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D M Sheehan

Publications and source records attributed to D M Sheehan.

At least 73 records · Page 4Linked to original sources
PubMed1988

Estrogen- and antiestrogen-induced ornithine decarboxylase activity and uterine growth in the rat.

The estrogen antagonists tamoxifen and monohydroxytamoxifen are also classified as partial estrogen agonists. In infantile rats, estradiol induced a single peak of uterine ODC activity at 6h following injection regardless of the extent of induction by various estradiol doses. By contrast, the timing of the ODC activity peak induced by tamoxifen and monohydroxytamoxifen was highly dependent upon the dosing conditions and was delayed to 18 h at lower tamoxifen doses. In immature rats, tamoxifen and monohydroxytamoxifen induced two peaks of uterine ODC activity resembling those induced by estradiol. Both ODC activity peaks were delayed by 9 h, without decreases in peak heights, by a 50-fold tamoxifen dose reduction. In all experiments the initial appearance of antiestrogen- and estradiol-induced ODC activity corresponded to initial uterine wet weight gain regardless of dosing condition. Thus, when dose-related temporal shifts are taken into account, tamoxifen and monohydroxytamoxifen are complete agonists with respect to induction of uterine weight gain and ODC activity.

Animals↗
PubMed1988

Uterine abnormalities in rats exposed neonatally to diethylstilbestrol, ethynylestradiol, or clomiphene citrate.

The toxicity of the synthetic estrogens diethylstilbestrol (DES), and ethynylestradiol (EE), and the antiestrogen clomiphene citrate (CC) was evaluated by assessing postnatal uterine growth and development prior to the onset of puberty in the rat. Both DES and EE, administered during the neonatal period (postnatal days 1-5), initially increased uterine weight and luminal epithelium hypertrophy. However, uterine weight declined in both DES- and EE-treated animals and fell below controls beyond day 11. Luminal epithelium stimulation generally paralleled uterine weight changes. Precocious development of uterine glands occurred after estrogenization (compared to untreated controls), but subsequently gland numbers were approximately 60% of control levels. Neonatal CC exposure induced only slight uterine weight gain but caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis. Luminal epithelium hypertrophy appears to be a useful measure of antiestrogen activity. These data demonstrate the toxicity of DES and EE as assessed by altered prepubertal uterine gland development. Additionally, the inhibition of uterine gland genesis after neonatal CC exposure occurs in conjunction with prolonged luminal epithelium hypertrophy.

Animals↗
PubMed1987

Cell cycle-specific effects of sodium arsenite and hyperthermic exposure on incorporation of radioactive leucine and phosphate by stress proteins from mouse lymphoma cell nuclei.

Cultured mouse lymphoma cells incorporated [3H]leucine and [32P]phosphate into nuclear stress proteins within 3 h after exposure to either elevated temperature (45 degrees C) or sodium arsenite. Radiolabeled proteins were detected by autoradiography after two-dimensional polyacrylamide gel electrophoresis. To determine the cell cycle stage specificity of labeling, nuclei were isolated and sorted into two cell cycle phases using a fluorescent activated cell sorter. After either heat shock or sodium arsenite treatment, the majority of [3H]leucine incorporation into stress proteins occurred during the G0 + G1 phase with minimal labeling in the G2 phase. On the other hand, 32P labeling of stress proteins occurred in both the G0 + G1 and G2 phases after exposure to sodium arsenite, while incorporation of 32P was limited after heat stress. Following sodium arsenite treatment, a distinct set of four stress proteins (80-84 kDa) was detected with [3H]leucine only in G0 + G1 phase, but with [32P]phosphate these stress proteins were labeled in both G0 + G1 and G2. There was differential [32P]phosphate labeling between proteins of the 80-84 kDa set during cell cycling. Individual proteins of this set were isolated from gel plugs after sodium arsenite or heat-shock treatment. Coelectrophoresis of proteins from the two treatment groups showed that they had similar electrophoretic mobilities. All four proteins of the 80-84 kDa set (sodium arsenite induced) possessed similar polypeptide maps after digestion with V8 protease. Cytofluorometric analysis demonstrated a reduction in the number of nuclei in both S and G2 phases of the cell cycle two h after heat shock, but not following sodium arsenite treatment. However, there was a significant depression in the number of nuclei in S and G2 4 h after exposure to sodium arsenite and very modest labeling with 32P of stress proteins was observed at this time.

