For decades, studies of endocrine-disrupting chemicals (EDCs) have challenged traditional concepts in toxicology, in particular the dogma of “the dose makes the poison,” because EDCs can have effects at low doses that are not predicted by effects at higher doses. Here, we review two major concepts in EDC studies: low dose and nonmonotonicity. Low-dose effects were defined by the National Toxicology Program as those that occur in the range of human exposures or effects observed at doses below those used for traditional toxicological studies. We review the mechanistic data for low-dose effects and use a weight-of-evidence approach to analyze five examples from the EDC literature. Additionally, we explore nonmonotonic dose-response curves, defined as a nonlinear relationship between dose and effect where the slope of the curve changes sign somewhere within the range of doses examined. We provide a detailed discussion of the mechanisms responsible for generating these phenomena, plus hundreds of examples from...

/pdf/hormones-and-endocrine-disrupting-chemicals-low-dose-effects-2vn32zoqc8.pdf

Hormones and endocrine-disrupting chemicals: Low-dose effects and nonmonotonic dose responses

Extracellular stimuli elicit changes in gene expression in target cells by activating intracellular protein kinase cascades that phosphorylate transcription factors within the nucleus. One of the best characterized stimulus-induced transcription factors, cyclic AMP response element (CRE)-binding protein (CREB), activates transcription of target genes in response to a diverse array of stimuli, including peptide hormones, growth factors, and neuronal activity, that activate a variety of protein kinases including protein kinase A (PKA), pp90 ribosomal S6 kinase (pp90RSK), and Ca2+/calmodulin-dependent protein kinases (CaMKs)[corrected]. These kinases all phosphorylate CREB at a particular residue, serine 133 (Ser133), and phosphorylation of Ser133 is required for CREB-mediated transcription. Despite this common feature, the mechanism by which CREB activates transcription varies depending on the stimulus. In some cases, signaling pathways target additional sites on CREB or proteins associated with CREB, permitting CREB to regulate distinct programs of gene expression under different conditions of stimulation. This review discusses the molecular mechanisms by which Ser133-phosphorylated CREB activates transcription, intracellular signaling pathways that lead to phosphorylation of CREB at Ser133, and features of each signaling pathway that impart specificity at the level of CREB activation.

CREB: a stimulus-induced transcription factor activated by a diverse array of extracellular signals.

A specific protein, bound to DNA, can activate transcription of a wide array of genes in many eukaryotes. Further analysis suggests a general outline for how eukaryotic transcriptional activators function and are controlled.

How eukaryotic transcriptional activators work

The Fos/Jun and ATF/CREB families of transcription factors function in coupling extracellular signals to alterations in expression of specific target genes. Like many eukaryotic transcription factors, these proteins bind to DNA as dimers. Dimerization is mediated by a structure known as the "leucine-zipper" motif. Although Fos/Jun and ATF/CREB were previously thought to interact preferentially with different DNA regulatory elements (the AP-1/TRE and ATF/CRE sites, respectively), we find that members of these two families form selective cross-family heterodimers. The resulting heterodimers display distinguishable DNA binding specificities from each other and from their parental homodimers. These findings indicate that the Fos/Jun and ATF/CREB families of transcription factors are not as distinct as was previously thought. We suggest that they can be grouped into a superfamily of transcription factors.

https://www.pnas.org/content/pnas/88/9/3720.full.pdf

Cross-family dimerization of transcription factors Fos/Jun and ATF/CREB alters DNA binding specificity.

