Clinical Studies on Stilbestrol

Diethylstilbestrol toxicity highlighted in 1940…

Study Abstract

In a previous paper we have reported our results on animal experiments concerning the toxicity and activity of stilbestrol.

We found that the close similarity of the action of natural and synthetic estrogenic substances on the female sex organs and the pituitary gland of immature, mature and castrated animals could be confirmed. Some evidence of toxicity could be observed in our animals after we administered doses of stilbestrol as low as 0.1 mg. three times a week, although the changes at higher dose levels were not as drastic and alarming as those reported by Loesser.

Clinically the drug has been used by numerous investigators of the Continent and recently also in the United States for the treatment of various ovarian disorders.

While the reports on the activity of the drug in the majority of cases are very encouraging, considerable discrepancy exists with regard to the untoward symptoms observed.

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DES DIETHYLSTILBESTROL RESOURCES

DES clinical experiment (prevention of late pregnancy complications), 1949

image of George and Olive Smith

The influence of diethylstilbestrol on the progress and outcome of pregnancy based on a comparison of treated with untreated primigravidas

1949 Study Abstract

In a clinical experiment aimed at determining the value of diethylstilbestrol in the prevention of the complications of late pregnancy, 387 primigravidous women in the prenatal clinic at the Boston Lying-in Hospital were given the drug in gradually increasing doses from the early part of pregnancy (weeks 12 to 20) to the thirty-sixth week. So far as was possible, alternate primigravidous women who presented themselves for prenatal care before the twentieth week were treated, the synchronous untreated patients, of whom there were 555, serving as controls. Except for stilbestrol administration, the obstetrical care of the two groups was identical.

The incidence of late pregnancy toxemia was very low (2.3 per cent) in the stilbestrol-treated patients. The difference between this figure and the 6.8 per cent incidence in the control series could not have occurred by chance. In the few cases that developed despite stilbestrol, the disease was later in onset and less severe than in the control group.

Analysis of the data on spontaneous premature delivery revealed that the premature infants of stilbestrol-treated mothers were unusually large and mature for their gestational ages. If prematurity is defined in terms of weight of the babies, the incidence of this abnormality was significantly less in the treated patients than in the controls. On the basis of week of delivery, on the other hand, there was no real difference between the two groups.

Postmaturity was significantly less frequent in the stilbestrol-treated patients than in the controls.

The incidence of unexplained stillbirth was 1.1 per cent in the controls and 0.5 per cent in the treated patients. This difference could have occurred by chance.

There were four fetal deaths in the stilbestrol-treated patients, an incidence of 1.0 per cent as against twenty-one, or 3.8 per cent, in the untreated patients; a highly significant difference. This reduction in fetal mortality would appear to be due largely to two factors: (1) the lower incidence and later onset of toxemia, and (2) the greater size and maturity of prematurely delivered infants.

A complete analysis of the data on the uncomplicated term pregnancies of the treated and control patients revealed no difference so far as the mothers were concerned (e.g., length of labor, uterine inertia, intrapartum or post-partum bleeding, weight gain). Analysis of the data on full-term infants, however, revealed that significantly more babies of stilbestrol-treated mothers weighed over eight pounds and were more than 21 inches long at birth.

Four years later, Dieckmann presented his prospective placebo controlled trial in an annual meeting of the American Gynecological Society (1953), the result of which contradicts the findings of Smith (above). During discussion time, Smith made a remark :

Our experience with the use of stilbestrol continues to be satisfactory … we are convinced that it has reduced the complications of late pregnancy and saved many babies“.

Smith was at this time an authority in the field and the objective evidence provided by Dieckman was largely ignored. Doctors continued prescribing DES to several million women over the next 20 years despite controlled studies with contrary findings…

Sources

  • American Journal of Obstetrics and Gynecology, 58, Issue 5 Pages 994–1009, November 1949.
  • Drs. George and Olive Smith featured image apache.be.
DES DIETHYLSTILBESTROL RESOURCES

Does the Administration of Diethylstilbestrol during Pregnancy have Therapeutic Value ?

Diethylstilbestrol usage was questioned by WJ Dieckmann in 1953

From the Department of Obstetrics and Gynecology of the University of Chicago and the Chicago Lying-in Hospital Chicago, Ill.

