Biomedical subjects

R R Newbold

Publications and source records attributed to R R Newbold.

At least 19 recordsLinked to original sources

PubMed1992

Characterization of murine cell lines from diethylstilbestrol-induced uterine endometrial adenocarcinomas.

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormone-dependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:cytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three- to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.

Adenocarcinoma↗

PubMed1992

Fluctuations of lactoferrin protein and messenger ribonucleic acid in the reproductive tract of the mouse during the estrous cycle.

The physiological role of lactoferrin (LF), the major estrogen-inducible protein in the murine uterus, is unclear; however, LF may be a useful marker for the study of estrogen action in the uterus. Thus, the expression of LF mRNA and the localization of the protein in genital tract tissues and secretions of female mice (6-8 wk old) at different stages of the estrous cycle were investigated. Uterine luminal fluid (ULF) was analyzed for LF by means of gel electrophoresis and Western blot techniques; LF mRNA and protein were identified in reproductive tract tissues through in situ hybridization and immunocytochemistry. At diestrus, the level of LF mRNA was low, and staining for the protein was very light in uterine epithelial cells; LF was undetectable in ULF. At proestrus, LF mRNA and protein increased in the uterine epithelium and LF was readily detectable in ULF. LF mRNA and protein reached the highest levels at estrus. At early metestrus as compared to estrus, LF mRNA and protein were detected in decreasing amounts in uterine epithelial cells; the protein was undetected in ULF. By late metestrus and diestrus, LF mRNA and protein returned to a low level, and the protein was undetectable in ULF. LF protein was also demonstrated by immunocytochemistry in the epithelium of the oviduct, cervix, and vagina. LF protein fluctuation similar to that observed in the uterus was seen in these tissues; however, the uterus demonstrated the most dramatic changes in the number of epithelial cells involved in LF production during the estrous cycle. In summary, LF mRNA and its expression in uterine epithelial cells of the mouse varied with the stage of the estrous cycle. These results, combined with previously reported findings that LF is a major constituent of mouse ULF under the influence of estrogen, suggest that LF may play an important role in normal reproductive processes.

Animals↗

PubMed1992

Developmental exposure to estrogens induces persistent changes in skeletal tissue.

Short-term exposure to estrogens during development has been reported to cause irreversible changes including neoplasia in estrogen target tissues, i.e. reproductive tract and mammary gland. Moreover, it has been established that estrogens have a dramatic effect on bone turnover. The recent demonstration of a low level of estrogen receptor (ER) in bone cells strongly suggests that these estrogenic effects are direct. This report was designed to evaluate whether neonatal exposure to diethylstilbestrol (DES) induces irreversible changes in bone tissue as demonstrated in other specific target organs. We show that short-term exposure of newborn mice (day 1-5) to DES (2 micrograms/pup/day) induces permanent changes in skeletal tissue in adulthood; femurs of DES-treated animals were significantly shorter than age-matched control mice. Furthermore, a significant increment (1.5 fold) in the amount of bone in the femurs (representative of long bone) and vertebrae (representative of short bone) was observed in DES-exposed animals. These data provide further evidence that bone tissue is a specific estrogen target tissue. Finally, we postulate that physiological exposure to estrogens in childhood might be one of the key factors in determining the final peak bone density in adulthood.

Animals↗

PubMed1991

Ontogeny of peroxidase activity in epithelium and eosinophils of the mouse uterus.

Outbred CD-1 mice treated for 1 or 4 days with 1 mg/kg of diethylstilbestrol (DES) at various ages after birth were examined for histochemical localization of peroxidase in the uterine epithelium. Peroxidase activity in uterine extracts was also measured by a radiometric assay and the conversion of [3H]DES to [3H]Z,Z-diensestrol (Z,Z-DIES). While no peroxidase activity was detected by a histochemical method in uterine epithelium from untreated 5-day old mice, the enzyme was apparent in mice treated for 4 days with DES; uterine eosinophils were absent at this age. By day 9, DES-induced staining for peroxidase in uterine epithelial cells and the number of uterine eosinophils had increased significantly. In addition, at this age, the biochemical assays for uterine peroxidase were sensitive enough to show that DES is converted to Z,Z-DIES and that [3H]estradiol gives rise to 3H2O and water-soluble radioactive metabolites. The peroxidase response to DES, determined by both histochemical and biochemical methods, increased with the age of the immature mice. These data indicate that the neonatal uterus, although deficient in eosinophils, demonstrates a peroxidase response to estrogen and that this response is localized primarily in the luminal epithelium. The role of this DES-induced peroxidase activity in converting DES to activated metabolites that may cause cell damage is discussed.

