth hormone found in female rats are not necessarily optimum for the expression of female-specific CYP2C12.

Animals↗
PubMed1994

Effects of neonatally administered monosodium glutamate on the sexually dimorphic profiles of circulating growth hormone regulating murine hepatic monooxygenases.

Neonatal male and female mice were treated with monosodium glutamate (MSG) at either 2.0 or 4.0 mg/g body weight on alternate days during the first 9 days of life. As adults, mice were catheterized to obtain unstressed, serial blood samples for the determination of ultradian profiles of circulating growth hormone. In addition, monooxygenase levels (i.e. steroid hydroxylases and drug-metabolizing enzymes) were measured in hepatic microsomes. Generally, both doses of MSG produced the same developmental defects. Mice neonatally exposed to the amino acid developed a syndrome characterized by retarded growth, obesity and reduced organ weights. While vehicle-treated mice secreted growth hormone in sexually dimorphic patterns defined by pulse frequency (i.e. F > M), hormone concentrations in plasma samples obtained during 8 continuous hr of serial blood collections from both male and female MSG-treated mice were barely detectable at best, and exhibited no pulsatility. Approximately 15% of the measured monooxygenases were male-predominant, 35% were female-predominant and 50% had no sex differences. The enhanced expression of the hepatic monooxygenases in response to MSG-induced depletion of plasma growth hormone indicates that the hormone basically functions as a suppressor of the murine enzyme system.

Age Factors↗
PubMed1994

Growth hormone-dependent and -independent sexually dimorphic regulation of phenobarbital-induced hepatic cytochromes P450 2B1 and 2B2.

Sexually dimorphic regulation of phenobarbital-induced cytochromes P450 2B1 and 2B2 (collectively referred to as P450 2B) as well as P450 2B-dependent monooxygenase activities was studied in multi-hormone-depleted hypophysectomized rats and in growth hormone (GH)-deficient monosodium glutamate (MSG)-treated rats. Our results indicate that endogenous GH suppresses phenobarbital induction of P450 2B and that the feminine pattern of continuous GH secretion is more suppressive than the masculine profile of episodic secretion. Moreover, we have found that it is the height of the GH pulse, and not necessarily its frequency nor the interpulse trough periods, that signals the suppressive effects of GH on P450 2B expression in male rats. Last, irrespective of the presence or absence of circulating GH, the magnitude of phenobarbital induction of P450 2B and associated monooxygenases was consistently lower in female rats, suggesting the presence of some degree of sex-dependent, but GH-independent regulation.

Animals↗
PubMed1994

Testicular regulation of sexual dimorphisms in the ultradian profiles of circulating growth hormone in the chicken.

Ultradian patterns in plasma growth hormone (GH) concentrations were determined in adult white Leghorn roosters, hens and capons. Serial blood samples were collected every 15 min over 8 h through surgically placed chronic indwelling right atrial catheters and assayed for GH content by a GH content by a homologous radioimmunoassay. In both sexes, GH levels fluctuated episodically, with peak and interpeak periods each lasting about 45 min in both roosters and hens. However, GH concentrations in the peaks and nadirs were 2.5-3.5 times greater in the plasma GH profiles of roosters as compared to hens, which resulted in roosters having higher mean GH concentrations. Caponizing completely feminized the episodic GH secretory profile. In contrast to chickens, the common sexually dimorphic feature in secretory GH profiles of mammals is the enhanced peak frequencing found in females.

Activity Cycles↗
PubMed1994

Over-expression of CYP2C11, the major male-specific form of hepatic cytochrome P450, in the presence of nominal pulses of circulating growth hormone in adult male rats neonatally exposed to low levels of monosodium glutamate.

Male rat pups were injected with monosodium glutamate (MSG), 4 mg/g b.wt., on either days 1 and 3 or days 1, 3, 5, 7 and 9 of life. Plasma samples obtained during 8.5 continuous hr of serial blood collections from adults, neonatally treated with five doses of MSG, contained no detectable concentrations of growth hormone. In addition, the livers of these animals contained no measurable expression levels of the major male-specific form of cytochrome P450 (CYP2C11) mRNA or its protein. In contrast, adult male rats treated with only two neonatal injections of MSG exhibited typical masculine profiles of growth hormone release, except that the amplitudes of the ultradian pulses were reduced to about 10 to 20% of normal male levels. Otherwise, like control males the peaks occurred about every 3.5 to 4 hr and the intervening 3-hr troughs had undetectable concentrations of growth hormone. Moreover, the livers of these twice-injected MSG-treated rats over-expressed CYP2C11 as indicated by a 200% increase in CYP2C11 mRNA and a concomitant 30 to 40% increase in CYP2C11 protein and its catalytic activities. The effectiveness of minipulses of growth hormone, mimicking the pattern found in MSG-treated animals, to induce over-expression of hepatic CYP2C11 in hypophysectomized rats, suggests that the supraexpression of CYP2C11 found in the MSG-treated animals may be explained, in part, by the abnormal profiles of circulating growth hormone induced by neonatal exposure to the amino acid. In conclusion, depending upon neonatal exposure levels, MSG can produce permanent, yet very different defects in the adult male patterns of growth hormone secretion and growth hormone-dependent CYP2C11 expression.

