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Biomedical subjects

D M Sheehan

Publications and source records attributed to D M Sheehan.

At least 55 records · Page 3Linked to original sources
PubMed1992

Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). I. Multireplicated dose-response studies in four inbred strains and one outbred stock of mice.

A large-scaled multireplicated developmental toxicity study was conducted in various strains/stocks of mice with the herbicide, 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), by gavage on Gestational Days 6 through 14. The most important attributes of the study design were replicated test groups, a minimum of four dose levels per replicate, use of multiple stocks/strains of animals to obtain an estimate of the range in sensitivities due to genotype, complete pathological evaluation of maternal animals, and histopathological as well as teratological evaluation of the fetuses. Developmental toxicity was observed at doses below those producing discernible or measurable maternal toxicity. Regression and/or probit analyses were conducted to determine whether a dose-response relationship existed. Reduced fetal weight and increased incidence of cleft palate and embryolethality were the most significant prenatal effects of 2,4,5-T exposure observed in this study. Each strain/stock exhibited a dose-related decrease in fetal weight with the CD-1 mice having the steepest slope and the A/J mice having the shallowest slope. There was a striking similarity among the slopes of the dose-response curves for the various strains/stocks. The mean incidence of embryolethality in the A/J strain was significantly greater than that of the other strains or stocks. There was substantial variation among replicates within strains. The use of the replicated study design was logistically necessary due to the magnitude of the study and it also served to increase the statistical power of the study.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1992

Developmental toxicity of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T). II. Multireplicated dose-response studies with technical and analytical grades of 2,4,5-T in four-way outcross mice.

A series of multireplicated developmental toxicity studies were conducted in four-way outcross mice and CD-1 outbred mice administered either analytical or technical grades of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T) by gavage on Gestational Days 6 through 14. The formulations of 2,4,5-T differed by a factor of 10-fold in 2,3,7,8-tetrachlorodibenzo-p-dioxin levels. Reduced fetal weight and increased incidences of cleft palate and embryolethality were the most significant prenatal effects of both formulations of 2,4,5-T observed in all strains/stocks of mice. Both the outcross and outbred mice exhibited a dose-response relationship with each of the above endpoints and the dose-response curves were parallel. There were no embryotoxic or fetotoxic differences between the technical and analytical grades of 2,4,5-T with regard to extent of fetal weight reduction, resorption rate, or cleft palate incidence. There was little difference in the results between the four-way outcross mouse and the CD-1 outbred mouse.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1992

Long-term effects of postnatal exposure to diethylstilbestrol on uterine estrogen receptor and growth.

Diethylstilbestrol (DES) treatment of female rats on postnatal days (PND) 1-5 reduces uterine growth, estrogen receptor (ER) level and gland number by PND 25, while daily DES treatment on PND 1-25 increases uterine growth 4-fold, further reduces ER level and completely suppresses gland formation. We now report the persistence of these effects in adults. By PND 60, uterine weight was 70% of controls in rats injected with DES on PND 1-5 but only 10% of controls in rats injected PND 1-10 or longer. In fact, uterine weights were the same on PND 10 and 60. Uterine gland numbers were reduced to 30% of controls in all DES-treated rats regardless of exposure length; however, luminal and glandular epithelial cell heights were reduced to less than 50 and 70%, respectively, of controls when DES was given on PND 1-25 but not when given on PND 1-5. Ovariectomy 7 days prior to sacrifice on PND 60 reduced uterine weight in controls by 67% and in rats injected with DES on PND 1-5 by 53%, but had no effect in rats injected with DES on PND 1-10. DES exposure at either PND 1-5 or 1-10 lowered ER levels by 35-50% at both 60 and 90 days. Treatment with a high dose of estradiol (E2) 1 week before sacrifice significantly down-regulated ER to the same concentration in all treatment groups at PND 60 and 90. Following E2 treatment, all groups also showed increased uterine weight at PND 60 and 90. These data show there is a short period of development (PND 5-10) in which further DES exposure indirectly inhibits uterine growth.