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DES DIETHYLSTILBESTROL RESOURCES

Transgenerational Epigenetic Inheritance: Focus on Endocrine Disrupting Compounds

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2014 Study Abstract

The classic case of an EDC is diethylstilbestrol (DES), an estrogen agonist and androgen receptor antagonist synthesized first in the 1930s and prescribed to at least 5 million women at risk for miscarriage or experiencing other reproductive problems, from 1938 up to 1975. Instead of the desired effects, use of this compound lead to increased incidence of breast, vaginal, and cervical cancers.

In addition, maternal exposure has documented adverse affects on daughters. These include the same types of cancers as well as a variety of difficulties conceiving and maintaining pregnancies, reproductive tract malformations, abnormal menstrual cycles, early puberty, and behavioral issues. The vast variety of effects is probably related to complexities introduced by the timing of DES treatment and doses. For example, a recent large study of women exposed prenatally to DES revealed a strong correlation between DES, particularly in the first trimester, and noncancerous uterine fibroids. Offspring of rodents exposed to DES during pregnancy recapitulate many of these effects.

Although the initial clinical studies were limited to female offspring, correlations between DES exposure and hypospadias, cryptorchidism, and testicular cancer have been reported in F1 and F2 sons and grandsons of women given DES. Analyses of the clinical studies suggest that the male reproductive illnesses are related to but not necessarily caused by estrogen actions. An alternative hypothesis is that DES produces low-birth-weight babies, and these infants are more prone to testicular dysgenesis syndrome. Multigenerational work in mice has demonstrated that high, but clinically relevant, doses of DES increase the incidence of uterine and other reproductive tract tumors in females and lesions in the male rete testes in F2 offspring. Few data on the critical F3 generation in humans are available nor are there experimental data from rodent models.

EDCs, such as DES, share many properties with steroid hormones: they act at low doses (picograms) and can act in a nonmonotonic manne. Like hormones, they are particularly effective during development, at which time they can modify the course of reproductive tract and brain development. Importantly, the EDCs are more promiscuous than steroids and bind to a larger variety of receptors than normal ligands, albeit with reduced affinities.

Sources

  • Transgenerational Epigenetic Inheritance: Focus on Endocrine Disrupting Compounds, Endocrinology, NCBI PubMed PMC4098001, 2014 Aug.
  • Featured image Matt Artz.
DES DIETHYLSTILBESTROL RESOURCES

Epigenomics in Environmental Health

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DES establishes altered epigenetic marks capable of transgenerational inheritance

2011 Study Abstract

Exposures to endocrine disrupting chemicals such as the model toxicant diethylstilbestrol (DES) and the omnipresent bisphenol A (BPA) are of particular concern in the context of development.

In utero exposure of mice to DES has been shown to result in the hypermethylation of the developmentally critical (specifically to uterine organogenesis) Hoxa10 gene.

Although not a genotoxic agent, epidemiologic evidence from individuals exposed to DES during the first 3 months in utero indicates an increase in vaginal clear cell carcinoma incidence and reproductive disorders as adults.

In addition, grandchildren of DES exposed women reported higher incidences of rare reproductive disorders; whether this reflects detection bias or possibly implicates a role for epigenetic transgenerational inheritance remains to be clarified.

Ruden et al. drawing similarities between DES and Hsp90 has proposed a mechanism through which DES establishes altered epigenetic marks capable of transgenerational inheritance, wherein DES plays a role in modifying H3K4 methylation by increasing the activity of the H3K4 methyltransferase SMYD3, thereby altering epigenetic control of various genes.

Sources

  • Full study (free access) : Epigenomics in Environmental Health, Journal of andrology, Frontiers in Genetics, NCBI PubMed PMC3268636, 2011.
  • Featured image by sciencemag.
DES DIETHYLSTILBESTROL RESOURCES

DES-induced change in AR immunoexpression

Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol

2001 Study Abstract

This study evaluated whether androgen action is altered in rats treated neonatally with diethylstilbestrol (DES) at a dose that induced reproductive tract abnormalities.