In 1991, a group of 21 scientists gathered at the Wingspread Conference Center to discuss evidence of developmental alterations observed in wildlife populations after chemical exposures. There, the term "endocrine disruptor" was agreed upon to describe a class of chemicals including those that act as agonists and antagonists of the estrogen receptors (ERs), androgen receptor, thyroid hormone receptor, and others. This definition has since evolved, and the field has grown to encompass hundreds of chemicals. Despite significant advances in the study of endocrine disruptors, several controversies have sprung up and continue, including the debate over the existence of nonmonotonic dose response curves, the mechanisms of low-dose effects, and the importance of considering critical periods of exposure in experimental design. One chemical found ubiquitously in our environment, bisphenol-A (BPA), has received a tremendous amount of attention from research scientists, government panels, and the popular press. In this review, we have covered the above-mentioned controversies plus six additional issues that have divided scientists in the field of BPA research, namely: 1) mechanisms of BPA action; 2) levels of human exposure; 3) routes of human exposure; 4) pharmacokinetic models of BPA metabolism; 5) effects of BPA on exposed animals; and 6) links between BPA and cancer. Understanding these topics is essential for educating the public and medical professionals about potential risks associated with developmental exposure to BPA and other endocrine disruptors, the design of rigorously researched programs using both epidemiological and animal studies, and ultimately the development of a sound public health policy.

/pdf/bisphenol-a-and-the-great-divide-a-review-of-controversies-1t5863axtp.pdf

Bisphenol-A and the Great Divide: A Review of Controversies in the Field of Endocrine Disruption

We have examined the relationship between sequence-specific DNA-binding proteins that activate transcription of E1A-inducible adenovirus early promoters. Factors previously referred to as E4F1 and E2A-EF bind to the E4 and E2A promoters, respectively. We demonstrate here that E4F1 and E2A-EF have identical DNA-binding specificity. Moreover, E4F1 and E2A-EF both activate transcription of the E4 and E2A promoters in vitro. These findings demonstrate that E4F1 and E2A-EF are the same factor, which we have designated activating transcription factor, or ATF. In addition to the E4 and E2A promoters, ATF binds to an important functional element of the E1A-inducible E3 promoter. Interaction of a common activator protein, ATF, with multiple E1A-inducible early viral promoters, suggests a significant role for ATF in E1A-mediated transcriptional activation.

http://www.pnas.org/content/84/23/8355.full.pdf

A cellular protein, activating transcription factor, activates transcription of multiple E1A-inducible adenovirus early promoters

Full-term pregnancy early in reproductive life is protective against breast cancer in women. Pregnancy also provides protection in animals against carcinogen-induced breast cancer, and this protection can be mimicked by using the hormones estrogen and progesterone. The molecular mechanisms that form the basis for this protective effect have not been elucidated. On the basis of our results, we propose a cell-fate hypothesis. At a critical period in adolescence the hormonal milieu of pregnancy affects the developmental fate of a subset of mammary epithelial cells and its progeny, which results in persistent differences in molecular pathways between the epithelial cells of hormone-treated and mature virgin mammary glands. These changes in turn dictate the proliferative response to carcinogen challenge and include a block in carcinogen-induced increase in mammary epithelial cell proliferation and an increased and sustained expression of nuclear p53 in the hormone-treated mammary gland. This hormone-induced nuclear p53 is transcriptionally active as evidenced by increased expression of mdm2 and p21 (CIP1/WAF1). Importantly, exposure to perphenazine, a compound that induces mammary gland differentiation but does not confer protection, does not induce p53 expression, indicating that p53 is not a differentiation marker. The proliferative block and induction of p53 are operative in both rats and mice, results that support the generality of the proposed hypothesis.

https://www.pnas.org/content/pnas/98/22/12379.full.pdf

p53 is a potential mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis

One of the most consistent results in the epidemiology of human breast cancer is the inverse relationship of risk and early full-term parity. The goal of this study was to investigate the molecular mechanisms through which early full-term pregnancy protects the breast from cancer development. We used Wistar-Furth (WF) rats as our experimental system and mimicked pregnancy using estrogen and progesterone (E/P). Sexually mature female rats were treated with steroid hormones for 21 days and after 28 days of gland involution, the rats were administered MNU. Rats that received a high dose of 20 microg E and 20 mg P exhibited an 82% reduction in the incidence of mammary adenocarcinomas as compared to the rats receiving only blank pellets. Decreasing doses of E/P were partially protective suggesting that complete differentiation of the gland was not required for refractoriness. We measured the RNA expression levels of several target genes involved in the regulation of mammary cell proliferation and/or differentiation including estrogen receptor (ER) and progesterone receptor (PR), cyclins D1 and D2, the cell cycle inhibitors p16, p21 and p27, and the tumor suppressor p53. At the time of MNU treatment we found no significant differences in the expression of these genes, with the possible exception of p21, indicating that hormone treatment did not result in constitutive changes in expression levels. The numbers of apoptotic cells were low and comparable in the hormone exposed and age-matched virgin gland (AMV) at the time of carcinogen challenge and remained low for 8 days after MNU treatment. The number of BrdU-labeled cells at the time of carcinogen challenge were also low in both the AMV (1.8%) and hormone exposed (0.8%) animals. In contrast, cell proliferation in the AMV (5.7%) was significantly different from both the parous involuted (1.2%) and the E/P-treated involuted (1.5%) animals 8 days after MNU treatment. We interpret these data to indicate that hormone treatment results in mammary epithelial cells that have persistent alterations in intracellular pathways governing proliferation responses to carcinogens.