Aging↗
PubMed1992

The hamster cheek pouch as a convenient ectopic site for studies of uterine morphogenesis and endocrine responsiveness.

The Syrian hamster cheek pouch was evaluated as a convenient transplantation site for studies of estrogen-dependent uterine growth and morphogenesis. At one month of age, hosts were either ovariectomized (Ovex) or ovariectomized and estrogen implanted (Ovex+E2), and at the same time the uterus from a 7-day old untreated donor was transplanted into the host's right cheek pouch. Periodic inspection (by simple eversion of the pouch) revealed viable transplants in the majority of hosts for both groups, and clear evidence of estrogen-dependent transplant growth that continued for at least 9 months. At that time, weight of the transplanted uterus was comparable to that of a given host's own in situ uterus, but uteri at both sites weighed six to eightfold more in Ovex+E2 hosts than in Ovex hosts. Histological analysis also revealed similar degrees of endometrial atrophy in Ovex hosts and hypertrophy/hyperplasia in Ovex+E2 hosts for both in situ and transplanted uteri. Furthermore, while only scant and rudimentary endometrial glands developed in both in situ and transplanted uteri within Ovex hosts, uteri at both sites within the Ovex+E2 hosts were riddled with cystic glandular structures and exhibited marked leukocytic infiltration. These data demonstrate that neonatal uteri transplanted to the hamster cheek pouch will grow, differentiate and follow an endocrine-responsive morphogenetic program that is quantitatively and qualitatively consistent with that of the host's in situ uterus. Lastly, we were able to cleanly separate epithelium from the stroma of 5-day old hamster uteri, reassociate the two tissues in vitro, transplant the recombinants into cheek pouches of adult female hamsters and subsequently observe growth and maintenance of a generally normal uterine morphology and differentiated function.

Animals↗
PubMed1992

Embryotoxicity of phenytoin in adrenalectomized CD-1 mice.

It has been proposed that the anticonvulsant drug phenytoin (PHT) and glucocorticoids induce orofacial clefting by the same mechanism. Previous work had demonstrated that PHT treatment significantly increased endogenous maternal corticosterone concentrations for approximately 48 hr after dosing in A/J mice. The purpose of the present investigation was to determine whether PHT is embryotoxic in the absence of endogenous maternal glucocorticoids. Maternal adrenal glands were removed on Day 7 of gestation, and the incidence of clefting after PHT treatment was determined. There was a high level of maternal toxicity following adrenalectomy (ADX) and PHT treatment at either 60 or 75 mg/kg. This increased toxicity did not appear to be due to altered maternal drug levels in ADX mice. There was a significant increase in the clefting incidence among offspring of ADX dams treated with PHT at 60 mg/kg. This dose of PHT did not elevate maternal corticosterone levels in ADX dams. These data suggest that PHT is capable of producing clefts in the absence of endogenous maternal corticosterone.

Abnormalities, Drug-Induced↗
PubMed1991

Target tissue specificity of retinoic acid-induced stress proteins and malformations in mice.

Retinoic acid (RA) is teratogenic in rodents and also induces the synthesis of stress proteins in fetal mouse limb buds. To determine if the RA induction of stress proteins is target tissue specific, pregnant CD-1 mice were gavaged with 100 mg/kg RA on day 11 of gestation, and nuclei isolated from tissues susceptible to RA-induced malformations (target tissues) as well as nuclei isolated from nontarget tissues were examined for stress protein synthesis and malformations. Forelimb and hindlimb (target tissues), as well as heart and tail (nontarget tissues), were removed from embryos 2.5 hours after RA treatment (1.5 hr after [3H]leucine labeling). Cell nuclei were isolated, stained with a DNA specific fluorochrome, propidium iodide, and sorted from the G0 + G1 and G2 + M phases of the cell cycle. Forelimb and hindlimb target tissues showed the synthesis in these embryonic nuclear proteins of an 84,000 relative molecular mass (Mr) protein and a 90,000 Mr protein following RA treatment. Two 20,000-25,000 Mr stress proteins were also labeled both in forelimb and hindlimb. Forelimb and hindlimb from untreated dams showed no stress protein labeling. Neither heart nor tail, nontarget tissues, showed any stress protein labeling following RA treatment. Classical teratological evaluation of embryos treated on GD 11 and sacrificed on GD 17 showed that 100% of the fetuses had forelimb and/or hindlimb malformations, while no malformations were observed in either the heart or tail. Based on the correlation of teratological anomalies with the identification of stress proteins in target tissue only, we postulate that stress proteins may be involved in the teratogenic process. Further work is necessary to establish whether a causal relationship exists.

