Prenatal exposure to DES associated to altered methylation of the gene at the molecular level

image of female-mice

Are estrogens carcinogenic during development of the testes?

1998 Abstract

Many chemicals in the environment mimic the female sex hormone, estrogen.

Exposure to environmental estrogens during early fetal development was proposed by Sharpe & Skakkebaek as a potential risk factor for subsequent testicular disease, including neoplasia and poor semen quality.

To understand the mechanisms of action of estrogenic chemicals during differentiation of the male genital tract, we have studied developmental exposure to the synthetic estrogen, diethylstilboestrol (DES). While DES is a much more potent estrogen than most environmental chemicals examined, several of these compounds share some of the same properties as DES, such as a relative lack of binding to serum estrogen carrying proteins.

Prenatal exposure to DES is associated with poor semen quality, prostatic disease, cryptorchidism and testicular neoplasia in mice. A rare form of testicular cancer, rete testis carcinoma, was observed in five percent of male mice treated in utero with DES. We also demonstrated altered regulation of an estrogen responsive gene, lactotransferrin (LTF) in the seminal vesicles of treated mice, but not the controls. Likewise, LTF was irreversibly altered in the uteri of developmentally treated females; at the molecular level altered methylation of the gene appears to be involved, thus, providing a potential marker for hormonal effects during development.

The induction of permanent or “imprinted” responses during the development of a relatively estrogen-free reproductive tract cell suggests that undifferentiated targets for estrogen action may be sites for subsequent growth and differentiation defects associated with neoplasia.

  • Are estrogens carcinogenic during development of the testes?, NCBI PubMed, APMIS : acta pathologica, microbiologica, et immunologica Scandinavica, PMID : 9331098, 1997 Oct.
  • White female mice featured image credit Steph Hillier.
DES DIETHYLSTILBESTROL RESOURCES

p53 protein expression and gene analysis in clear cell adenocarcinoma of the vagina and cervix

image of P53-Protein

Prenatal DES exposure associated with generalized DNA damage and p53 gene mutations

1996 Study Abstract

OBJECTIVE
p53 is the most commonly mutated gene in human cancers. The objective of this study was to determine if clear cell adenocarcinomas (CCAs) of the vagina and cervix are associated with p53 gene mutations or alterations in p53 tumor-suppressor protein expression.

METHODS
Paraffin-embedded tissue specimens from 21 women (median age 22 years) with clear cell adenocarcinoma of the vagina or cervix were studied. Fifteen women had a prior history of in utero exposure to diethylstilbestrol. p53 protein expression was detected by immunohistochemical (IHC) analysis with monoclonal antibody DO-7 (Dako Corp.) which recognizes both wild-type and mutant p53 proteins. For p53 gene analysis, genomic DNA from malignant tissue was isolated and exons 4-10 were amplified by PCR and subjected to mutation screening by single-stranded conformation polymorphism (SSCP) analysis.

RESULTS
p53 protein was detected by IHC in tumors from 14 of 21 cases (67%). The observed p53 staining patterns were heterogeneous in both the proportion and intensity of tumor cells stained but were clearly overexpressed relative to the surrounding benign stroma. Metastatic tumors from 3 women with metastatic disease were also positive for p53 staining. SSCP analysis did not identify p53 mutations in any of the cases and strongly suggests that the tumors contained only wild-type p53 alleles.

CONCLUSIONS
Recent studies have demonstrated that wild-type p53 may accumulate in response to DNA damage which normally leads to growth arrest or programmed cell death. Our observations are consistent with the hypothesis that p53 overexpression in CCAs of the vagina and cervix is a response to generalized DNA damage, rather than a result of p53 protein half-life prolongation resulting from mutational inactivation of p53. Overexpression of wild-type p53 protein in vaginal and cervical CCA may relate to the more favorable prognosis of this subset of tumors in comparison to other gynecologic tumors containing mutated p53 genes.