Rats were treated on alternate days 2-12 with 10 microg DES and studied on Day 18. DES-induced abnormalities included a 70% reduction in testis weight, distension and overgrowth of the rete, distension and reduction in epithelial height of the efferent ducts, underdevelopment of the epididymal duct epithelium, reduction in epithelial height in the vas deferens, and convolution of the extra-epididymal vas.  In DES-treated rats, androgen receptor (AR) immunoexpression was virtually absent from all affected tissues and the testis, whereas AR expression in controls was intense in epithelial and stromal cells. The DES-induced change in AR immunoexpression was confirmed by Western analysis for the testis.

In rats treated neonatally with 1 microg DES, reproductive abnormalities were absent or minor, except for a 38% reduction in testis weight; loss of AR immunoexpression also did not occur in these rats. Treatment-induced changes in AR expression were paralleled by changes in Leydig cell volume per testis (91% reduction in the 10-microg DES group; no change in the 1-microg DES group).

To test whether suppression of androgen production or action alone could induce comparable reproductive abnormalities to 10 microg DES, rats were treated neonatally with either a potent gonadotropin-releasing hormone antagonist (GnRHa) or with flutamide (50 mg/kg/day). These treatments reduced testis weight (68% for GnRHa, 40% for flutamide), and generally retarded development of the reproductive tract but failed to induce the abnormalities induced by 10 microg DES. GnRHa and flutamide caused no detectable change in AR immunoexpression in target tissues, with the exception of minor changes in the testes of flutamide-treated males. GnRHa treatment caused a reduction (83%) in Leydig cell volume comparable to that caused by 10 microg DES. Immunoexpression of estrogen receptor alpha (ER alpha) in the efferent ducts and of ER beta in all tissues studied were unaffected by any of the above treatments. Neonatal coadministration of testosterone esters (TE; 200 microg) with 10 microg DES prevented most of the morphological abnormalities induced by 10 microg DES treatment alone, though testis weight was still subnormal (46% reduction in DES + TE vs 72% in DES alone and 49% with TE alone) and some lumenal distension was still evident in the efferent ducts. Coadministration of TE with DES prevented DES-induced loss of AR immunoexpression (confirmed for testis by Western blot analysis).

It is concluded that

  1. reproductive tract abnormalities induced in the neonatal male rat by a high (10 microg) dose of DES are associated with reduced AR expression and Leydig cell volume;
  2. these changes are largely absent with a lower dose of DES (1 microg);
  3. treatments that interfere with androgen production (GnRHa) or action (flutamide) alone failed to induce reproductive tract abnormalities or alter AR expression as did 10 microg DES;
  4. a grossly altered androgen:estrogen balance (low androgen + high estrogen) may underlie the reproductive tract abnormalities, other than reduced testis weight, induced by high doses of DES.

Sources

  • Suppression of androgen action and the induction of gross abnormalities of the reproductive tract in male rats treated neonatally with diethylstilbestrol, Journal of andrology, NCBI PubMed PMID: 11229807, 2001 Mar-Apr.
  • Featured image by Lurm.
DES DIETHYLSTILBESTROL RESOURCES

DES multigenerational assay data interpretation

Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning

2002 Study Abstract

The objective of the study was to determine which period of exposure produces the most marked effects on the reproductive capacity and sexual development of the rat, with particular emphasis on the relative sensitivity of in utero and postnatal exposures.

The endocrine active chemical, diethylstilbestrol (DES) was used as an agent known to affect many of the endpoints examined. Hitherto, such comparisons have been made between studies, rather than within a study. Our data will be helpful in the interpretation of future multigenerational assay data.

