DES mediates immune suppression via modulation of microRNA expression

DES-mediated immune suppression may be due to mechanisms involving apoptosis, dysregulation in gene expression, changes in miR profile and regulation of T cell differentiation, 2014

Study Abstract

Diethylstilbestrol (DES) is a synthetic compound and considered as an endocrine disruptor that interferes with the normal development of human organs and even immune tissues.

DES has been investigated for its immunosuppressive effects, thymic atrophy, and various types of cancer including breast cancer, ovarian cancer, uterine cancer, etc.

In this study, we investigated DES regulated mechanisms in T cells and examined whether DES exposure of T cells leads immune suppression and affects immune functions. To this end, we investigated the effect of DES on T cells in vitro and specifically examined DES-induced apoptosis in T cells, expression of genes involved in apoptosis, and differentiation of T helper cells in vitro.

We observed significant increase in apoptosis in activated T cells in dose-dependent manner in vitro. Upon examination of FasL expression in activated T cells, there was a significant increase in FasL expression in activated T cells post DES treatment. Furthermore, upon examination of microRNA (miR) profile in activated T cells, there was a dysregulation in a large number of miRs by DES. Out of a total of 885 miRs screened, there were 217 miRs showing more than 1.5 fold and 101 miRs showing 2.0 fold or more differential expression in DES group when compared to vehicle-treated group.

Next, the immunosuppressive effect of DES was tested using an mouse model of delayed type hypersensitivity (DTH). Interestingly, DES treatment decreased the DTH responses and reversed the inflammation triggered by methylated bovine serum albumin (mBSA). Also, there was a significant increase in regulatory T (Tregs) cells but suppression of both Th1 and Th17 cells in mice that received mBSA associated with DES [mBSA+DES] treatment when compared to mBSA associated with vehicle (VEH) [mBSA+VEH] treatment.

Taken together, the results obtained from this study demonstrate that DES-mediated immune suppression may be due to mechanisms involving apoptosis, dysregulation in gene expression, changes in miR profile and regulation of T cell differentiation.

Reference

DES DIETHYLSTILBESTROL RESOURCES

Molecular mechanisms through which DES triggers thymic atrophy, specifically autophagy

Diethylstilbestrol (DES) induces autophagy in thymocytes by regulating Beclin-1 expression through epigenetic modulation

2018 Study Abstract

Diethylstilbestrol (DES) is an endocrine disruptor that was used to prevent adverse effects of pregnancy in women in late 1940s until early 1970s. Its use was banned following significant toxicity and negative effects not only in the mothers but also transgenerationally.

Previous studies from our laboratory showed that DES induces thymic atrophy and immunosuppression in mice.

In this study, we investigated the molecular mechanisms through which DES triggers thymic atrophy, specifically autophagy.

To that end, we treated C57BL/6 mice with DES, and determined expression of two autophagy-related proteins, microtubule-associated protein-1 light chain 3 (LC3) and Beclin-1 (Becn1).

We observed that DES-induced thymic atrophy was associated with increased autophagy in thymocytes and significant upregulation in the expression of both Becn1 and LC3. DES also caused downregulation in the expression of miR-30a in thymocytes, and transfection studies revealed that miR-30a targeted Becn1. Upon examination of methylation status of Becn1, we noted hypomethylation of Becn1 in thymocytes of mice exposed to DES.

Together, these data demonstrate for the first time that DES induces autophagy in thymocytes potentially through epigenetic changes involving hypomethylation of Becn1 and down-regulation of miR-30a expression.

Reference

  • Diethylstilbestrol (DES) induces autophagy in thymocytes by regulating Beclin-1 expression through epigenetic modulation, Toxicology, NCBI PubMed PMID: 30153466, 2018 Dec.
  • Featured image sweattlab/daves-art.
DES DIETHYLSTILBESTROL RESOURCES

Demographic, lifestyle, and reproductive risk factors for ectopic pregnancy

DES Daughters have a much higher risk of EP,
Fertility and sterility, 2018

Abstract

OBJECTIVE
To evaluate the relationship between demographic, lifestyle, and reproductive factors and the risk of ectopic pregnancy (EP).

DESIGN
Prospective cohort.

SETTING
United States.

PATIENT(S)
Nurses’ Health Study II cohort comprising 41,440 pregnancies from 22,356 women.