Abnormalities, Drug-Induced↗
PubMed1991

Detection of estrogen receptor (ER) in the rat brain using rat anti-ER monoclonal IgG with the unlabeled antibody method.

Application of Sternberger's unlabeled antibody enzyme method for detection of the estrogen receptor (ER) using a rat primary antibody with rat tissues has been discouraged, presumably because nonspecific staining of endogenous IgG was expected with the required anti-rat IgG bridging antibody. Because the blood-brain barrier greatly reduces immunoglobulin infiltration into the brain, we hypothesized that rat brain tissue could be specifically immunostained using rat IgG primary antibodies. A rat monoclonal anti-ER antibody (H222) specifically stained ERs in the brains of ovariectomized but not in ovariectomized estrogen-treated rats. In contrast, the uterus, a well-perfused target organ stained intensely in a nonspecific fashion. Dense populations of estrogen receptors were observed in the medial preoptic area, the bed nucleus of the stria terminalis, and the arcuate and ventromedial nuclei of the hypothalamus. A monoclonal rat IgG directed against alpha-tubulin labeled primarily cortical dendrites quite distinct from the neuronal nuclei that are the primary antigenic sites for the estrogen receptor antibody. These results confirm that the sensitive unlabeled antibody method can be applied to rat brain tissues, even when the primary antibody is rat IgG and that labeling of endogenous IgG may be used as a simple method to evaluate the integrity of the blood brain barrier.

Animals↗
PubMed1991

Growth of neonatal rat uterine luminal epithelium on extracellular matrix.

We have developed a tissue culture system using an extract of basement membrane (extracellular matrix) which promotes the in vitro growth and development of uterine luminal epithelium from the 5-day-old rat. Uterine luminal epithelium, free of stroma, was obtained as short tubes by trypsinization of uterine segments followed by mechanical separation. Epithelial segments were grown in a serum-free medium on culture dishes coated with an extracellular matrix. After 2 days, rapid cell growth resulted in monolayer cultures, which subsequently formed organoid structures similar to differentiated uterine glands present in uterine tissue taken from older rats. Electron microscopy of cultures revealed columnar cells with basally located nuclei, apical microvilli, lateral membranes with interdigitations, desmosomes, and secretory Golgi complexes, all features found in functioning uterine epithelium in vivo. This model will allow the in vitro investigation of the development of uterine epithelium-specific functions free of the influence of stromal cell factors.

Animals↗
PubMed1991

Short-term effects of physiological and pharmacological doses of estradiol on estrogen receptor and uterine growth.

Estradiol (E2) regulation of estrogen receptor (ER) concentrations has been shown to be both time- and dose-dependent. E2 concentrations of 0.5 mg/ml or greater contained in Silastic capsules suppressed uterine ER concentrations after one day's exposure. In this study, we looked at the effects of physiological (1.0 and 10.0 micrograms subcutaneous injections) and pharmacological (5.0 mg/ml implants) doses of E2 on ER concentrations at times less than 24 hours. The implanted rats had maximum E2 plasma levels of approximately 2000 pg/ml for at least six hours which fell to around 800 pg/ml by 12 hours where they remained up to 24 hours. The physiological doses resulted in plasma levels at one hour of 2000 pg/ml (10 micrograms dose) and 250 pg/ml (1 microgram dose) both of which fell to less than 60 pg/ml by six hours. All treatments caused maximal ER suppression by six hours; however, the implants caused a greater reduction in ER levels than either of the physiological doses. The reduction of ER levels was due primarily to a decrease in the "cytosolic" receptor. Despite the decrease in ER, all doses caused a significant and equivalent increase in uterine weight at six hours, however, only the implanted animals maintained the maximal uterine weight gain through 24 hours. This maintenance of uterine weight appears to be correlated with a small but significant increase in the nuclear ER level over this same time period. Thus, while E2 can cause a short-term suppression of its receptor concentration with no effect on short-term uterine weight gain, uterine growth is positively correlated with the level of nuclear ER.