Animals↗
PubMed1987

Dissociation of estrogen-induced uterine growth and ornithine decarboxylase activity in the postnatal rat.

Estrogens are teratogens and developmental carcinogens in several species. We have used uterine growth to quantitate the potency of three estrogens [estradiol (E2), diethylstilbestrol (DES), ethynylestradiol (EE2)] during four postnatal periods (days 1-5, 10-14, 20-24, and 60-64) in the rat. Alphafetoprotein (AFP), present at high levels in neonatal serum, is thought to regulate estrogen bioavailability. Association constants for DES and EE2 were 2.7% and 4.9% of that for E2 binding to AFP, determined in a batch Sephadex equilibrium binding assay. On days 1-5, DES and EE2 were about 80-fold more potent than E2 in increasing uterine weight. As AFP levels fell, potency differences between E2 and the synthetic estrogens decreased. In the adult, which essentially lacks AFP, the three estrogens were nearly equipotent. These data are consistent with AFP regulation of estrogen potency. On days 10-14, uterine growth was less sensitive than at other ages to all three estrogens, perhaps related to uterine differentiation and/or the high endogenous serum E2 levels reported at this age. However, when we examined another uterine estrogen response, ornithine decarboxylase (ODC) induction at 6 h following estrogen injection, all three hormones were about equipotent in both neonatal and adult animals. This apparently AFP-independent event shows dissociation of ODC induction and uterine growth, which could be due to separate mechanisms for hormone entry to target tissue or subsequent intracellular events.

Aging↗
PubMed1987

The in vitro embryotoxicity of 5-fluorouracil in rat embryos.

The fluorinated pyrimidine 5-fluorouracil (5-FU) is an effective chemotherapeutic agent that is teratogenic in a number of species. The mechanism for the embryopathic effect of the drug is unknown. We examined the effects of this compound on gestation day 10.5 rat embryos cultured for 48 hours in a rodent whole embryo culture system. Embryos were exposed for 1-4 hours to various doses of 5-FU. Embryolethality was minimal in all treatment groups. The malformation frequency increased with higher doses; within a dose, the malformation frequency increased with longer exposure to the drug. The tail and hindlimb bud were the most commonly affected structures in vitro; tail and leg defects are produced in several species by exposure to the drug in vivo. The embryopathic drug concentration in the culture media (2-8 micrograms/ml) is similar to the plasma level of 2-17 micrograms/ml, which is associated with embryopathy in vivo. Results from this study suggest that the whole embryo culture system is an appropriate model for developmental toxicity studies of 5-FU.

Abnormalities, Drug-Induced↗
PubMed1986

Identification of uterine nuclear type II estrogen binding sites in estrogen treated rats.

Uterine nuclear fractions from estrogen-treated rats contain both the estrogen receptor and a lower affinity estrogen binding site (type II site). In Scatchard plots of estrogen binding, two types of curves are seen. The hook-shaped form is composed of a linear component (the estrogen receptor) and a convex component (the type II site) while the curvilinear form is resolvable into two linear binding species (the estrogen receptor and a secondary site). To clarify the relationship between the two forms, we examined the curvilinear form from immature rats injected for 4 days with estradiol (E2) for type II site properties. Like the hook-shaped type II, this form could be detected in a nuclear exchange assay at both 37 and 4 degrees C, but at neither temperature in the presence of reducing agent. Additionally, the steroid specificity of the curvilinear form was identical to the hook-shaped form. The hook-shaped form was found in both immature and ovariectomized adult rats implanted for 6 days with an E2-releasing Silastic capsule to provide pharmacological E2 levels. When uteri from implanted animals displaying the hook-shaped form were mixed in various ratios with uteri lacking type II sites, the curvilinear form was produced. Animals given an E2 implant for 3 days, followed by a 3 day hormone-free period showed a curvilinear form. In vivo E2 dose-response experiments showed the curvilinear form at low E2 doses and the hook-shaped form at the high dose and in implanted animals. We conclude that curvilinear Scatchard plots result from the presence of authentic type II at lower concentrations than those giving rise to the hook-shaped form.

Animals↗
PubMed1986

Insulin stimulation of ornithine decarboxylase activity in developing rat heart.

