DES Gene Changes and Proposed Mechanisms of Action

image of Mechanisms involved in breast cancer etiology

Exposure to Diethylstilbestrol during Sensitive Life Stages: A legacy of heritable health effects

2013 Study Abstract

The fact that DES causes developmental changes in the second generation through gestational exposure has required evaluation of the mechanisms involved in several target tissues. DES is classified as a carcinogen by the World Health Organization, U.S. Environmental Protection Agency, National Toxicology Program, and the International Agency for Cancer Research. Studies on the genotoxicity of DES in humans have not revealed striking outcomes; to date, it does not change ploidy patterns, cause specific mutations known to induce high risk of breast cancer, or induce loss of heterozygosity of allelic imbalance. In directed in vitro tests, the data on induction of sister chromatid exchange, induction of micronuclei, and unscheduled DNA synthesis, were negative or equivocal. However, DES caused aneuploidy, induced adduct formation in mitochondrial DNA, and altered the ability of microtubules to form.

DES is also known to affect endocrine sensitive tissues and may have hereditary effects due to DNA modifications ; like many other breast cancer risk factors, it may have multiple mechanisms of action, depending on the target tissue. Through molecular studies many potential mechanisms of action have been proposed among them are different genetic and epigenetic pathways that have been implicated in the DES-induced carcinogenesis and reproductive developmental abnormalities seen in humans and animals. These effects appear to occur in specific target tissues and may be related to gene expression of the ER-α at the time of exposure. DES may create an environment conducive to the development of cancer over time.

In mouse models, pre-and neonatal DES exposure induces a wide range of gene expression changes that persist into adulthood. Molecular mechanistic studies have shown that many of the changes caused by DES, including structural and cellular abnormalities, are caused by altered programming of hox and wnt genes which play roles in reproductive tract differentiation. DES potentially inhibits the expression of wnt7a, hoxa10, and hoxa11 during critical periods of reproductive tract development. Changes in hox gene expression have led to abnormalities in tissues that depend on their expression for normal developmental signaling. Down regulation of hoxa11 (which is found in the stroma and epithelial cells of the uterus) may be partly responsible for DES-induced uterine malformations, as similar malformations are seen in hoxa11-null mice. Hoxa10 (which is expressed in the uterine horns) controls uterine organogenesis and its expression is increased in cultured human endometrial cells but repressed in mice after in utero exposure to DES. Female mice exposed to DES in utero had aberrant methylation in the promoter and intron of hoxa10, which persisted into adulthood.

Genetic modifications by DES have also been implicated in the initiation and progression of neoplasms and cancer. Neonatal DES exposure in mice can reprogram uterine differentiation by changing genetic pathways controlling uterine morphogenesis and/or altering gene expression in stem cells. DES affects the methylation patterns of genes that are associated with proliferation (c-jun, c-fos, c-myc, ltf); genes associated with apoptosis (bcl-2, bcl-x); and growth factors associated with proliferation and differentiation (EGF, TGF-α). This change in methylation is referred to as estrogen imprinting. Estrogen imprinting is an epigenetic mechanism where early-life exposure to estrogens (i.e., DES, Bisphenol A) permanently alters DNA methylation and gene expression of estrogen-responsive genes. Once changed, the altered gene profiles can continue to be expressed without further hormonal stimulation.

Proto-oncogenes help regulate normal cell proliferation and differentiation. When these genes are changed through mutation or methylation they can cause neoplastic cell transformation. Studies have shown changes in patterns of expression of estrogen-related proto-oncogenes in the genital tract of female mice exposed to DES. Changes in the proto-oncogenes and growth factors that cause elevations in their expression are associated with increased proliferation in the tissues (like the uterus and vagina) which can lead to cancer. Genetic modifications of the apoptotic genes that cause decreases in apoptosis are also associated with an increased incidence of cancer.