Animals↗
PubMed1991

Estradiol down-regulation of the rat uterine estrogen receptor.

We have previously shown that neonatal exposure of rats to pharmacologic doses of diethylstilbestrol via daily injections resulted in a significant decrease in the estrogen-binding capacity of the uterine estrogen receptor (ER). In this study, we examined the effects of physiologic and pharmacologic doses of estradiol (E2) administered to adult ovariectomized rats via Silastic implants. Two days after implantation, uteri were removed, weighted, and homogenized, and ER levels were determined in the supernatant (hydroxylapatite assay) and low-speed pellet (nuclear exchange assay). Implants containing E2 concentrations of 0.005 or 0.05 mg/ml increased cytosolic but not total ER-binding capacity, whereas 0.5 or 5.0 mg of E2/ml implants decreased the binding capacity of cytosol ER to 40% and total ER to 50% of control values. The 0.005-mg/ml dose increased cytosol ER without increasing uterine weight; all higher doses significantly increased uterine weight. Determination of ER protein by an ER radioimmunoassay showed the same extent of reduction of ER concentration as the binding assays, demonstrating that the loss in E2 binding capacity is homologous down-regulation. The down-regulation of ER was maximal at 24 hr and was completely reversible after implant removal, although the time required to recover from down-regulation was dose dependent. Uterine weight also returned to control levels slowly after implant removal. Neither the sedimentation rate of the down-regulated ER nor the Kd of the cytosolic ER changed following long-term implantation; however, the Kd of the nuclear ER decreased significantly. This is the first demonstration of in vivo homologous down-regulation of uterine ER. ER down-regulation may play a role in several biologic processes.

Animals↗
PubMed1989

Transplacental estrogen responses in the fetal rat: increased uterine weight and ornithine decarboxylase activity.

The synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE2), are more potent than 17 beta-estradiol (E2) in inducing uterine weight gain in the neonatal rat, due to the binding of E2 to serum alpha-fetoprotein (AFP). However, all three hormones are equipotent in inducing neonatal uterine ornithine decarboxylase (ODC) activity. The present study assessed estrogen potency in fetal rats. Pregnant CD rats were injected sc daily on gestation days (GD) 16-20 with DES, EE2, or E2 in sesame oil. Both DES and EE2, but not E2, significantly increased uterine weight at birth, to more than twice that of controls. In addition, implants which continuously release E2 only slightly increased uterine weight at birth. Alternatively, dams were given a single estrogen injection on GD 20 and were sacrificed at various times after injection. Peak fetal uterine ODC activity occurred at 6-8 hours after maternal injection for all three estrogens. E2 had a relative potency about tenfold less than either DES or EE2 in stimulating fetal ODC activity, in contrast to equal potencies of the three estrogens in the postnatal rat uterus. Similar patterns were found following direct fetal injection with E2 or DES. In summary, these data demonstrate a transplacental induction of fetal uterine ODC activity and uterine weight gain by both DES and EE2. In addition, the lack of correlation between these endpoints in response to E2 suggests that they may be useful as selective indicators of potential toxicity of both natural and synthetic estrogens.

Animals↗
PubMed1989

Workshop on risk assessment in reproductive and developmental toxicology: addressing the assumptions and identifying the research needs.