Animals↗
PubMed1993

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.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1993

Irreversible suppression of growth hormone-dependent cytochrome P450 2C11 in adult rats neonatally treated with monosodium glutamate.

Neonatal exposure to monosodium glutamate (MSG) permanently blocks growth hormone (GH) secretion, which results in the development of a well-defined syndrome characterized by stunted body growth, obesity and impaired drug metabolism. We have found that restoration of the normal masculine circulating profile of GH (i.e., six daily pulses) by use of an external pumping apparatus is ineffective in restoring the normal expression of hepatic cytochrome P450 2C11, a major GH-dependent drug and steroid metabolizing enzyme that is eliminated by MSG treatment. Moreover, administering GH at two, four or seven plasma pulses per day with amplitudes ranging from physiologic to 7 times normal were similarly ineffective in restoring the expression (at both an activity and mRNA level) of the cytochrome. Additionally, multicytochrome P450-dependent hexobarbital hydroxylase was also unresponsive to GH administration in the MSG-treated rats. Because GH replacement was unable to correct the enzyme defects, our results suggest that the developmental abnormalities produced by neonatal MSG are not simply a result of a GH deficiency per se, but are due to an irreversible insensitivity of the target cell to GH.

Animals↗
PubMed1992

Changes in the expression of cytochrome P450c17 associated with ovarian cystic follicles. An immunocytochemical and enzymatic analysis of porcine ovaries.

The steroidogenic activity of normal preovulatory and cystic follicles, and corpora lutea of porcine ovaries was investigated by immunocytochemical and radioenzymatic techniques. Using a specific antibody to porcine cytochrome P450c17, immunocytochemical staining was specifically localized in the theca interna layer of normal follicles and undetectable in the granulosa layer. The theca interna layers of non-luteinized cystic follicles were immunoreactive while those of luteinized follicles were not. Corpora lutea cells were essentially negative. The 3 beta-hydroxysteroid dehydrogenase/delta 5-delta 4 isomerase activity was similar in luteinized cystic follicular and corpora lutea tissues, which had 8 times higher activity than found in normal preovulatory follicles. The formation of either corpora lutea or luteinized cysts led to a profound decline (12- to 15-fold) in 17 alpha-hydroxylase and 17,20 lyase activities compared to normal preovulatory follicles. In agreement with these enzyme findings, radioimmunoassays revealed very high levels of progesterone with nearly undetectable levels of androgens in the luteinized cysts. These studies demonstrate the functional similarities between cells of luteinized cysts and those of normal corpora lutea and suggest a pathology associated suppression of P450c17 expression in porcine cystic follicles.

17-alpha-Hydroxyprogesterone↗
PubMed1991

Interpulse interval in circulating growth hormone patterns regulates sexually dimorphic expression of hepatic cytochrome P450.

Plasma growth hormone (GH) profiles are sexually differentiated in many species and regulate the sex-dependence of peripubescent growth rates and liver function, including steroid hydroxylase cytochrome P450 expression, by mechanisms that are poorly understood. By use of an external pump to deliver to hypophysectomized rats pulses of rat GH of varying frequency and amplitude, a critical element for liver discrimination between male and female GH patterns was identified. Liver expression of the male-specific steroid 2 alpha (or 16 alpha)-hydroxylase P450, designated CYP2C11, was stimulated by GH at both physiological and nonphysiological pulse amplitudes, durations, and frequencies, provided that an interpulse interval of no detectable GH was maintained for at least 2.5 hr. This finding suggests that hepatocytes undergo an obligatory recovery period after stimulation by a GH pulse. This period may be required to reset a GH-activated intracellular signaling pathway or may relate to the short-term absence of GH receptors at the hepatocyte surface after a cycle of GH binding and receptor internalization. These requirements were distinguished from those necessary for the stimulation by GH of normal male growth rates in hypophysectomized rats, indicating that different GH responses and, perhaps, different GH-responsive tissues recognize distinct signaling elements in the sexually dimorphic patterns of circulating GH.