  • p53 protein expression and gene analysis in clear cell adenocarcinoma of the vagina and cervix, Gynecologic oncology, NCBI PubMed PMID : 8774636, 1996 Mar.
  • P53 Protein featured image credit bioquest.
DES DIETHYLSTILBESTROL RESOURCES

Cellular and molecular effects of developmental exposure to diethylstilbestrol

cellular image

Low doses of DES demonstrate alterations in both male and female exposed offspring

1995 Study Abstract

Concerns have been raised regarding the role of environmental and dietary estrogens as possible contributors to an increased incidence of various abnormalities in estrogen-target tissues of both sexes.

These abnormalities include breast cancer, endometriosis, fibroids, and uterine adenocarcinoma in females, as well as alterations in sex differentiation, decreased sperm concentrations, benign prostatic hyperplasia, prostatic cancer, testicular cancer, and reproductive problems in males.

Whether these concerns are valid remains to be determined; however, studies with the potent synthetic estrogen diethylstilbestrol (DES) suggest that exogenous estrogen exposure during critical stages of development can result in permanent cellular and molecular alterations in the exposed organism. These alterations manifest themselves in the female and male as structural, functional, or long-term pathological changes including neoplasia.

Although DES has potent estrogenic activity, it may be used as a model compound to study the effects of weaker environmental estrogens, many of which may fit into the category of endocrine disruptors.

Conclusions

There are many possible cellular and molecular mechanisms that may be involved in the toxic response to DES and other environmental estrogens if an organism is exposed during critical stages of development.

Several mechanisms have been discussed in this report. Studies with the developmentally DES-exposed murine model have duplicated and predicted many of the lesions seen in similarly DES-exposed humans and in the wildlife population.

Currently, there is increased interest in the effects of other environmental estrogens and antiandrogens on reproductive tract differentiation and development.

Whether these compounds have effects similar to DES is uncertain, but since low doses of DES demonstrate alterations in both male and female exposed offspring, the possibility of adverse effects from other compounds with estrogenic and/or antiandrogenic activity must be considered.

  • Read and download the full paper (free access) Cellular and molecular effects of developmental exposure to diethylstilbestrol: implications for other environmental estrogens, Environmental health perspectives, NCBI PubMed PMC1518878, 1995 Oct.
  • Featured image credit Rick-Clement Bootha.
DES DIETHYLSTILBESTROL RESOURCES

Neonatal DES exposure effects on the expression of genes EGF and lactoferrin

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Exposure to diethylstilbestrol during a critical developmental period of the mouse reproductive tract leads to persistent induction of two estrogen-regulated genes

1994 Study Abstract

Exposure to estrogens during critical periods of development induces teratogenic and carcinogenic lesions in the reproductive tracts of humans and experimental animals. It is important to determine the molecular and cellular targets of estrogenic chemicals and to establish the mechanisms by which interactions of estrogens with the developing genital tract results in permanent lesions of growth and differentiation.

The experiments presented here were designed to examined the effects of neonatal estrogen exposure on the expression of two genes, lactoferrin and epidermal growth factor (EGF), that are subject to steroid hormone regulation.

Using in situ and Northern RNA hybridization, immunoblotting, and immunohistochemistry, our data demonstrate that exposure to the synthetic estrogen, diethylstilbestrol, during a critical neonatal period results in the persistent ovary-independent induction of mRNA and protein encoded by these two genes in the mouse uterus and vagina.

The constitutive expression of lactoferrin and EGF, and probably other estrogen-regulated genes, may contribute to the establishment of a permanently “estrogenized” phenotype which is then instrumental in the development of abnormal tissue morphogenesis, function, and neoplasia in the rodent reproductive tract.