The risk assessment process is an imprecise procedure aimed at determining a toxicant exposure level with an acceptable risk to the human population. The lack of precision is due to the uncertainties in the assumptions that must be made due to the lack of specific scientific information or knowledge of how to use certain types of data. Unfortunately, every necessary piece of information cannot be obtained for every chemical requiring a risk assessment. In order to better identify and understand some of the assumptions that are made in the risk assessment of reproductive and developmental toxicants, a workshop was organized to specifically define the assumptions underlying the risk assessments for seven specific toxicants (dibromochloropropane, dioxin, glycol ethers, heptachlor, lead, tetrahydrocannabinol, and vitamin A) and to determine the potential research which would reduce the uncertainty associated with making those assumptions. The major assumptions discussed centered around the topics of heterogeneous populations, thresholds, safety factors, exposure assessment, quantitative structure-activity relationships, and mechanisms. This report is the summary of the workshop discussions.

Animals↗
PubMed1989

Estradiol-stimulated increases in uterine eosinophils and nuclear type II estrogen-binding sites are prevented by pertussis toxin.

Previous studies from several laboratories have demonstrated that estradiol treatment resulted in an increase in nuclear type II binding sites. Our previous data suggest that this increase was due to the estradiol-stimulated influx of circulating eosinophils. Therefore, we suggested that the uterine nuclear type II estrogen-binding sites were not of uterine origin. In this report we present further evidence to support this hypothesis. Treatment of immature rats with estradiol resulted in the stimulation of several uterine parameters, namely wet weight, protein synthesis, eosinophil number, peroxidase activity, nuclear type II binding sites, and the synthesis and secretion of a 180-kDa protein. The coadministration of pertussigen had no effect on the estradiol-stimulated increase in wet weight, protein synthesis, or the synthesis and secretion of the 180-kDa protein. However, pertussigen did prevent the estradiol-stimulated increase in eosinophils, peroxidase activity, and nuclear type II binding sites, demonstrating a coordinated response. Since peroxidase activity is known to be contained int he eosinophil, these data are consistent with our earlier demonstration that the type II sites are of eosinophil origin. These data also support and extend our previous findings in neonatal animals that estradiol can stimulate a growth response without a corresponding increase in the nuclear type II binding sites. These results further indicate that the estradiol-stimulated increase in eosinophils does not appear to play a key role in the control of uterine growth.

Animals↗
PubMed1989

Estrogen regulation of an eosinophil chemotactic factor in the immature rat uterus.

Associated with the generalized uterine growth stimulated by estradiol in the rat are specific responses including messenger RNA (mRNA) synthesis, protein synthesis, and peroxidase activity. The increase in peroxidase activity, although sensitive to inhibitors of RNA and protein synthesis, results from an estradiol-stimulated influx of eosinophils into the uterus. We postulated the existence of an estradiol-regulated uterine chemotactic factor, testing this by an in vitro chemotactic assay with eosinophils isolated from mature rats. Treatment of immature rats with 1 microgram estradiol for 24 h resulted in a significant increase in eosinophil chemotaxis compared to uterine extracts of vehicle-treated rats. This increase was seen as early as 3 h after estradiol administration and was maximal at 24 h. The magnitude of the chemotactic response was dependent on the dose of estradiol administered (1-100 micrograms). Estrone or estriol treatment resulted in chemotactic activity greater than control but less than estradiol. Direct addition of estradiol to extracts of control animals did not increase chemotaxis. The estradiol-stimulated chemotaxis was blocked by in vivo treatment with the antiestrogen tamoxifen and by inhibitors of RNA and protein synthesis. Analysis of extracts from estradiol-treated uteri shows that the chemotactic factor is heat labile, pronase sensitive, and has a mass of approximately 20 kilodaltons (kDa). These data suggest that the estradiol-stimulated influx of eosinophils into the rat uterus is mediated by the synthesis, modification, or release of a protein whose synthesis is estradiol receptor mediated.

Animals↗
PubMed1988

Postnatal uterine development in the rat: estrogen and antiestrogen effects on luminal epithelium.