Age Factors↗

PubMed1991

Characteristics of estrogen-induced peroxidase in mouse uterine luminal fluid.

Peroxidase activity in the uterine luminal fluid of mice treated with diethylstilbestrol was measured by the guaiacol assay and also by the formation of 3H2O from [2-3H]estradiol. In the radiometric assay, the generation of 3H2O and 3H-labeled water-soluble products was dependent on H2O2 (25 to 100 microM), with higher concentrations being inhibitory. Tyrosine or 2,4-dichlorophenol strongly enhanced the reaction catalyzed either by the luminal fluid peroxidase or the enzyme in the CaCl2 extract of the uterus, but decreased the formation of 3H2O from [2-3H]estradiol by lactoperoxidase in the presence of H2O2 (80 microM). NADPH, ascorbate, and cytochrome c inhibited both luminal fluid and uterine tissue peroxidase activity to the same extent, while superoxide dismutase showed a marginal activating effect. Lactoferrin, a major protein component of uterine luminal fluid, was shown not to contribute to its peroxidative activity, and such an effect by prostaglandin synthase was also ruled out. However, it was not possible to exclude eosinophil peroxidase, brought to the uterus after estrogen stimulation, as being the source of peroxidase activity in uterine luminal fluid.

Animals↗

PubMed1991

Androgen metabolism in control and neonatally estrogenized male mice.

Reduction, oxidation, and aromatization of androgens were studied in the male genital tract of untreated control and neonatally estrogenized mice. This study shows regional differences in 5 alpha-reductase and 17 beta-hydroxysteroid oxidoreductase activities in untreated male genital tract; 3 alpha/3 beta-hydroxysteroid oxidoreductase (3 alpha/3 beta-HSOR) activity varied little between tissues. Neonatal treatment with diethylstilbestrol (DES, 2 micrograms/pup/day on days 1 through 5) caused an alteration in the androgen metabolism of the male genital tract, resulting in apparent decreased net accumulation of dihydrotestosterone (DHT). This developmentally-induced 5 alpha-reductase deficiency may play a role in the long-term inhibitory effects of early estrogenization by DES in the growth and function of male sex accessory glands. No aromatase activity could be demonstrated in the male genital tract of control or neonatally estrogenized mice.

Androgens↗

PubMed1990

Uterine adenocarcinoma in mice following developmental treatment with estrogens: a model for hormonal carcinogenesis.

In order to study the effects of perinatal exposure to estrogens on the developing reproductive tract, outbred female mice were treated neonatally (days 1 to 5) with varying doses of diethylstilbestrol (DES) and sacrificed from 1 to 18 months of age. Uterine adenocarcinoma was observed in a time- and dose-related manner after DES treatment; at 18 months, neoplastic lesions were seen in 90% of the mice exposed neonatally to 2 micrograms/pup of DES/day, while none was observed in the corresponding control mice. These DES-induced uterine tumors were estrogen dependent; when DES-treated mice were ovariectomized before puberty, no uterine tumors developed. As a marker for neoplasia, uterine tumors were transplanted and carried as serial transplants in nude mice. The transplanted tissue retained some differentiated uterine gland structure and function and also required estrogen supplementation for maintenance. Additional groups of neonatal mice were treated with various DES analogues (hexestrol and tetrafluorodiethylstilbestrol) and steroidal estrogens. The compounds were ranked according to developmental estrogenic potency (hexestrol greater than trifluorodiethylstilbestrol greater than DES greater than 17 beta-estradiol). The combined prevalence of uterine atypical hyperplasia and adenocarcinoma follows the order of estrogenic potency. The experimental induction of these tumors will provide the basis for additional studies in mechanisms of hormonal carcinogenesis.

Adenocarcinoma↗

PubMed1990

The role of the estrogen receptor in uterine epithelial proliferation and cytodifferentiation in neonatal mice.