INTERVENTION(S)
Demographic, lifestyle, and reproductive factors self-reported in 1989 then updated every 2 years. Multivariable log-binomial regression models with generalized estimating equations were used to estimate adjusted risk ratios (aRR).

MAIN OUTCOME MEASURE(S)
Ectopic pregnancy.

RESULT(S)
Incident EP was reported in 411 (1.0%) pregnancies. Former and current smokers had 1.22 (95% confidence interval [CI], 0.97-1.55) and 1.73 (95% CI, 1.28-2.32) times, respectively, the risk of EP compared with never smokers. The risk of EP 10 years after quitting was similar to never smokers (aRR 0.90; 95% CI, 0.60-1.33). Women consuming ≥10 g/day of alcohol had 1.50 (95% CI, 1.08-2.09) times the risk of EP compared with never consumers. In utero exposure to diethylstilbestrol (aRR 3.55; 95% CI, 2.51-5.01), earlier initiation of oral contraceptives (aRR 2.64; 95% CI, 1.70-4.09 for <16 years vs. never), intrauterine device use (aRR 3.99; 95% CI, 2.06-7.72), or history of infertility (aRR 3.03; 95% CI, 2.48-3.71) or tubal ligation (aRR 16.27; 95% CI, 11.76-22.53) also were associated with a higher risk of EP.

CONCLUSION(S)
Women who were current or former smokers, consumed ≥10 g/day of alcohol, were exposed to diethylstilbestrol in utero, initiated oral contraceptives at earlier than age 16 years (which may be a marker of riskier sexual behaviors), and who had a history of infertility, intrauterine device use, or tubal ligation had a higher risk of EP.

Reference

  • Demographic, lifestyle, and reproductive risk factors for ectopic pregnancy, Fertility and sterility, NCBI PubMed PMID: 30503132, 2018 Dec.
  • Featured image : Transvaginal ultrasound images of an intrauterine pregnancy (IUP) and ectopic pregnancy. (A) An IUP at 6 weeks. The central dark area is the intrauterine gestational sac and within the sac is a circular ringed structure that is the yolk sac. The small oval structure below the yolk sac is the fetus. (B) An ectopic pregnancy. To the right of the image is the normal uterus and to the left of the uterus is the doughnut-shaped ectopic pregnancy – credit BMJ Faculty of Sexual & Reproductive Healthcare of the Royal College of Obstetricians & Gynaecologists.
DES DIETHYLSTILBESTROL RESOURCES

Pharmacologic and Environmental Endocrine Disruptors in the Pathogenesis of Hypospadias

a Review – Current environmental health reports, 2018

Abstract

PURPOSE OF REVIEW

Endocrine disrupting chemicals (EDCs) potentially have a role in causing hypospadias malformation through modifiable in-utero exposure. Considering the emerging literature on the role of potential endocrine disrupting substances on the occurrence of hypospadias and the potential to inform public health efforts to prevent the occurrence of these malformations, we have summarized the current literature, identified areas of consensus, and highlighted areas that warrant further investigation.

RECENT FINDINGS

Pharmaceuticals, such as diethylstilbestrol, progestin fertility treatments, corticosteroids, and valproic acid, have all been associated with hypospadias risk. Data on exposure to dichlorodiphenyltrichloroethane and hexachlorobenzene pesticides, as well as non-persistent pollutants, particularly phthalates, is less consistent but still compelling. Improving exposure assessment, standardizing sample timing to relevant developmental windows, using clear case identification and classification schemes, and elucidating dose-response relationships with EDCs will help to provide clearer evidence. Promising directions for future research include identification of subgroups with genetic hypospadias risk factors, measurement of intermediate outcomes, and study of EDC mixtures that will more accurately represent the total fetal environment.

Exogenous Sex Steroids

Estrogens were the first chemicals to be studied in the context of maternal exposure and hypospadias. The synthetic nonsteroidal estrogen, diethylstilbestrol (DES), is a known carcinogen formerly administered to pregnant women to prevent miscarriage prior to evidence of adverse health effects and a lack of efficacy for that indication.

An early cohort study conducted in the Netherlands identified four cases of hypospadias among 205 sons of women exposed to DES in utero (~ 2% prevalence) versus 8 cases out of 8729 sons of mothers without DES exposure (0.09% prevalence). This study observed a strong association (prevalence odds ratio (pOR) 21.3; 95% CI 6.5–70.1) between maternal in utero DES exposure and hypospadiac son.