Activity Cycles↗
PubMed1991

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.

Animals↗
PubMed1991

Sex- and dose-dependent effects of neonatally administered aspartate on the ultradian patterns of circulating growth hormone regulating hexobarbital metabolism and action.

Rats, neonatally treated with monosodium aspartate (MSA), exhibited developmental defects through adulthood that were characterized by stunted growth, obesity, and reduced size of the liver, kidney, adrenals, and pituitary. Adult male and female rats treated with 4 mg of MSA had no detectable plasma growth hormone as determined from serial blood samples taken every 15 min for 8 consecutive hr. Associated with this loss of circulating growth hormone in the males was a dramatic decline in in vivo and in vitro hexobarbital metabolism and hepatic cytochrome P450 to female levels. The loss of plasma growth hormone in the females had no effect on the already low levels of hepatic monooxygenases. At 2 mg/g body weight, MSA produced both sex- and dose-dependent effects that were far more subtle than the full-blown obesity and growth retardation associated with the larger 4-mg dose. While the mean concentration of circulating growth hormone was reduced 70 to 90% in 2-mg-MSA-treated rats, the sexually dimorphic, ultradian patterns of growth hormone secretion were undisturbed. Affected males continued to secrete a pulse of growth hormone every 3 hr, albeit at greatly reduced amplitudes, interposed by normally undetectable baselines. Similarly, 2-mg-MSA-treated females had greatly reduced mean levels of plasma growth hormone, but with the usual secretion of multiple pulses never dropping to baseline. Surprisingly, the sex-dependent, hepatic monooxygenases, which are normally regulated by the ultradian secretions of growth hormone, were unaffected by the 2-mg MSA treatment. Our results suggest that while an ultradian pulse of circulating growth hormone is necessary for the characteristically male profile of hepatic monooxygenases, neither the amplitude of the secretory peaks nor their total growth hormone content is critical.

Animals↗
PubMed1991

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.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1991

Developmental levels and androgen responsiveness of hepatic mono-oxygenases of male rats perinatally exposed to maternally administered cimetidine.

Cimetidine, a widely prescribed, potent histamine H2-receptor antagonist, is unrelatedly a mild antiandrogen. Since perinatal androgens are essential for normal masculine imprinting, we wanted to determine if maternally administered cimetidine interfered with the development of the male offspring's sex-dependent, hepatic drug-metabolizing enzymes. Cimetidine was administered in the mothers' drinking water at several levels reflecting both human and rat therapeutic-like doses from day 17 of gestation through day 7 of lactation. With the exception of a 30% decline in the Vmax for hexobarbital hydroxylase during adulthood, the developmental profile of microsomal drug-metabolizing enzymes, when measured at 1, 4 and 18 weeks of age, was generally unaffected by perinatal exposure to cimetidine. Furthermore, the levels of various components of the hepatic mono-oxygenase system (i.e. heme, cytochromes P-450 and b5, hexobarbital hydroxylase and p-chloro-N-methylaniline demethylase) following orchidectomy and androgen replacement at physiologic and inductive concentrations, were basically the same in control and cimetidine-exposed rats.

Aminopyrine N-Demethylase↗
PubMed1991

Renaturalizing the sexually dimorphic profiles of circulating growth hormone in hypophysectomized rats.

Hypophysectomy resulted in a total elimination of measurable circulating growth hormone with an associated loss of body weight gain. The typical sexually dimorphic plasma growth hormone patterns: pulsatile profiles in male rats and tonic-like secretion in female rats, were lost. The male- and female-dependent profiles of plasma growth hormone, monitored from serial blood collections, were restored by administering the hormone through a single electrically controlled external pump attached to an indwelling catheter, and by implanting osmotic pumps intraperitoneally, respectively. Restoring the natural patterns of plasma growth hormone in animals devoid of pituitaries, re-initiated body growth. However, the body weight gains in both sexes of hypophysectomized rats were much greater when rat growth hormone was introduced to the animals in a masculine (pulsatile) pattern that appeared to be independent of pulse frequency, rather than in a continuous feminine profile. Subcutaneous injections, the most commonly reported method of administration, produced low-amplitude, long-lasting plasma peaks that were not as effective as pulse infusion in restoring growth. The procedure allows manipulation of the hormone profile (i.e. number of pulses/day, pulse amplitude, and through duration in the pulsatile pattern, and plasma concentration in the tonic pattern) in order to identify, and thus study the presumed salient components of the pattern regulating growth hormone responses.

Animals↗