We have examined the relationship between localization of estrogen receptor (ER) and selected cell responses induced by estrogen in the neonatal CD-1 mouse uterus. The following simultaneous staining techniques were used to determine whether only uterine epithelial cells with ER are capable of showing proliferative and secretory activities after estrogen treatment: 1) ER localization and [3H]thymidine incorporation using immunohistochemistry and autoradiography; and 2) immunohistochemical double staining of ER and an estrogen-induced secretory protein lactoferrin (LF). Uterine tissues from two strains (CD-1 and BALB/c) mice on day 4 of age showed detectable ER by immunostaining in both epithelial and stromal cells. Day 4 CD-1 mice received a single injection of diethylstilbestrol (DES; 20 micrograms/kg BW). The percentage of ER-immunostained uterine epithelial cells and the intensity of the staining rapidly increased from 6-36 h, indicating that estrogen stimulates the expression and detectability of ER during the course of hormone treatment. Twenty-eight and 81% of epithelial cells showed positive ER immunostaining 6 and 24 h, respectively, after the DES treatment. The intensity of ER-immunostained stromal cells, however, was slightly decreased by the treatment. DES administration elicited epithelial cell proliferation. Labeling indices of epithelial cells reached a maximum (approximately 44%) 12 h after an injection of DES, and a second small peak was recognized 24 h after the treatment. Labeling indices of positively ER-immunostained epithelial cells and those of negatively stained epithelial cells showed almost the same pattern for 6-18 h after treatment. The respective maximum values were 45% and 43%. When day 4 mice were given three daily injections of DES in saline and killed 12 and 24 h after the last injection, significant amounts of LF were recognized in the apical cytoplasm of the epithelial cells. The epithelial cells that showed LF immunostaining always exhibited ER immunostaining in the nuclei. These results suggest that exogenous estrogen elicits increased expression of ER, DNA synthesis, and cell proliferation in neonatal uterine epithelial cells associated with low levels of ER, while there was a direct relationship with the presence of ER in epithelial cells and the induction of LF.

Animals↗

PubMed1989

Lactotransferrin gene expression in the mouse uterus and mammary gland.

The mouse mammary gland and uterus expressed the gene for the secretory protein lactotransferrin under various physiological conditions. Lactotransferrin, however, was induced by estrogen in a time- and dose-dependent fashion in the uterus of the immature mouse, but was not affected by estrogen in the mammary gland. Differences were also found in the expression of lactotransferrin in mammary glands and uteri of adult females during lactation. A high level of the protein was detected by immunocytochemistry in uterine epithelial cells 1 day after parturition, but immunoreactivity disappeared quickly thereafter. Lactotransferrin message was, however, relatively abundant in the mammary gland at the end of the lactation period. The presence of lactotransferrin in various tissues also was investigated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Two forms of immunoreactive material was detected by this method; a 70K band was found in uterine luminal fluid from the estrogen-stimulated immature mouse and in homogenates of lung, vagina, mammary gland, oviduct, spleen, lymph node, and uterus of the adult female mouse, and a 65K band was detected in submaxillary gland, kidney, ovary, and all of the above tissues. Brain and duodenum had no detectable immunoreactive material. A transient appearance of lactotransferrin was observed in the uterine luminal fluid of pseudopregnant mice. These changes in the level of lactotransferrin in the uterus and mammary gland under various physiological conditions suggest that the regulation of this protein's expression is tissue specific.

Animals↗

PubMed1989

Female gene expression in the seminal vesicle of mice after prenatal exposure to diethylstilbestrol.

Previous studies from our laboratory on the feminization of the male mouse reproductive tract after prenatal exposure to diethylstilbestrol (DES) showed that the mRNA for the major estrogen-inducible uterine secretory protein, lactoferrin (LF), was constitutively expressed in the seminal vesicle of male mice exposed prenatally to DES, but not in the seminal vesicle of control mice. After castration, treatment with 17 beta-estradiol (20 micrograms/kg.day) for 3 days induced the LF mRNA in the seminal vesicle of both control and prenatally DES-exposed mice; however, the levels in DES-treated tissues were approximately 6-fold higher than those in control tissue. This report describes the presence of LF in seminal vesicle tissues and secretions of prenatally DES-exposed mice, as determined by immunohistochemistry and Western blot analysis. Further, these data are correlated with immunolocalization of the estrogen receptor in the seminal vesicle tissue. We conclude that the seminal vesicle of prenatally DES-exposed male mice has acquired two key characteristics of female tissues, namely LF production/regulation and estrogen receptor localization/distribution similar to that in uterine tissues.

Animals↗

PubMed1989

Developmental pattern of estrogen receptor expression in female mouse genital tracts.