In a US cohort study, maternal DES exposure was related to a higher but not statistically significant increase in risk of offspring hypospadias with ten cases per 2552 live births from exposed mothers and three cases per 1336 live births from unexposed mothers (pOR 1.7; 95% CI 0.4–6.8).

A case-control study surveying 834 mothers with 251 hypospadiac children observed that women exposed to DES in utero were nearly five times more likely to have infants with hypospadias (OR 4.9; 95% CI 1.1–22.3).

A French, multigenerational cohort study also observed a relation between maternal DES exposure during pregnancy and increased prevalence of hypospadias for the next two generations, suggesting that the underlying biological mechanism may be epigenetic

Thus, studies on DES raise the possibility of an association with hypospadias. While no longer prescribed, DES is similar in chemical structure with other xenobiotic compounds and thus, the epidemiologic findings are still of relevance. It is important to note, however, that the studies of DES exposure all suggest a possible epigenetic effect on the development of hypospadias in later generations. While DES is historically important and chemically relevant to this discussion, its effect may derive from interaction with the maternal oocyte rather than the developing male penis itself.

References

  • Pharmacologic and Environmental Endocrine Disruptors in the Pathogenesis of Hypospadias: a Review, Current environmental health reports, NCBI PubMed PMID: 30578470, 2018 Dec.
  • Featured image springer.
DES DIETHYLSTILBESTROL RESOURCES

Pregnancy Drugs, Fetal Germline Epigenome, and Risks for Next-Generation Pathology

A Call to Action, Environmental and Molecular Mutagenesis,
Escher J, Robotti S, 19 March 2019.

Abstract

Drugs taken during pregnancy can affect three generations at once:

  • the gestating woman (F0),
  • her exposed fetus (F1),
  • and the fetal germ cells that confer heritable information for the grandchildren (F2).

Unfortunately, despite growing evidence for connections between F0 drug exposures and F2 pathology, current approaches to risk assessment overlook this important dimension of risk.

In this commentary, we argue that the uniquemolecular vulnerabilities of the fetal germline, particularly with regard to global epigenomic reprogramming, combined withempirical evidence for F2 effects of F1 in utero drug and other exposures, should change the way we consider potential long-term consequences of pregnancy drugs and alter toxicology’s standard somatic paradigm.

Specifically, we

  1. suggest that pregnancy drugs common in the post-war decades should be investigated as potential contributorsto the “missing heritability” of many pathologies now surging in prevalence;
  2. call for inclusion of fetal germline risks in pregnancy drugsafety assessment;
  3. and highlight the need for intensified research to ascertain generational impacts of diethylstilbestrol (DES), a vanguard question of human germline toxicity.

Only by fully addressing this important dimension of transplacental exposure can we responsibly evaluate safety of drug exposures during pregnancy and convey the full scope of risks, while also retrospectively comprehending the generational legacy of recent history’s unprecedented glut of evolutionarily novel intrauterine exposures.

The imperative to intensify research on diethylstilbestrol F2s

“Finally, we ask that research to ascertain generational impacts of diethylstilbestrol (DES) should be intensified and broadened in scope. The DES disaster presents a paradigmatic question of human germline toxicity, and a unique opportunity to better understand generational impacts of this drug, and also the broader phenomenon of hormone disruption in humans. But while research on DES F2s has linked the exposure to a variety of pathologies, including increased risks of infertility, hypospadias, tumor growth, ovarian cancer, menstrual irregularities, and ADHD, by and large the research has been limited compared to the magnitude of exposure and the breadth of possible ensuing pathologies.

For example, the issue of neurodevelopmental outcomes and socio-sexual behavior strikes us as very important and mostly unexplored. Only one study has attempted to ascertain F2 neurodevelopmental impairments, and it indeed detected a link. Though a recent study found no evidence of same-sex orientation in female F2s borne of female DES exposed F1s, no other study has probed socio-sexual outcomes in F2s other either sex, whether through male or female F1s. We note that a recent animal study found these types of F2 effects when F0 dams were exposed to EDCs. In other words, research must think more broadly about F2 pathologies precipitated by DES exposure to also encompass the brain, cognitive ability,behavior, sexuality, and other crucial endpoints beyond the standard teratogenesis paradigm.

The urgency of more F2 research is critical not just for our affected families, but for the science of endocrine disruption generally and an entire population increasingly worried about generational effects of hormone-disrupting chemicals. DES is the bellwether of hormone disruption—given the combined existence of exposure records and possibilities for ascertainment of F2 outcomes, it uniquely shines a light on this crucial scientific question of germline and heritable effects of disrupted hormone signaling.