/pdf/hormone-induced-refractoriness-to-mammary-carcinogenesis-in-39wcocgrzg.pdf

Hormone-induced refractoriness to mammary carcinogenesis in Wistar-Furth rats.

Reproductive history is a consistent risk factor for human breast cancer. Epidemiological studies have repeatedly demonstrated that early age of first full-term pregnancy is a strong protective factor against breast cancer and provides a physiologically operative model to achieve a practical mode of prevention. In rodents, the effects of full-term pregnancy can be mimicked by exposure to low doses of estrogen and progesterone or treatment with human chorionic gonadotropin. The cellular and molecular mechanisms that underlie hormone-induced refractoriness are largely unresolved. Several hypotheses have been proposed to explain the protective effects of hormones. These involve the induction of differentiation of the mammary gland to provide a less responsive cell population to carcinogens, a decrease in proliferative activity in the parous gland compared to the age-matched virgin, an altered hormonal environment mediated by a decrease in circulating growth hormone, and an alteration in cell fate mediated by specific molecular changes induced by estrogen and progesterone. The evidence for and against these hypotheses is discussed along with recent results on possible molecular alterations that may underlie the refractory state. One central question that is still unresolved is whether the refractoriness is intrinsic to the mammary epithelial cells and/or mediated by persistent alterations in the host environment.

/pdf/hormone-induced-protection-against-breast-cancer-41qphlme7p.pdf

Hormone-Induced Protection Against Breast Cancer

Utilizing the gel electrophoresis/DNA binding assay, a factor specific for the upstream transcriptional control sequence of the EIA-inducible adenovirus EIIA-early promoter has been detected in HeLa cell nuclear extract. Analysis of linker-scanning mutants of the promoter by DNA binding assays and methylation-interference experiments show that the factor binds to the 17-nucleotide sequence 5' TGGAGATGACGTAGTTT 3' located between positions -66 and -82 upstream from the cap site. This sequence has been shown to be essential for transcription of this promoter. The EIIA-early-promoter specific factor was found to be present at comparable levels in uninfected HeLa cells and in cells infected with either wild-type adenovirus or the EIA-deletion mutant dl312 under conditions in which the EIA proteins are induced to high levels [7 or 20 hr after infection in the presence of arabinonucleoside (cytosine arabinoside)]. Based on the quantitation in DNA binding assays, it appears that the mechanism of EIA-activated transcription of the EIIA-early promoter does not involve a net change in the amounts of this factor.

http://www.pnas.org/content/83/16/5914.full.pdf

Identification of a factor in HeLa cells specific for an upstream transcriptional control sequence of an EIA-inducible adenovirus promoter and its relative abundance in infected and uninfected cells.

Lakshmi Sivaraman

Papers

A cellular protein, activating transcription factor, activates transcription of multiple E1A-inducible adenovirus early promoters

p53 is a potential mediator of pregnancy and hormone-induced resistance to mammary carcinogenesis

Hormone-induced refractoriness to mammary carcinogenesis in Wistar-Furth rats.

Hormone-Induced Protection Against Breast Cancer

Identification of a factor in HeLa cells specific for an upstream transcriptional control sequence of an EIA-inducible adenovirus promoter and its relative abundance in infected and uninfected cells.