Ornithine decarboxylase (ODC; EC 4.1.1.17) is an important enzyme in the synthesis of polyamines and is associated with growth and differentiation. Insulin stimulation of cardiac ODC has been proposed as a marker of the functional completion of the sympathetic pathway to the rat heart. However, earlier studies, using subsaturating substrate concentrations and a single time point measurement after insulin treatment, have been inconsistent concerning the postnatal age at which significant insulin stimulation of ODC occurs. The present study, using a validated near-saturating substrate assay, examines more thoroughly early neonatal insulin induction of cardiac ODC with respect to both the magnitude and the time course of response. Insulin (20 IU/kg s.c.) significantly increased ODC activity at several time points at each postnatal age measured (days 2, 5, 8, 15 and 22), with maximum ODC activity occurring by 2.5 to 3 hr after insulin injection at all ages. Insulin-stimulated ODC activity was increased over control levels by 86, 84, 87, 150 and 127% on days 2, 5, 8, 15 and 22, respectively. These results demonstrate that age is not a variable in the time of peak insulin stimulation of ODC activity and, in contrast to earlier reports, show that significant insulin induction of cardiac ODC activity occurs reliably across ages in the early postnatal period. The inconsistency of earlier studies may be due to a number of factors, including the use of subsaturating enzyme assays only, known to be subject to several types of error.

Age Factors↗
PubMed1985

The postnatal ontogeny of rat uterine glands and age-related effects of 17 beta-estradiol.

In the uterus of the newborn rat, only the luminal epithelium is differentiated. Differentiation of musculature and glandular epithelium occurs postnatally, the latter originating as invaginations of the luminal epithelium into the stroma. Using unambiguous criteria for quantification of uterine glands, we find that uterine glands first appear on postnatal day 9 after which the increase in the number of glands is rapid and synchronous, with approximately 4.4 glands per uterine section reached by day 15. Between days 15 and 35, the number of glands per uterine section varied in a cyclic manner with an amplitude of approximately one gland per uterine section and a period of 6-7 days. Although exogenous 17 beta-estradiol (E2) administered on postnatal days 1-5 induced slight premature gland genesis, the number of glands per uterine section was approximately 30% lower between days 15-26 compared to untreated animals. Administration of E2 during the period of normal gland genesis (days 10-14) induced a dose-related delay in the onset of appearance of glands. After this, gland genesis proceeded at a normal rate; however, the maximum levels reached were again generally below those observed in untreated controls. E2 administered after uterine glands were established (days 20-24) induced a small increase in gland number compared to controls. E2 also induced temporary hypertrophy, hyperplasia, and cellular degeneration in the luminal epithelium during each of the dosing periods without corresponding changes in the stroma or myometrium. These data demonstrate that uterine gland genesis occurs between postnatal days 9-15 and that exogenous estrogen can alter, in an age-specific manner, both uterine gland genesis and the number of glands per uterine section.

Age Factors↗
PubMed1985

Inhibition of rat uterine gland genesis by tamoxifen.

We have previously shown that rat uterine gland genesis occurs rapidly and synchronously between postnatal days 9-15. Exogenous estrogens either stimulate or inhibit gland genesis depending on dose and age at administration. We therefore examined the developmental effects of the triphenylethylene antiestrogen tamoxifen, which exhibits both estrogen agonist and antagonist properties, in the postnatal rat uterus. Tamoxifen administered sc in oil on postnatal days 1-5 or days 10-14 caused dose-related inhibition of uterine gland genesis which persisted to day 26 or day 60, respectively. Tamoxifen administered on postnatal days 20-24, which is after the age of normal gland genesis, did not alter the number of preexisting glands. A 24-h exposure to tamoxifen inhibited 17 beta-estradiol (E2)-induced ornithine decarboxylase (ODC) activity measured 6 h after E2 administration in 14-day-old rats. Treatment with tamoxifen before or during the period of gland genesis also reduced uterine responsiveness to a single dose of E2 as measured by both uterine weight gain (after a 24-h exposure on days 14, 19, 22, and 26) and the pattern of E2-induced ODC activity in 26-day-old rats. Control rats respond to E2 with peaks of ODC activity at 6 and 18 h after administration. Treatment with tamoxifen on either postnatal days 1-5 or 10-14 reduced the 18-h peak to approximately half of controls but did not affect the 6-h E2-induced ODC peak. Analysis of both nuclear and translocatable cytosol estrogen receptor in uteri from 26-day-old rats indicate that neither the dissociation constant (KD) nor the number of binding sites was affected by tamoxifen treatment on postnatal days 1-5 or 10-14.

Age Factors↗
PubMed1984

Eosinophils as the source of uterine nuclear type II estrogen binding sites.