We examined the effects of the synthetic estrogens, diethylstilbestrol (DES) and ethynylestradiol (EE), and the triphenylethylene antiestrogen, clomiphene citrate (CC), on uterine growth and development in the rat. These compounds, unlike estradiol, do not bind significantly to rat serum alphafetoprotein (AFP). Administration of DES or EE during the period of normal uterine gland genesis (postnatal days 10-14) induced luminal epithelium hypertrophy and increased uterine wet weight. The durations of these responses were dose-related. By day 26, luminal epithelium cell numbers were significantly depressed, compared to controls. Uterine gland development was delayed 6 to 9 days, depending upon estrogen dose, and the numbers of uterine glands ultimately achieved were generally less than in untreated control animals. While a daily dose of 0.1 micrograms CC/rat did not alter uterine development, 10 micrograms CC/rat caused prolonged luminal epithelium hypertrophy and inhibited uterine gland genesis without inducing the large increases in uterine weight or the decreases in luminal epithelium cell number seen after estrogen exposure. The number of stromal cells was significantly increased on day 26 after CC exposure. Together with previous studies, these data demonstrate the greater potency and developmental stage specificity of non-AFP-bound estrogens with respect to altering uterine gland development. In addition, these data suggest that the disruptive influence of antiestrogens on gland genesis may be mediated through an indirect influence on the uterine stroma.

Animals↗
PubMed1988

Alterations in developing rat uterine cell populations after neonatal exposure to estrogens and antiestrogens.

Exposure of rats to either estrogens or antiestrogens during early postnatal development reduces subsequent uterine growth as measured by uterine weight. However, individual uterine cell types respond differently to these agents and uterine weight alone cannot discern subtle or even large alterations in individual cell populations. Using a computerized planimetric technique, we estimated the prepubertal growth of the uterine luminal epithelium, endometrial stroma, glands, and circular and longitudinal muscle after exposure of neonatal rats (postnatal days 1-5) to the estrogens 17 beta-estradiol (E2), diethylstilbestrol (DES), or ethynylestradiol (EE), and the antiestrogens tamoxifen or clomiphene citrate. On postnatal day 26, the cross-sectional areas of the luminal epithelium, endometrial stroma, and circular muscle were reduced after estrogen exposure, compared to untreated controls, while longitudinal muscle cross-sectional area was not affected. Since cell densities (cell number/unit area) were increased, these estrogen-induced area reductions demonstrate a decrease in cell size. Total cell numbers, estimated as the product of cell type areas and their respective cell densities, were also reduced by neonatal estrogen exposure. The synthetic estrogens DES and EE were more potent than E2 with respect to reduction of uterine growth. Neonatal antiestrogen exposure caused large area reductions only in the uterine glands and luminal epithelium. Little change in cell density occurred in any cell population exposed to antiestrogen. These data demonstrate that the decreased uterine growth resulting from estrogen exposure during early postnatal development is a consequence of combined hypotrophy and hypoplasia in all cell types except longitudinal muscle while antiestrogen-induced morphological alterations were limited to hypoplasia having epithelial cell specificity.

Aging↗
PubMed1988

Effects of postnatal DES treatment on uterine growth, development, and estrogen receptor levels.

The neonatal rodent appears to be an appropriate animal model for estrogen toxicity in the developing reproductive tract. Newborn rats were treated with diethylstilbestrol (DES) at human therapeutic doses (approx 1 mg/kg) during two ontogenetic periods (postnatal days 1-5 and 1-25). Treatment on days 1-5 doubled uterine wt by day 5; however, these uteri failed to grow after discontinuation of DES treatment. In contrast, uterine wt was 4-fold higher and DNA content was 2-fold higher than controls on days 10-25 with continued DES treatment. Total uterine estrogen receptor levels, depressed 60% by day 5 of DES treatment, partially recovered after discontinuation of DES treatment but remained 25% below controls on day 25. Receptor levels following DES on days 1-25 decreased to about 15% of the controls by day 15. Short-term DES treatment approximately halved uterine gland content while continued treatment almost completely inhibited gland appearance. DES effects on glands appear related to continued hypertrophy of the luminal epithelium, from which uterine glands are derived. Subsequent failure of uterine growth caused by DES treatment on days 1-5 is similar to clinical findings of hypoplastic uteri in DES-treated patients. Disruption of the normal ontogenetic patterns of estrogen receptor by DES may be involved. These data demonstrate abnormal patterns of growth, estrogen receptor levels and morphogenesis in uteri of rats treated postnatally with DES.

Age Factors↗