The primary research study looking at DES F2 outcomes in the United States is the National Cancer Institute (NCI) DES Combined Cohort Follow-Up Study formed in 1992. It consists of eight different cohort studies which totaled about 21,000 participants at inception and included DES-exposed F0 mothers, DES F1 daughters, and DES F1 sons, as well as unexposed mothers, daughters and sons. The oldest cohort started in 1971, the most recent in 1984, and two are British studies. Almost all the studies look at F2 outcomes via F1 exposed females, but not the F1 exposed males. In the initial decade, all participants of the largest of the eight cohorts, the Diethylstilbestrol Adenosis Project (DESAD), were physically examined yearly. Since that time the research inputs consist of questionnaires every five years.
While some subjects have been lost to the study due to location mobility and death, extensive attempts are made to seek all participants. Decreasing ability to do robust follow-up concerns us. We are just beginning to appreciate the effects on F2s, and indeed many effects on F1s as well, and while several other teams have published significant DES studies, many of those other efforts lack access to a database of people with confirmed medical histories of DES exposure, which presents a limitation.

A withering of efforts from what had been the primary source of research on the effects of DES represents a loss of an opportunity to learn critical lessons for understanding what may be at stake when early germ cells undergo programming in a foreign hormonal milieu.”

References

DES DIETHYLSTILBESTROL RESOURCES

The Effect of Structural Diversity on Ligand Specificity and Resulting Signaling Differences of Estrogen Receptor α

High mobility of the H9-H10 loop induced by DES is correlated with the significant up-regulation of specific gene expression

2019 Study Abstract

Numerous chemicals have been reported to exert estrogen-like endocrine disrupting effects via a receptor binding mechanism that directly interacts with the ligand binding domain (LBD) of estrogen receptor α (ERα). However, not only their binding affinities to ERα but also their interference in specific cell and tissue functions are clearly different. In this regard, significant regulation differences among three representative estrogenic chemicals (diethylstilbestrol (DES), bisphenol A (BPA) and diarylpropionitrile (DPN)), well-known ERα agonists with very similar structures, have been observed. Molecular dynamics (MD) simulation is used to explore the underlying mechanism of different regulation effect induced by the similar estrogen-like chemicals. DES induced 12 Å motion of the H9-H10 loop markedly expands the negative electrostatic potential surface of the AF-2 domain, which is consistent with the over-regulation effect of the agonist. In comparison, the 3 Å motion induced by BPA and DPN corresponds to the low-regulation effect of the chemicals. Cross-correlation analysis indicates that the different ERα motions and resulting surface feature of AF-2 domain is brought by the distinguished binding modes of the agonists. Moreover, only hydrophobic DES with estrogen-like size and flexibility has high binding affinity of -23.47 kcal/mol binding free energy. Both the hydrophilic group in DPN and the small molecular size of BPA dramatically decrease the agonist binding ability, and their binding free energies are only -12.43 kcal/mol and -11.82 kcal/mol, respectively. Our study demonstrates that similar chemicals interact differently with ERα and induce different allosteric effects, which explains the observed regulation diversity.

Reference

  • The Effect of Structural Diversity on Ligand Specificity and Resulting Signaling Differences of Estrogen Receptor α, Chemical Research in Toxicology, NCBI PubMed PMID: 30924335.
DES DIETHYLSTILBESTROL RESOURCES

Grandmaternal DES and ADHD in Children

Dr Kioumourtzoglou‘s reply to comments in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

We appreciate the interest that Drs Ryan and Smith and Dr Costas have expressed in our article.

Drs Ryan and Smith raise the issue of exposure misclassification and the potential problem of overreporting of grandmaternal diethylstilbestrol (DES) exposure by mothers whose children have attention-deficit/hyperactivity disorder (ADHD).

First, we were not clear in the article, but the κ of 0.74 for nurse and grandmother reporting was specific for DES.

We also found that the DES-specific κ value did not vary by ADHD status.

Concern for biased recall does need to be considered, and our results should indeed be interpreted cautiously, while hopefully spurring more work in this area.

That said, some aspects of our study differ from the military study Drs Ryan and Smith reference.

When the nurses reported their mothers’ DES exposure (1993), there had been no human studies of any third-generation effects and possibly only 1 mouse study of tumors. Thus, the level of attention to potential anthrax effects on those directly exposed was not there for third-generation DES effects in 1993.