  • Read and/or download the full report Exposure to diethylstilbestrol during a critical developmental period of the mouse reproductive tract leads to persistent induction of two estrogen-regulated genes, Cell growth and differentiation : the molecular biology journal of the American Association for Cancer Research, NCBI PubMed Vol. 5, 595-606, 1994 June.
  • Featured image credit cgd.aacrjournals.
DES DIETHYLSTILBESTROL RESOURCES

Altered protein response, atypical gene expression following DES exposure early in development

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Molecular feminization of mouse seminal vesicle by prenatal exposure to diethylstilbestrol : altered expression of messenger RNA

1994 Study Abstract

Exposure to estrogens during critical stages of development has been reported to cause irreversible changes in estrogen target tissues such as the reproductive tract. In fact, recent studies using mice describe prenatal estrogen exposure resulting in the expression of the major estrogen-inducible uterine secretory protein, lactoferrin (LF), by the seminal vesicles of the male offspring.

Thus, we have studied the role of estrogens in abnormal and normal gene expression in the developing male reproductive tract using LF and seminal vesicle secretory protein IV (SVS IV), an androgen-regulated murine seminal vesicle secretory protein, as markers.

Lactoferrin and SVS IV protein and mRNA expression were studied in histological samples by using the techniques of in situ hybridization (ISH) and immunohistochemistry (IHC). Seminal vesicle secretory protein IV was expressed in all (100%) epithelial cells of the control seminal vesicle, but this protein was decreased by castration. However, LF expression was undetectable by ISH or IHC in control seminal vesicle epithelium. Lactoferrin was inducible in 2% of the seminal vesicle epithelial cells from adult castrated mice treated with estradiol 17 beta (E2; 20 micrograms/kg/day for 3 days), indicating that a small percentage of the seminal vesicle cells could be induced to secrete LF after modification of the endocrine environment.

Prenatal DES treatment (100 micrograms./kg. maternal body weight on days 9 through 16 of gestation) resulted in the male offspring exhibiting constitutive expression of LF in 5% of the seminal vesicle epithelial cells, while expression of the androgen-regulated protein SVS IV was slightly decreased. The maximal contrast between LF and SVS IV expression was observed in prenatally DES-treated mice that were subsequently castrated as adults and further treated with E2; LF was detected in 40% of the epithelial cells in these mice. Double immunostaining techniques revealed that epithelial cells which were making LF had ceased production of SVS IV. Since a large percentage of the epithelial cells in the intact prenatal DES exposed male was capable of expressing the normal gene product, SVS IV, it was concluded that DES treatment during prenatal development appears to imprint or induce estrogenic sensitivity in the adult seminal vesicle, causing increased production of LF.

The results suggest that this altered protein response may be an example of atypical gene expression in male reproductive tract tissues following hormonal manipulation early in development.

  • Molecular feminization of mouse seminal vesicle by prenatal exposure to diethylstilbestrol: altered expression of messenger RNA, The Journal of urology, NCBI PubMed PMID/ 8158792, 1994 May.
  • Featured image credit forum.hairsite.
DES DIETHYLSTILBESTROL RESOURCES

Effects of neonatal exposure to DES on protein expression

Neonatal DES exposure induces organ specific alterations in the synthesis of proteins in vagina and uterus

1992 Study Abstract

Neonatal treatment of female mice with diethylstilbestrol (DES) results in genital tract abnormalities including ovary-independent vaginal proliferation and cornification.

Protein profiles were examined in vagina and uterus from 45-day-old, ovariectomized C57BL/Tw mice which had been given 5 daily injections of 2 micrograms DES or oil vehicle alone from the day of birth, and in those from 45-day-old, ovariectomized mice given 3 daily injections of 0.1 microgram DES from 42 days of age. Proteins extracted were analyzed by two-dimensional polyacrylamide gel electrophoresis.

