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.