DES treatment affects male mice at the genomic level. DES altered Insl3 mRNA expression in male mice exposed in utero. Emmen et al. found a threefold decrease in Insl3 mRNA, which is expressed in fetal Leydig cells and is associated with the transabdominal phase of testis descent and development of the gubernaculum. This finding may provide a mechanism for DES-induced cryptorchidism. Another group found that gestational DES exposure in C57Bl/6 mice decreased the expression of two transcription factors (GATA4 and ID2) in the testes of adult males. GATA4 (expressed in Sertoli cells, Leydig cells, and other testicular somatic cells) is required for the correct expression of Sry and all the steps in testicular organogenesis that follow. ID2 is associated with the inhibition of differentiation of different cell types, and the decrease in GATA4 and ID2 may be associated with fertility problems later in life.

The research into tissue-specific mechanisms of action for DES is still underway. There are other unique attributes of DES that likely lead to its long-term effects following brief periods of exposure. A study of metabolism and disposition of DES in the pregnant rat, demonstrated enhanced disposition of DES and DES oxidative metabolites to the fetal reproductive tissues vs. liver following a single maternal exposure. Studies in mice demonstrate an accumulation of DES in the fetal reproductive tract, where it can reach levels three times higher than fetal blood . These findings of accumulated DES in reproductive tissues relate specifically to the location of ER-α, the known receptor for DES. The fact that there are multiple metabolic DES products has complicated the understanding of its effects. DES metabolites (especially quinines) are reactive ; they are formed in vivo, bind DNA and have been found in mammary tissue of rat, adult mouse reproductive tract, and mouse fetal tissues. These oxidative metabolites affect CYP gene activation and likely play a role in cancer mediation.

DES is no longer used in the human population which makes research less of a priority for funding organizations. However, for individuals/families already exposed, DES seems to be an initiating event in an initiation/promotion model for hormonal carcinogenesis and there is ample reason to fund research on effects in their unexposed children. Therefore, thoughtful follow-up of all generations and justified/planned use of stored samples (blood) will be critical in the future to determine those at highest risk for adverse health consequences.

Sources

  • Exposure to Diethylstilbestrol during Sensitive Life Stages: A legacy of heritable health effects, Birth defects research. Part C, Embryo today : reviews, NCBI PubMed PMC3817964, 2013 Nov 5.
  • Mechanisms involved in breast cancer etiology featured image PMC3817964/figure/F2.
DES DIETHYLSTILBESTROL RESOURCES

Prenatal DES-exposure may interfere with testicular Insl3 mRNA expression

Involvement of insulin-like factor 3 (Insl3) in diethylstilbestrol-induced cryptorchidism

2000 Study Abstract

Recently, it has been shown that targeted inactivation of the Insl3 gene in male mice results in cryptorchidism. The Insl3 gene encodes insulin-like factor 3 (Insl3), which is expressed in fetal Leydig cells. The testicular factor Insl3 appears to play an important role in the transabdominal phase of testis descent, which involves development of the gubernaculum.

Other studies have demonstrated that in utero exposure to diethylstilbestrol (DES), a synthetic estrogen, can lead to cryptorchidism both in humans and in animal models.

The present study was undertaken to investigate whether prenatal DES-exposure might interfere with testicular Insl3 mRNA expression. Furthermore, the effect of DES on steroidogenic factor 1 (SF-1) mRNA expression level was determined, since it has been shown that SF-1 plays an essential role in transcriptional activation of the Insl3 gene promoter.

Timed pregnant mice were treated with DES (100 microg/kg body weight) or vehicle alone on days E9 (gestational day 9) through E17. Control and DES-exposed mouse fetuses were collected at E16, E17 and E18, when transabdominal testis descent is taking place. Lack of gubernaculum development in DES-exposed animals was confirmed by histological analyses at E17. Expression of Insl3 and SF-1 mRNAs was studied in testes of control and DES-exposed fetuses at E16 and E18 by RNase protection assay.

Prenatal DES-exposure resulted in a three-fold decrease in Insl3 mRNA expression level (P<0.005), at both E16 and E18. In contrast, DES treatment had no effect on the expression of SF-1 mRNA.

These results support our hypothesis that DES may interfere with gubernaculum development by altering Insl3 mRNA expression, providing a possible mechanism by which DES may cause cryptorchidism.