  • In the vagina,
    • expression of 37 non-keratin proteins was altered by postpubertal injections of DES as compared with the controls.
    • In the neonatally DES-exposed vagina, expression of 26 of 37 proteins was altered as compared with controls.
  • In the uterus,
    • expression of 22 proteins was altered in the postpubertally DES-exposed group as compared with that in the control;
    • however, the protein expression pattern of the neonatally DES-exposed group closely resembled that of the control except for one protein (no. 23, pI = 6.1, MW = 39.5 kDa) which was specifically increased in the neonatally DES-exposed group.
    • By immunoblot analysis, 6 keratin polypeptides (49.5, 50, 52, 53, 57 and 58 kDa) were identified in vaginae of ovariectomized mice exposed neonatally and postpubertally to DES and of the controls.

These results indicate that neonatal DES exposure induces organ specific alterations in the synthesis of proteins in mouse vagina and uterus.

  • Effects of neonatal exposure to diethylstilbestrol on protein expression by vagina and uterus in mic, In vivo, NCBI PubMed PMID : 1378305, 1992 Jan-Feb.
  • Keratin polypeptides featured image credit slideplayer.
DES DIETHYLSTILBESTROL RESOURCES

Presence of lactoferrin protein in seminal vesicle tissues and secretions of prenatally DES-exposed mice

image of lactoferrin

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

1989 Study Abstract

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.

  • Female gene expression in the seminal vesicle of mice after prenatal exposure to diethylstilbestrol, Endocrinology, NCBI PubMed PMID : 2707167, 1989 May.
  • Lactoferrin featured image credit researchgate.
DES DIETHYLSTILBESTROL RESOURCES

DES induce permanent, irreversible disturbances in lymphocyte populations and functions

Short-term and long-term effects of estrogen on lymphoid tissues and lymphoid cells with some remarks on the significance for carcinogenesis

1984 Study Abstract

Estrogens have long been thought to play a role in regulating the immune system. The difference in some types of immune responses between males and females is well-known, as is the pronounced thymic involution induced by exogenous estrogens. Estrogens stimulate some aspects of macrophage activity and, depending on dose and mitogen, inhibit or stimulate lymphocyte proliferative response in vitro. Another example is the estrogen effect on the delayed type hypersensitivity response. A broad review is given of such estrogen effects on lymphoid tissue and immune response. Most of the studies published so far are phenomenological. However, the recent description of estrogen receptors in the thymus and in some lymphocyte subpopulations, as well as a deeper understanding of regulating factors in the immune system, open the possibility of a more detailed understanding of the estrogen mechanism of interference. Estrogen effects in adults are reversible.

After treating neonatal mice with the synthetic estrogen diethylstilbestrol (DES), disturbances are induced in lymphocyte populations and lymphocyte functions which are permanent and irreversible. Lymphocytes from adult, neonatally DES-treated female mice have a reduced mitogen response to ConA and LPS (T and B cell mitogen) and the delayed type hypersensitivity response is depressed. A detailed analysis demonstrated a decreased T helper cell population. The activity of Natural Killer cells is permanently reduced and this functional impairment is related to a decreased number of these cells, in turn determined at the bone marrow level. The same animals have an increased sensitivity to chemical carcinogens (methylcholanthrene) and they spontaneously develop epithelial changes in the uterine cervix which morphologically are similar to adenocarcinoma.

The association between estrogen-associated malignancy and estrogen effects in lymphocyte functions deserves further study.

  • Short-term and long-term effects of estrogen on lymphoid tissues and lymphoid cells with some remarks on the significance for carcinogenesis, US National Library of Medicine National Institutes of Health, Archives of toxicology, NCBI PubMed PMID : 6477127, 1984 Jul.
  • Featured image credit khanacademymedicine.
DES DIETHYLSTILBESTROL RESOURCES

Prenatal exposure to DES may affect T-cell differentiation and cause long-term effects on the immune functions

Prenatal exposure of mice to diethylstilbestrol disrupts T-cell differentiation by regulating Fas/Fas ligand expression through estrogen receptor element and nuclear factor-κB motifs

2012 Study Abstract

Prenatal exposure to diethylstilbestrol (DES) is known to cause altered immune functions and increased susceptibility to autoimmune disease in humans.