The distribution of the estrogen receptor (ER) was investigated in neonatal female genital tracts (uterus, oviduct, cervix, and vagina) from days 1-22 after birth, using immunohistochemistry employing an anti-ER monoclonal antibody. In uteri, the ER in epithelial cells began to be observed by day 4. The number of positive epithelial cells and the staining intensity gradually increased until day 22 of age. On the other hand, uterine stroma cells gave a strong ER immunostaining even on day 1. The staining intensity reached a maximum by days 4-7 and then slightly decreased with age. In the oviduct, cervix, and vagina, epithelial cells showed positive ER immunostaining on day 1, and the intensity increased gradually until day 22. ER immunostaining in stroma cells was almost constant during the development period. The ER in both epithelial and stroma cells from these younger animals showed similar biochemical properties, i.e. an increased affinity for nuclei and resistance to extraction with PBS. Thus, during neonatal development of the female reproductive tract, ER is present not only in stroma cells but also in epithelial cells. This ER protein exhibits properties and characteristics similar to those of adult mice. The presence of ER suggests that some of the estrogen actions of cell proliferation, differentiation, and tissue abnormalities resulting from prenatal and postnatal estrogen administration may be mediated by receptor interactions.

Aging↗

PubMed1988

Immunodetection of estrogen receptor in epithelial and stromal tissues of neonatal mouse uterus.

The tissue distribution and levels of estrogen receptor in neonatal mouse uterine tissue were determined in epithelial and stromal fractions separated by mild enzymatic treatment. Proteins of the isolated fractions were separated by gel electrophoresis and receptor was detected on immunoblots with monoclonal antibody H-222. Estrogen receptor protein was detectable in samples of reproductive tract tissue from 5- and 10-day-old mice. The level of receptor in 5-day-old animals was lower per unit DNA in epithelial cells than in stroma. Receptor levels were increased in both tissue types after treatment with diethylstilbestrol, but not with progesterone. Receptor protein present in these neonatal tissues was able to bind steroid as evidenced by affinity labeling with tamoxifen aziridine. Immunohistochemistry on sections of uteri from 4- and 10-day-old mice confirmed the biochemical results and indicated lower nuclear straining in epithelial cells than in stromal cells of uteri of 4-day-old mice. These results demonstrated that estrogen receptor protein is present in both epithelium and stroma of the neonatal mouse uterus, but at a higher level in stromal cells.

Animals↗

PubMed1988

Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism.

Nafoxidine (NAF) acts as an estrogen agonist or antagonist depending on the animal model used. In the CD-1 mouse uterus, a three-day uterine bioassay of NAF produced a bell-shaped dose response curve with a maximal uterine wet weight increase at 200 micrograms/kg; this dose produced only a fractional increase in uterine dry weight. Combination treatment with NAF and estradiol antagonized estradiol stimulation of both wet and dry weight parameters. The time course of uterine wet weight stimulation following a single injection of NAF had an early pattern (0-10 h) similar to that of estradiol. However, at later times after stimulation, the patterns changed dramatically: the low NAF dose (200 micrograms/kg) returned to control levels by 24 h; estradiol and the high dose NAF (1.7 mg/kg) showed sustained stimulation, which peaked at 36 h with NAF compared to 24 h for estradiol. Nuclear estrogen receptor (ER) levels were measured after a single injection of 1.7 mg/kg NAF and showed a bimodal pattern similar to that seen with estradiol, with increases at 1 h and 8 h, although the overall ER levels were elevated above those seen with estradiol. Cytosolic ER levels with NAF decreased by 1 h and remained low up to 48 h. NAF treatment did stimulate uterine DNA and RNA synthesis, with a delayed time course compared to estradiol. DNA synthesis following a single 1.7 mg/kg dose of NAF was 2.5 times higher than that produced by 20 micrograms/kg estradiol. NAF treatment resulted in hypertrophy and hyperplasia in the luminal epithelium but not in the glandular epithelium. Long-term exposure to estradiol for 5 wk resulted in development of uterine cystic glandular hyperplasia and increased secretory activity; long-term exposure to NAF produced a more significant tissue hyperplasia but no secretions. These studies show that NAF stimulates some of the receptor-mediated responses attributed to an estrogen agonist in the mouse uterus; but, when co-administered with estradiol, NAF antagonizes some aspects of estrogen action.

Animals↗

PubMed1988

Prenatal exposure of male mice to diethylstilbestrol alter the expression of the lactotransferrin gene in seminal vesicles.

We have previously isolated an estrogen-inducible secretory protein, lactotransferrin (LTF), and a cDNA to its messenger RNA from the uterus of mice. In this report we determined that the level of LTF mRNA is minimal in the seminal vesicles of normal mice. In contrast, expression of LTF mRNA in the seminal vesicles of developmentally estrogenized males was both constitutive and estrogen inducible. The results suggested that this alteration may be an example of atypical gene expression after hormonal manipulation early in development.