Sources and more information
  • Involvement of insulin-like factor 3 (Insl3) in diethylstilbestrol-induced cryptorchidism, Endocrinology, NCBI PubMed PMID: 10650968, 2000 Feb.
  • Leydig cells in the adult testis featured image credit media.nature.
DES DIETHYLSTILBESTROL RESOURCES

DES drastically reduces Insl3 gene expression

image of Protein_INSL3

DES exerts its anti-androgen effects mainly through classical ER signaling, particularly via ERα

2007 Study Abstract

Failure of the testes to descend into the scrotum (cryptorchidism) is one of the most common birth defects in humans.

In utero exposure to estrogens, such as 17beta-estradiol (E2) or the synthetic estrogen diethylstilbestrol (DES), down-regulates insulin-like 3 (Insl3) expression in embryonic Leydig cells, which in turn results in cryptorchidism in mice.

In humans, the offspring of pregnant women treated with diethylstilbestrol (DES),  exhibited an increased incidence of cryptorchidism and hypoplastic testes. In rodents, we and others have shown that administration of DES or E2 during the second half of gestation resulted in a drastic reduction of Insl3 gene expression and testosterone levels, even though the expression of Sf-1 was unaltered.

To identify the molecular mechanism whereby xenoestrogens block Insl3 gene transcription, we performed a microarray analysis of wild-type or estrogen receptor (ER) alpha-mutant testes exposed in utero to pharmacological doses of E2 or DES.

Six and 31 genes were respectively down-regulated and up-regulated by estrogen exposure (> or =4-fold). All six genes down-regulated by estrogen exposure, including Insl3 and the steroidogenic genes steroidogenic acute regulatory protein and cytochrome P450 17alpha-hydroxylase/17,20-lyase, were done so by an ERalpha-dependent mechanism. In contrast, up-regulation was mediated either by ERalpha for 12 genes or by an independent mechanism for the 19 remaining genes. Finally, we show that Insl3 gene expression and testicular descent were not affected by in utero exposure to E2 or DES in ERalpha mutant mice, whereas absence of ERbeta did not influence the effect of these estrogens.

Collectively, these data demonstrate that xenoestrogens inhibit the endocrine functions of fetal Leydig cells through an ERalpha-dependent mechanism.

Sources and more information
  • Full text (free access) : Estrogen receptor alpha is a major contributor to estrogen-mediated fetal testis dysgenesis and cryptorchidism, Endocrinology, NCBI PubMed PMID: 17673513, 2007 Nov.
  • Protein INSL3 featured image credit wikipedia.
DES DIETHYLSTILBESTROL RESOURCES

DES inhibits transabdominal testicular descent dose-dependently via down-regulating the expression of INSL3

image of INSL3 protein

The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats

2009 Study Abstract

OBJECTIVE
To study the effect of diethylstilbestrol (DES) at different doses on transabdominal testicular descent in rats and the expression of INSL3 in the testis and HOXA10 in the gubernaculum.

METHOD
Fifty E13.5 (embryonic day 13.5) pregnant female SD rats were randomly divided into five groups that received a subcutaneous injection of DMSO, 2.5, 5.0, 10.0 and 20.0 mg/kg DES (group A, B, C, D and E), respectively. Male offspring were killed at E19.5, and then fetal mortality, the degree of transabdominal testicular ascent (DTA) was determined by a stereomicroscope. The mRNA expressions of INSL3 in the testis and HOXA10 in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting.