In addition, the nurses were not queried about ADHD in their children until, at earliest, 12 years after the question about their mothers’ DES exposure (2005). So the possibility of priming the nurse to think about a possible DES-ADHD association by asking about exposure and outcome at the same time was avoided.

Additionally, the specificity of elevated effects in the first trimester would argue against recall bias. In our models, we simultaneously included terms for unknown trimester DES exposure and exposure in other trimesters as a check for confounding, which also works as a check for recall bias in this case.

Indeed, one might expect that nurses falsely recalling grandmaternal DES exposure would be more likely to report that they did not know the trimester of exposure, yet that group did not exhibit increased odds of ADHD.

In addition to these points, in responding to this letter we conducted the same analysis restricted to the 18 792 F0 women (42 097 F2 children) who provided information themselves about DES use during pregnancy with F1, instead of F1 reporting. In these analyses, we found virtually the same result, although with wider 95% CI as expected with the smaller numbers: adjusted odds ratio, 1.31 (95% CI, 1.00-1.71).

References

DES DIETHYLSTILBESTROL RESOURCES

Grandmaternal DES and ADHD in Children – Costas’ Question

Dr Costas editorial comment in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

“I read with interest the article “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits” by Kioumourtzoglou et al,  published in JAMA Pediatrics.

Using a valuable cohort of 47 450 women in the Nurses’ Health Study II, the authors found that exposure to the potent synthetic estrogen diethylstilbestrol during pregnancy was associated with increased risk of attention-deficit/hyperactivity disorder (ADHD) in the grandchildren.

They reasonably conclude that “diethylstilbestrol exposure is associated with multigenerational neurodevelopmental deficits.”

This result is a valuable contribution to the literature on etiology of neurodevelopmental disorders, adding further evidence for the role of endocrine-disrupting chemicals as risk factors for ADHD.

The etiology of ADHD is complex, involving a combination of genetic as well as environmental risk factors.

Heritability for ADHD has been estimated as 70% to 80%.

Ehe genetics basis of ADHD is partly owing to many variants of individual low effect scattered along the genome.” …

References

DES DIETHYLSTILBESTROL RESOURCES

Grandmaternal DES and ADHD in Children – Ryan’s Question

Drs Ryan and Smith editorial comment in ref to “Association of Exposure to Diethylstilbestrol During Pregnancy With Multigenerational Neurodevelopmental Deficits“, 2018

“We read with interest the publication by Kioumourtzoglou et al, identifying a link between grandmaternal diethylstilbesterol (DES) exposure and attention-deficit/hyperactivity disorder in children.

We certainly agree with the JAMA Pediatrics Editorial that this transgenerational association is novel and provocative.” …

References

  • Continue readingGrandmaternal Diethylstilbesterol and Attention-Deficit/Hyperactivity Disorder in Children, on JAMA Pediatrics doi:10.1001/jamapediatrics.2018.3737, December 2018.
  • Featured image by psycom.
DES DIETHYLSTILBESTROL RESOURCES

Possible links to the development of obesity

Diethylstilboestrol—A long-term legacy, School of Biosciences, 2012

Abstract

Obesity and type 2 diabetes levels have risen over the past century, the incidence being more marked in recent years. Both these conditions have adverse consequences and are significant public health issues. Even pets, laboratory animals and urban rats have increased in average body weight over the past decades. These trends in both humans and animals are not necessarily explicable by diet and exercise; prenatal exposure to environmental triggers (‘obesogens’), has been suggested as a possible factor, particularly to oestrogenic compounds such as DES, bisphenol A and phthalates.

Adipose tissue acts as an endocrine organ, releasing
hormones controlling appetite and energy metabolism and is a site for oestrogen synthesis. This mechanism, found in both animals and human beings, ensures a link between the food supply and the capacity to reproduce, since starvation and pregnancy are not a good combination. When low doses of DES were administered to mice pre- or neo-natally, the adult animals gained weight with altered expression of obesity-related genes and altered hormone levels. There was no difference in the number of fat cells but the cells already present increased in size.

Although it is not known at present whether DES acts as an obesogen in man, raised urinary concentrations of other environmental chemicals, such as phthalates, have been linked with the increased body weight and insulin resistance which lead to ‘metabolic syndrome’.

References

DES DIETHYLSTILBESTROL RESOURCES