In preliminary studies, DES was shown to be active in the immature rat uterotrophic assay with a lowest detected dose of 0.05 mg DES/kg body weight by sc injection and 10 mg DES/l (1.6 mg DES/kg body weight) by administration in drinking water. A dose of 60 microg DES/l drinking water ( approximately 6.5mg DES/kg body weight/day) was selected for the main study since this represented the midpoint of the drinking water uterotrophic dose response and produced no overt maternal toxicity. The study used 10 groups of concomitantly pregnant animals, including 2 control groups. The first comparison was between the effects of exposure to DES in utero, and exposure from conception to weaning. Another group of animals was exposed to DES in utero and cross-fostered to untreated pregnant females to prevent lactational transfer of DES to pups. Two groups were exposed to DES neonatally, either from birth to postnatal day (PND) 10 (pups thus having only lactational exposure), or from birth until weaning (PND 21; pups thus having both lactational exposure and self-exposure via drinking water). In addition, a dose response study to DES was conducted on animals exposed from weaning to PND 100, when the first phase of the study was terminated. Pups exposed to DES in utero and pups exposed from weaning to PND 100 were bred to assess fertility of the F1 animals and the sexual development of F2 offspring. This last comparison was to determine the extent to which weanling rats could be used in endocrine toxicity studies to assess their potential to show activity in utero. The most sensitive period of exposure for inducing developmental effects in F1 animals was from weaning onwards. The neonatal to weaning period (PND 1-21) was the next most sensitive. Essentially no effects were induced in F1 animals exposed in utero. No effects of any kind were observed in animals only exposed over the early neonatal period of PND 1-10. The mean day of vaginal opening, testes descent, and prepuce separation was only altered in groups where postnatal exposure to DES continued beyond PND 10, or was started at weaning. No changes were observed in anogenital distance or caudal sperm counts. Some changes in organ weights were observed, but the interpretation of these was often confused by concomitant changes in body weight. In general, histopathological examination of tissues yielded no additional information. In breeding studies with animals exposed to DES in utero, or from weaning, reduced litter sizes and marginal advances in the day of vaginal opening were observed in the offspring, together with changes in organ weights. However, no unique sensitivity was noted for exposure in utero. Evaluation of the several exposure periods and the many markers monitored in this study may have individual strengths in individual cases, but when rigorously compared using the reference estrogen DES, many preconceptions regarding their absolute or relative value were not upheld. Further, each of these markers is subject to natural variability, as demonstrated by comparisons made among the 5 separate control groups available in parts of the present study. This variability increases the chance that small changes observed in endocrine toxicity studies employing small group sizes and a single control group, or no concomitant control group, may be artifactual. The most marked effects observed in this study were on the developmental landmarks in the F1 animals induced by exposures after PND 10. Some effects on developmental landmarks and organ weights were observed in F2 animals following exposure either in utero or postweaning.

This study therefore does not establish a unique role for exposures in utero or during the early neonatal period.

Sources

  • Comparison of the developmental and reproductive toxicity of diethylstilbestrol administered to rats in utero, lactationally, preweaning, or postweaning, Toxicological sciences : an official journal of the Society of Toxicology, NCBI PubMed PMID: 12075118, 2002 Jul.
  • Featured image NASA.
DES DIETHYLSTILBESTROL RESOURCES

DES alteration of StAR protein and P450c17 gene

Diethylstilbestrol inhibits the expression of the steroidogenic acute regulatory protein in mouse fetal testis

2004 Study Abstract

This study investigated the early deleterious effects of an in-utero exposure to diethylstilbestrol (DES) on mouse testicular development.

To that purpose, pregnant mice were injected daily with up to 100 microg/kg DES from 10.5 to 17.5 days postcoitum (dpc). At 18.5 dpc, testes were removed from fetuses for RNA (RT-PCR) and protein (Western blot, immunohistochemistry) analysis.

Twenty-two genes were selected among which transcription factors, markers of differentiation of the different testicular cell lineages, steroidogenic enzymes and hormone receptors.

The Steroidogenic Acute Regulatory (StAR) protein produced by the fetal Leydig cells was dramatically reduced in the DES-exposed testes.

The P450c17 was the other gene modified following DES exposure.

The alteration of these two genes is consistent with the decrease observed in the intratesticular testosterone levels, in the DES-exposed testes.

Collectively, we demonstrated that DES did not alter testicular cell lineage specification but that it strongly inhibited the major function of the fetal Leydig cells.

Sources

  • Diethylstilbestrol inhibits the expression of the steroidogenic acute regulatory protein in mouse fetal testis, Molecular and cellular endocrinology, NCBI PubMed PMID: 15196701, 2004 May 31.
  • Cells featured image by Henry de Valence.
DES DIETHYLSTILBESTROL RESOURCES

Hypospadias in DES grandsons : a cohort study

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Hypospadias: a transgenerational effect of diethylstilbestrol ?

2002 Study Abstract

BACKGROUND
Transgenerational effects of diethylstilbestrol (DES) have been reported in animals, but effects in human beings are unknown. Alerted by two case reports, we aimed to establish the risk of hypospadias in the sons of women who were exposed to DES in utero.