Rat uterine nuclei have been reported to contain two types of estrogen binding sites (I and II). Type I is the classical high affinity, low capacity translocatable estrogen receptor, while type II is a lower affinity non-translocatable binding site. Correlation of data on hormonal specificity of induction and inhibition, tissue and cellular localization, and ontogenic appearance for type II binding sites and eosinophils suggested the hypothesis that these binding sites are associated with eosinophils. Although type II sites are reported to be highly correlated with uterine growth, premature growth can be elicited in newborn rats by five daily estradiol injections without the appearance of type II sites. Under these conditions, no eosinophils, as measured by peroxidase activity, are found. However, multiple estradiol injections on postnatal days 6-10 or 10-14 increased the levels of both type II sites and eosinophils. Eosinophils, purified from a peritoneal lavage, were found to contain a low affinity binding site with characteristics similar to type II binding sites. Short term estrogen-treated rat uteri contain only type I nuclear receptor and low levels of eosinophils while long term estrogen-treated rat uteri contain both type I and type II nuclear binding sites as well as approximately 6 X 10(5) eosinophils/uterus. The addition of this number of purified eosinophils to short term treated uteri resulted in saturation curves and Scatchard plots identical to those seen in long term treated uteri. These data indicate that the type II nuclear binding site is transported into the uterus with eosinophils following estrogen treatment.

Animals↗
PubMed1984

Estrogenic activity of zearalenone and zearalanol in the neonatal rat uterus.

Fusarium sp. contaminated feedstuffs elicit adverse estrogenic effects in several commercially important animal species via the mycotoxin zearalenone. An estrogenically active synthetic derivative, zearalanol, is used as an anabolic agent in cattle. Since estrogens can irreversibly alter target tissue development, we investigated the estrogenic activity of these compounds in the neonatal rat uterus. Both induced dose-dependent premature uterine growth when injected daily on postnatal days 1-5 (ED50 = 1.3 mg/kg BW). Nuclear estrogen receptor levels dramatically increased 1 hour after either a single injection on day 5 or after five daily injections. In 5-day-old animals, the translocated nuclear receptor was characterized as a single class of binding sites with a dissociation constant (KD) for estradiol (E2) of 1 nM. At 15 days, zearalanol-treated animals showed greater uterine nuclear receptor retention than zearalenone-treated animals. In 5-day-old animals, single mycotoxin doses induced five fold elevations of ornithine decarboxylase (ODC) at 6 hours. Unlike the growth response, ODC dose-response studies showed zearalanol to be about 20-fold more effective than zearalenone. Time course studies revealed that a low dose of zearalenone, but not of zearalanol, resulted in a shift in peak activity from 6 to 8 hours. These data suggest that metabolism of zearalenone may be important in short-term pharmacodynamics. In a competitive binding assay, neither compound competed [3H]E2 from the E2 binding site on alpha-fetoprotein. We conclude that the uterine growth response and ODC induction demonstrate the neonatal estrogenic action of these mycotoxins, apparently mediated via the estrogen receptor. The greater effectiveness of zearalanol in inducing ODC may be related to nuclear retention and/or zearalenone metabolism.

Animals↗
PubMed1984

Spleen cell phosphorylation of salt soluble nuclear protein from isoproterenol treated and sorted nuclei.

The effect of isoproterenol (IPR) on phosphorylation of acidic nuclear proteins was investigated by two-dimensional gel autoradiography. Mouse spleen cells stimulated to divide by the mitogen concanavalin A (Con A) were separated according to cell cycle stage by flow microfluorometric technique. Exposure of cells for 48 h to 4 micrograms IPR/ml culture medium produced no significant change in the proportion of S and G2 phase cells, while a cumulative dose of 8 micrograms IPR/ml caused a significant repression in DNA synthesis and a reduction in the number of nuclei in G2 + M phase. Four micrograms IPR/ml stimulated the greatest amount of G0 + G1 phosphorylation of nuclear protein. Several proteins from G0 + G1 and S nuclei incorporated 32P after Con A + IPR administration, and one protein from S phase nuclei revealed intensified labeling at the 8 micrograms cumulative IPR dose but not at the 4 micrograms dose. The isolated proteins (W, X, Y, and Z) were reassociated with homologous DNA, centrifuged in a sucrose gradient and shown to co-sediment with DNA. S phase nuclear protein X-S, which was found to be a mixture of proteins (X0 and X1), was the only exception. One component of X-S, X0 bound to DNA, while component X1 failed to bind. Chymotryptic and V8 protease digests of all isolated proteins were made and analyzed by autoradiography. Proteins X0 and X1, recovered from the sucrose gradient, possessed dissimilar fragment patterns. It is concluded that protein X-S is composed of two proteins (X0 and X1), one of which (X1) appears during S phase during the 8 micrograms IPR induced nuclear repression.