RESULTS
Male fetal mortality in group A, B, C, D, and E were 3.57%, 6.90%, 12.00%, 19.23% and 36.36%, respectively, which showed a dose-effect relationship between DES and the male fatal mortality (r=0.999, P<0.01). DTA in group B, C, D and E were (23.7+/-1.7) U, (38.8+/-1.9) U, (49.3+/-1.8) U and (58.6+/-2.1) U that were significantly larger than that in group A [(8.5+/-1.3) U] (q=46.12, 88.53, 120.44 and 141.37, respectively, P<0.01). There was also a dose-effect relationship between DES and DTA. In group B, C, D, and E, the expression of INSL3 mRNA were 0.9570+/-0.1490, 0.6760+/-0.1380, 0.0170+/-0.0040 and 0.0013+/-0.0003, respectively; the expressions of INSL3 protein were 0.8360+/-0.1520, 0.5310+/-0.1070, 0.0140+/-0.0020 and 0.0011+/-0.0003, respectively, which were significantly larger than the expression of INSL3 mRNA (1.801+/-0.126) and INSL3 protein (1.612+/-0.134) in group A (qmRNA=40.4840, 52.4402, 83.1585 and 82.0582, respectively, and qprotein=38.6151, 52.2747, 77.2756 and 76.1983, respectively, P<0.01). The expression of HOXA10 mRNA in group A, B, C, D, and E were 0.945+/-0.125, 0.940+/-0.119, 0.656+/-0.115, 0.544+/-0.118 and 0.463+/-0.114, respectively. Compared with the expression of HOXA10 mRNA in group A, the expression of group B was not significantly different (q=0.2213, P>0.05), those in other groups were down-regulated significantly (q=12.4304, 17.2477 and 20.2789, respectively, P<0.01).

CONCLUSION
DES inhibited transabdominal testicular descent dose-dependently via down-regulating the expression of INSL3. HOXA10 may play no role in low-dosage DES induced intra-abdominal cryptorchidism, but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones.

Sources and more information
  • The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats, Chinese journal of preventive medicine, NCBI PubMed PMID: 19534997, 2009 May.
  • Featured image Protein INSL3 credit wikipedia.
DES DIETHYLSTILBESTROL RESOURCES

INSL3 and SF-1 mRNA down-regulated expression in a dose-dependent manner following DES exposure

image of INSL3

Prenatal exposure to diaethylstilbestrol in the rat inhibits transabdominal testicular descent with involvement of the INSL3/LGR8 system and HOXA10

2009 Study Abstract

BACKGROUND
Prenatal exposure to diethylstilbestrol (DES) has been found to lead to intra-abdominal cryptorchidism, but the mechanism is still not completely clear. This study investigated the roles of the INSL3/LGR8 system and HOXA10 in DES-induced intra-abdominal cryptorchidism (DIIAC). The effect of DES on steroidogenic factor-1 (SF-1), that has been reported to control transcription of insulin-like factor 3 (INSL3), was also investigated.

METHODS
Fifty pregnant female SD rats at embryonic day 13.5 (E13.5) were randomly assigned to five groups that received a subcutaneous injections of dimethyl sulfoxide (control), 2.5 mg/kg, 5 mg/kg, 10 mg/kg, or 20 mg/kg of DES. Male offspring were sacrificed at E19.5, and fetal mortality and the degree of transabdominal testicular ascent (DTA) were determined under a stereomicroscope. The mRNA expression of INSL3 and SF-1 in the testis and leucine rich repeat-containing G protein-coupled receptors 8 (LGR8) and homeobox-A10 (HOXA10) in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting.

RESULTS
Higher fetal mortality and DTA were induced by DES in a dose-dependent manner (P < 0.01). Compared with the control group, the expression of INSL3 and SF-1 mRNA were down-regulated in a dose-dependent manner (P < 0.01), as was INSL3 protein; HOXA10 in the 2.5 mg/kg group and LGR8 mRNA in the 2.5 mg/kg and 5 mg/kg groups were not significantly different (P > 0.05); HOXA10 mRNA in groups C, D, and E decreased significantly and LGR8 mRNA levels in groups D and E increased significantly (P < 0.05, P < 0.01, respectively).

CONCLUSIONS
DES can inhibit transabdominal testicular descent in a dose-dependent manner via down-regulating the expression of INSL3, which is induced by down-regulating the expression of SF-1. HOXA10 may not be involved in DES induced intra-abdominal cryptorchidism at 2.5 mg/kg, but is involved at 5, 10 and 20 mg/kg. LGR8 may not be responsible for DES-induced transabdominal testicular maldescent.

Sources and more information
  • Prenatal exposure to diaethylstilbestrol in the rat inhibits transabdominal testicular descent with involvement of the INSL3/LGR8 system and HOXA10, Chinese medical journal, NCBI PubMed PMID: 19493424, 2009 Apr 20.
  • Featured image Protein INSL3 credit wikipedia.
DES DIETHYLSTILBESTROL RESOURCES