Altered gene expression patterns during the initiation and promotion stages of neonatally diethylstilbestrol-induced hyperplasia/dysplasia/neoplasia in the hamster uterus
2014 Study Abstract
Neonatal treatment of hamsters with diethylstilbestrol (DES) induces uterine hyperplasia/dysplasia/neoplasia (endometrial adenocarcinoma) in adult animals. We subsequently determined that the neonatal DES exposure event directly and permanently disrupts the developing hamster uterus (initiation stage) so that it responds abnormally when it is stimulated with estrogen in adulthood (promotion stage). To identify candidate molecular elements involved in progression of the disruption/neoplastic process, we performed:
- immunoblot analyses
- and microarray profiling (Affymetrix Gene Chip System) on sets of uterine protein and RNA extracts, respectively,
- and immunohistochemical analysis on uterine sections; all from both initiation stage and promotion stage groups of animals.
Here we report that:
- progression of the neonatal DES-induced hyperplasia/dysplasia/neoplasia phenomenon in the hamster uterus involves a wide spectrum of specific gene expression alterations
- and the gene products involved and their manner of altered expression differ dramatically during the initiation vs. promotion stages of the phenomenon.
Comparisons among gene expression studies
When we compared studies regarding differences in uterine gene expression related to perinatal DES exposure, we found the following limited correlations among them and with our analyses. In mice treated with DES on days 1–5 of life and killed 6 hr later [Developmental diethylstilbestrol exposure alters genetic pathways of uterine cytodifferentiation], Stat5a was upregulated at the mRNA level (upregulated at the protein level in our IS uteri). In rats exposed to DES during late gestation and killed on postnatal day 5 [Analysis of gene expression profiles in the offspring of rats following maternal exposure to xenoestrogens], IRF-1 was upregulated at the mRNA level (upregulated at the protein level in our PS uteri). In mice treated with three different doses of DES on days 1–5 of life and killed on postnatal day 19 (prior to puberty) [Developmental exposure to diethylstilbestrol alters uterine gene expression that may be associated with uterine neoplasia later in life], connexin-43 was upregulated only in the highest dose group (upregulated at the mRNA and protein level in our IS uteri). In mice also treated with DES on days 1–5 of life but killed at 10 weeks of age [Developmental exposure to diethylstilbestrol alters uterine gene expression that may be associated with uterine neoplasia later in life], upregulation at the mRNA level was detected for connexin-43 (upregulated at the mRNA and protein level in our IS uteri) and for PR (upregulated at the mRNA and protein level in our IS uteri but downregulated at the protein level in our PS uteri). Consequently, such comparisons fail to provide clear insights into the key biological pathways that drive the phenomenon of endocrine disruption.
Summary and conclusions
The observations presented here demonstrate that:
- progression of the neonatal DES-induced hyperplasia/dysplasia/neoplasia phenomenon in the hamster uterus involves a wide spectrum of specific gene expression alterations at both the mRNA and protein levels
- and the gene products involved and their manner of altered expression differ dramatically during the initiation vs. promotion stages of the phenomenon. Particularly interesting changes included members in the functional categories of nuclear receptors (progesterone receptor), cell-cell interactions (E-cadherin, connexins), cytokine action (IRF-1, Stat5A), growth factor action (IRS-1), extracellular matrix component (tenascin-C), transcription factors (Nrf2, Sp1), and multi-functional nuclear protein (SAFB1). Rather unexpected was the very limited matches in gene expression differences detected at both the mRNA and protein levels. Part of the explanation may be technical and due to the fact that commercially available hamster genome-based microarrays were not available. Thus, despite positive claims for the strategy, our default use of the mouse genome-based microarray likely resulted in missed detection of some relevant hamster mRNA targets because of interspecies differences at the genomic DNA level. Of course, a remaining fundamental question is how is it that perinatal exposure to DES and other suspected endocrine disruptor agents exerts pathophysiological consequences into adulthood. An emerging and increasingly compelling answer to that question is the paradigm of epigenetics [Physiological effects and mechanisms of action of endocrine disrupting chemicals that alter estrogen signaling], [Persistently altered epigenetic marks in the mouse uterus after neonatal estrogen exposure]. In fact, we are now assessing epigenetic endpoints in the neonatally DES-exposed hamster uterus and have observed alterations at the DNA methylation and micro RNA levels (manuscripts in preparation). Lastly, we and other investigators still need to define which specific molecular alterations, genetic and/or epigenetic, actually drive a given endocrine disruption phenomenon and precisely how it does so.
Sources and more information
- Full text (free access) : Altered Gene Expression Patterns During the Initiation and Promotion Stages of Neonatally Diethylstilbestrol-Induced Hyperplasia/Dysplasia/Neoplasia in the Hamster Uterus, Reproductive Toxicology, NCBI PubMed PMC4261067, 2014 Sep 19.
- Negative control IHC featured image credit PMC4261067/figure/F6.