Animals↗
PubMed1983

The postnatal ontogeny of rat uterine ornithine decarboxylase: acquisition of a second peak of estrogen-induced enzyme activity.

Uterine ornithine decarboxylase (ODC) activity is reported to increase after estrogen administration to fetal, neonatal, immature, and adult rats, suggesting that it may be a useful marker in studies of the development of estrogen responsiveness. Standard conditions were validated for enzyme assay of uterine cytosols from 5-day-old rats, and it was demonstrated that full activity was retained after freezing cytosol in liquid N2. Maximal activity, obtained 6 h after the injection of 10 micrograms estradiol (E2) to 5-day-old rats, was also elicited by the same dose of mestranol, ethynylestradiol, diethylstilbestrol, or moxestrol. Progesterone, testosterone, and low doses of the antiestrogens clomiphene and tamoxifen failed to alter background ODC levels, while high antiestrogen doses induced small increases in enzyme activity. The glucocorticoid prednisolone lowered ODC activity. Dose-response curves established that E2 was more effective in increasing adult ODC levels (ED50 = 0.2 micrograms/kg E2) than neonatal ODC levels (ED50 = 2 micrograms/kg E2). Time-course measurements were conducted over 24 h in control and E2-injected animals on postnatal days 5, 10, 14, 20, and 28 and in 60-day-old ovariectomized adults. While an age-dependent decrease in control and 6 h E2-induced ODC levels was observed, there was an unexpected progressive development by day 28 of a second peak of E2-induced ODC at 15-18 h. The 6 h neonatal and 6 and 15-18 h adult ODC peaks had apparent Km values for ornithine near 0.2 mM. The potential origin of the second peak and its relationship to other uterine events are discussed.

Animals↗
PubMed1982

Evidence for estradiol promotion of neoplastic lesions in the rat vagina after initiation with N-methyl-N-nitrosourea.

The hypothesis that estrogens are tumor promoters was tested by tumor induction in the rat vagina. Ovariectomized rats were given a single dose of N-methyl-N-nitrosourea (MNU) by vaginal instillation, followed one week later by long-term release Silastic implants containing estradiol (E2). After 16 months, a significant incidence (4/9) of benign vaginal stromal polyps was found in the MNU-E2 group, but no vaginal polyps were seen in groups given either MNU or E2 alone. A number of non-neoplastic changes were also seen and were due to E2 treatment either with or without MNU. The incidence of stromal polyps and their restriction to animals receiving the initiator-promoter regimen alone suggests that estrogens promote tumorigenesis in the rat vagina.

Animals↗
PubMed1981

Uterine responses to estradiol in the neonatal rat.

Although single doses of estrogens are known to be ineffective in stimulating complete uterine responses in newborn rats, repeated doses elicit toxic responses that become evident in adulthood. In this study, neonates injected daily from birth with 10 micrograms 17 beta-estradiol (E2) demonstrated significant uterine wet weight gain by day 3 and near-maximum growth (230% of control) by day 5. Elevated uterine weight can be maintained by repeated daily injections at least through day 13. Other uterine growth responses after 5 days of E2 (as percent of control) are: dry weight, 163%; DNA content, 193%; protein content, 211%; and nuclear estrogen receptor, 890%. Ornithine decarboxylase activity increased to 520% of control when measured 6 h after a single E2 injection on day 5. Histological examination of uteri from animals treated for 5 days reveals an altered stroma with evidence of circular muscle differentiation, while the lumenal epithelium, which is cuboidal in controls, becomes columnar after E2 injections. These data suggest that estrogens act in fundamentally the same manner in the neonatal uterus as in the adult uterus, although the appearance of the complete response appears to e slower in the neonate. The estrogen-induced precocious development we describe suggests that an estrogen may be involved in the normal postnatal development of uterine estrogen responsiveness. Adult toxicity, resulting from repeated neonatal estrogen dosing, may partly be a consequence of continual hormone action inducing developmentally inappropriate responses.

Aging↗