In the current study, we investigated the effect of prenatal exposure to DES on thymocyte differentiation involving apoptotic pathways.

Prenatal DES exposure caused thymic atrophy, apoptosis, and up-regulation of Fas and Fas ligand (FasL) expression in thymocytes. To examine the mechanism underlying DES-mediated regulation of Fas and FasL, we performed luciferase assays using T cells transfected with luciferase reporter constructs containing full-length Fas or FasL promoters. There was significant luciferase induction in the presence of Fas or FasL promoters after DES exposure. Further analysis demonstrated the presence of several cis-regulatory motifs on both Fas and FasL promoters. When DES-induced transcription factors were analyzed, estrogen receptor element (ERE), nuclear factor κB (NF-κB), nuclear factor of activated T cells (NF-AT), and activator protein-1 motifs on the Fas promoter, as well as ERE, NF-κB, and NF-AT motifs on the FasL promoter, showed binding affinity with the transcription factors. Electrophoretic mobility-shift assays were performed to verify the binding affinity of cis-regulatory motifs of Fas or FasL promoters with transcription factors. There was shift in mobility of probes (ERE or NF-κB2) of both Fas and FasL in the presence of nuclear proteins from DES-treated cells, and the shift was specific to DES because these probes failed to shift their mobility in the presence of nuclear proteins from vehicle-treated cells.

Together, the current study demonstrates that prenatal exposure to DES triggers significant alterations in apoptotic molecules expressed on thymocytes, which may affect T-cell differentiation and cause long-term effects on the immune functions.

  • Download the full study (free access) Prenatal exposure of mice to diethylstilbestrol disrupts T-cell differentiation by regulating Fas/Fas ligand expression through estrogen receptor element and nuclear factor-κB motifs, US National Library of Medicine National Institutes of Health, THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS,, NCBI PubMed PMC3477208, 2012 Nov.
  • Featured image credit bbc news health.
DES DIETHYLSTILBESTROL RESOURCES

Premature ovarian failures (POF)

DES exposure in utero is an obvious cause of POF

2013 Study Abstract

Premature ovarian failure (POF) is clinically suspected by amenorrhea and confirmed by an elevated FSH serum level above 40 mUI/L (even 20 mUI/L) twice, in a woman before the age of 40. Prevalence of POF is between 1 to 2% in women. In 90% of cases, no aetiology is identified.

Obvious causes are chemotherapy, pelvic radiotherapy, ovarian surgery and diethylstilbestrol exposure in utero.

A karyotype should be performed as Turner Syndrome is the most frequent genetic cause of POF. Some X abnormalities such as X deletion or X autosome translocation can be found. FMR1 pre-mutation (fragile X syndrome) should be searched for, even though no cases of mental retardation are known, in the family. Other genetic abnormalities can be suggested by associated symptoms (i.e.: FOXL2, SF1 mutations). Auto-immune aetiology can be suspected if other auto-immune features are present, however, there are no reliable auto-antibodies to confirm auto-immunity in POF. Treatment of POF is based on hormonal replacement therapy in order to avoid estrogen deficiency, suppress vasomotor symptoms and avoid bone loss as well as cardiovascular risk. Estrogens should be associated with progesterone or a progestin, at least up to the age of 51. Patients with POF should be informed that spontaneous pregnancies may occur (in 5% of cases).

In case of desire of pregnancy, the patient should be oriented to a specialized unit for in vitro fertilization with oocyte donation. Psychological support is essential and should be part of the treatment. POF is associated with an increased risk of emotional distress and depression. No preventive treatment of POF is available so far.

  • Premature ovarian failures, US National Library of Medicine National Institutes of Health, Presse médicale, NCBI PubMed PMID : 24157186, 2013 Nov.
  • Featured image credit sridevifertility.
DES DIETHYLSTILBESTROL RESOURCES