Androgens↗

PubMed1988

Lesions of testis and epididymis associated with prenatal diethylstilbestrol exposure.

Cryptorchidism and retention of Müllerian duct structures occur with high frequency among the male offspring of CD-1 mice treated with 100 micrograms diethylstilbestrol/kg body weight on days 9 through 16 of pregnancy. Hyperplasia of the rete testis and Müllerian duct structures were found in many of the DES-treated male mice, as was a low but significant number of reproductive tract neoplasms.

Animals↗

PubMed1987

Müllerian remnants of male mice exposed prenatally to diethylstilbestrol.

Prenatal exposure of males to diethylstilbestrol (DES) results in reproductive tract teratogenesis, ie, retention of Müllerian duct remnants. The potential of these remnants to develop pathological changes has not been studied. Therefore, pregnant outbred CD-1 mice were subcutaneously injected with daily doses of DES (100 micrograms/kg) on days 9 through 16 of gestation. DES-exposed male offspring and age-matched control male mice were sacrificed at 10 to 18 mo of age and examined for reproductive tract abnormalities. Prominent Müllerian remnants were observed in 268 out of 277 (97%) of the DES-exposed male mice. These remnants differentiated into "femalelike structures" homologous to oviduct and uterus. The Müllerian remnants were often enlarged and cystic and shared supporting connective tissue with adjacent male structures. Previously reported lesions, termed "epididymal cysts," were determined histologically to be cystic "oviductlike" structures and were, therefore, considered a Müllerian duct abnormality. Pathological changes in these male oviductal and uterine homologs included benign and malignant lesions. In addition, epididymal structures were altered. Inflammation and sperm granulomas were prevalent in DES-treated mice as young as 10 mo old but were only observed in control mice at 18 mos. Cysts of epididymal duct origin, hyperplasia, and adenoma of the epididymal duct were also observed. No comparable abnormalities were noted in 122 control males of corresponding ages. The data presented in this report demonstrated that transplacental exposure to DES affected the differentiation and normal development of the male genital tract involving both the Müllerian (paramesonephric) and Wolffian (mesonephric) ducts. The long-term changes in these tissues include lesions, some of which resembled neoplasia although the natural history of the lesions is not known. Moreover, some previously described abnormalities referred to as "epididymal cysts" were associated with tissues derived from embryonic female origin.

Animals↗

PubMed1987

Testicular tumors in mice exposed in utero to diethylstilbestrol.

Treatment of pregnant women with diethylstilbestrol (DES) is associated with the subsequent development of reproductive tract abnormalities such as epididymal cysts, retained hypotrophic testes and sperm abnormalities in their male offspring. It recently has been suggested that prenatal DES exposure is associated with development of testicular seminoma in humans. Studies of in utero exposure of laboratory animals to DES are few, but previous reports from our laboratory have described several abnormalities in the reproductive tract of the mouse following prenatal DES exposure. To study the possible association of testicular tumors and prenatal DES exposure in mice, pregnant outbred CD-1 mice were injected subcutaneously with daily doses of DES (100 micrograms./kg.) on days nine through 16 of gestation. DES-exposed and age-matched control male mice were sacrificed at 10 to 18 months of age and examined for testicular lesions. In addition to the nonmalignant abnormalities reported in previous studies such as 91% cryptorchidism and degenerative changes, interstitial cell tumors were observed in nine mice among 277 mice treated prenatally with DES. Two of these lesions were benign tumors and five were interstitial cell carcinomas. Rete testis adenocarcinoma was seen also in 5% of these DES-treated animals and is described in another report. The overall incidence of testicular tumors is 8% in DES-exposed male mice. No comparable lesions were seen in 122 control male mice. These results suggest that the testicular lesions that can occur following prenatal DES exposure include neoplasia. The combined prevalence of DES-induced tumors of the corpus testis and rete testis in mice suggests the male offspring may be more at risk for developing carcinoma of the reproductive tract than the female offspring.

Animals↗

PubMed1987

Estrogens and development.

The normal development of the genital organs of mammals, including humans, is under hormonal control. A role for the female sex hormone estrogen in this process is still unclear. However, exposure of experimental animals or humans to the potent exogenous estrogen, diethylstilbestrol (DES), results in persistent differentiation effects. Since many chemicals in the environment are weakly estrogenic, the possibility of hormonally altered differentiation must be considered.

Animals↗