METHODS
We did a cohort study of all sons of a Dutch cohort of 16284 women with a diagnosis of fertility problems. We used a mailed questionnaire assessing late effects of fertility treatment to identify boys with hypospadias. We compared the prevalence rate of hypospadias between boys with and without maternal DES exposure in utero.

FINDINGS
16284 mothers (response rate 67%) reported 8934 sons. The mothers of 205 boys reported DES exposure in utero. Four of these children were reported to have hypospadias. In the remaining 8729 children, only eight cases of hypospadias were reported (prevalence ratio 21.3 [95% CI 6.5-70.1]). All cases of hypospadias were medically confirmed. Maternal age or fertility treatment did not affect the risk of hypospadias. Children conceived after assisted reproductive techniques such as in-vitro fertilisation were not at increased risk of hypospadias compared with children conceived naturally (1.8, 0.6-5.7).

INTERPRETATION
Our findings suggest an increased risk of hypospadias in the sons of women exposed to DES in utero. Although the absolute risk of this anomaly is small, this transgenerational effect of DES warrants additional studies.

Sources

  • Hypospadias in sons of women exposed to diethylstilbestrol in utero: a cohort study, Lancet, NCBI PubMed PMID: 11943257, 2002 Mar 30.
  • Featured image Donna Borzyskowski.
DES DIETHYLSTILBESTROL RESOURCES

Epigenetic Transgenerational Actions of Endocrine Disruptors

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Evidence that DES has effects in multiple generations have been reported

2006 Study Abstracts

Endocrine disruptors have recently been shown to promote an epigenetic transgenerational phenotype involving a number of disease states (e.g. male infertility). The anti-androgenic fungicide vinclozolin was found to act transiently at the time of embryonic sex determination to promote in the F1 generation a spermatogenic cell defect and subfertility in the male. When the animals were allowed to age up to 1 yr, a number of other disease states developed. This phenotype was transferred through the male germ line to all subsequent generations analyzed (F1–F4). The ability of an environmental factor (i.e. endocrine disruptor) to promote an epigenetic transgenerational phenotype impacts the potential hazards of environmental toxins, mechanisms of disease etiology, and evolutionary biology. The biological importance of the epigenetic actions of environmental agents is reviewed in the context of the primordial germ cell and development of epigenetic transgenerational phenotypes.

Transgenerational Phenomena and Environmental Factors

… Examples of environmental factors during embryonic development that influence the F1 generation include the effects of heavy metals causing cancer, abnormal nutrition that causes diabetic and uterine defects, chemical exposure (i.e. ethosuximide and benzpyrene) causing brain and endocrine defects, and endocrine disruptors such as diethylstilbestrol , phthalates, and dithiothreitol causing reproductive tract and endocrine defects. … Therefore, exposure to a number of environmental factors in utero can cause abnormal phenotypes in the F1 generation in a number of different species. Because the F1 generation is exposed to the environmental factor, the F1 effect is not a transgenerational phenotype.

Transgenerational effects of environmental factors require effects minimally on the F3 generation. This is because the F3 generation is the first generation not directly exposed to the environmental factor. The ability of an external agent to induce a transgenerational phenotype requires a genetic (i.e. DNA sequence) or an epigenetic (i.e. DNA methylation) phenomenon mediated through the germ line . Transgenerational inheritance of an epigenetic state has been shown to occur using several mouse genetic lines and markers and more recently with the use of monozygotic twins with epigenetic differences. … Environmental toxins such as endocrine disruptors have also been shown to influence the F1 generation after parental exposure, but few have demonstrated transgenerational effects on multiple generations. Some evidence that diethylstilbestrol has effects in the F2 generation have been reported.

Endocrine Disruptors and Reproductive Toxicology

… Neonatal exposure to estrogen alters the ER-α and ER-β expression during postnatal testis and hypothalamic/pituitary development Interestingly, neonatal exposure to the estrogenic compound diethylstilbestrol promotes abnormal testis and male reproductive tract development and leads to changes in gene expression. Therefore, actions of estrogenic endocrine disruptors on estrogen receptors may impair normal fetal gonadal development and lead to infertility. …

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DES DIETHYLSTILBESTROL RESOURCES