Estrogen influences the physiology of many target tissues in both women and men. The long-term effects of estrogen are mediated predominantly by nuclear estrogen receptors (ERs) functioning as DNA-binding transcription factors. Tissue-specific responses to estrogen therefore result from regulation of different sets of genes. However, it remains perplexing as to what regulatory sequence contexts specify distinct genomic responses. First, this review classifies estrogen response sequences in mammalian target genes. Of note, around one third of known human target genes associate only indirectly with ER, through intermediary transcription factor(s). Then, computational approaches are presented both for refining direct ER-binding sites and for formulating hypotheses regarding the overall genomic expression pattern. Surprisingly, limited evolutionary conservation of specific estrogen-responsive sites is observed between human and mouse. Finally, consideration of the cellular functions of regulated human genes suggests links between particular biological roles and specific types of estrogen response elements, although with the important caveat that only a restricted set of target genes is available. These analyses support the view that specific, hormone-driven gene expression programs can result from the interplay of environmental and cellular cues with the distinct types of estrogen-response sequences.

SignificanceStandard in vitro assays to assess genotoxicity frequently generate positive results that are subsequently found to be irrelevant for in vivo carcinogenesis and human cancer risk assessment. Currently used follow-up methods, such as animal testing, are expensive and time-consuming, and the development of approaches enabling more accurate mechanism-based risk assessment is essential. We developed an in vitro transcriptomic biomarker-based approach that provides a robust biomarker reflecting stress-signaling responses. The biomarker correctly identifies the vast majority of irrelevant genotoxicity results from in vitro chromosome damage assays. TGx-DDI, a multigene biomarker for DNA damage-inducing agents, is the first biomarker that not only shows convincing interlaboratory and intralaboratory reproducibility, but also performs accurately in a system suitable for high-throughput screening.

Estrogen plays an important role in the regulation of vascular tone and in the pathophysiology of cardiovascular disease. Physiological effects of estrogen are mediated through estrogen receptors alpha (ERalpha) and beta (ERbeta), which are both expressed in vascular smooth muscle and endothelial cells. However, the molecular pathways mediating estrogen effects in blood vessels are not well defined. We have performed gene expression profiling in the mouse aorta to identify comprehensive gene sets the expression of which is regulated by long-term (1 wk) estrogen treatment. The ER subtype dependence of the alterations in gene expression was characterized by parallel gene expression profiling experiments in ERalpha-deficient [ERalpha knockout (ERalphaKO)] and ERbeta-deficient (ERbetaKO) mice. Importantly, these data revealed that ERalpha- and ERbeta-dependent pathways regulate distinct and largely nonoverlapping sets of genes. Whereas ERalpha is essential for most of the estrogen-mediated increase in gene expression in wild-type aortas, ERbeta mediates the large majority (nearly 90%) of estrogen-mediated decreases in gene expression. Biological functions of the estrogen-regulated genes include extracellular matrix synthesis, in addition to electron transport in the mitochondrion and reactive oxygen species pathways. Of note, the estrogen/ERbeta pathway mediates down-regulation of mRNAs for nuclear-encoded subunits in each of the major complexes of the mitochondrial respiratory chain. Several estrogen-regulated genes also encode transcription factors. Computational analysis of promoters from coexpressed genes revealed overrepresentation of binding sites for such factors, lending support for an estrogen-regulatory transcriptional network in the vasculature. Overall, these findings provide a foundation for understanding the molecular basis for estrogen effects on vasculature gene expression.

A framework of “Common Criteria” (i.e. a series of questions) has been developed to inform the use and evaluation of biomonitoring data in the context of human exposure and risk assessment. The data-rich chemical benzene was selected for use in a case study to assess whether refinement of the Common Criteria framework was necessary, and to gain additional perspective on approaches for integrating biomonitoring data into a risk-based context. The available data for benzene satisfied most of the Common Criteria and allowed for a risk-based evaluation of the benzene biomonitoring data. In general, biomarker (blood benzene, urinary benzene and urinary S-phenylmercapturic acid) central tendency (i.e. mean, median and geometric mean) concentrations for non-smokers are at or below the predicted blood or urine concentrations that would correspond to exposure at the US Environmental Protection Agency reference concentration (30 µg/m3), but greater than blood or urine concentrations relating to the air concentration at the 1 × 10−5 excess cancer risk (2.9 µg/m3). Smokers clearly have higher levels of benzene exposure, and biomarker levels of benzene for non-smokers are generally consistent with ambient air monitoring results. While some biomarkers of benzene are specific indicators of exposure, the interpretation of benzene biomonitoring levels in a health-risk context are complicated by issues associated with short half-lives and gaps in knowledge regarding the relationship between the biomarkers and subsequent toxic effects.