Information-dependent enrichment analysis reveals time-dependent transcriptional regulation of the estrogen pathway of toxicity.

The twenty-first century vision for toxicology involves a transition away from high-dose animal studies to in vitro and computational models (NRC in Toxicity testing in the 21st century: a vision and a strategy, The National Academies Press, Washington, DC, 2007). This transition requires mapping pathways of toxicity by understanding how in vitro systems respond to chemical perturbation. Uncovering transcription factors/signaling networks responsible for gene expression patterns is essential for defining pathways of toxicity, and ultimately, for determining the chemical modes of action through which a toxicant acts. Traditionally, transcription factor identification is achieved via chromatin immunoprecipitation studies and summarized by calculating which transcription factors are statistically associated with up- and downregulated genes. These lists are commonly determined via statistical or fold-change cutoffs, a procedure that is sensitive to statistical power and may not be as useful for determining transcription factor associations. To move away from an arbitrary statistical or fold-change-based cutoff, we developed, in the context of the Mapping the Human Toxome project, an enrichment paradigm called information-dependent enrichment analysis (IDEA) to guide identification of the transcription factor network. We used a test case of activation in MCF-7 cells by 17ß estradiol (E2). Using this new approach, we established a time course for transcriptional and functional responses to E2. ERα and ERß were associated with short-term transcriptional changes in response to E2. Sustained exposure led to recruitment of additional transcription factors and alteration of cell cycle machinery. TFAP2C and SOX2 were the transcription factors most highly correlated with dose. E2F7, E2F1, and Foxm1, which are involved in cell proliferation, were enriched only at 24 h. IDEA should be useful for identifying candidate pathways of toxicity. IDEA outperforms gene set enrichment analysis (GSEA) and provides similar results to weighted gene correlation network analysis, a platform that helps to identify genes not annotated to pathways.

Formation of two novel estrogen guanine adducts and HPLC/MS detection of 4-hydroxyestradiol-N7-guanine in human urine.

Estrogen-DNA adducts are potential biomarkers for assessing the risk of developing of a number of hormonally modified cancers, including breast cancer. Formation of the 4-hydroxyestradiol-N(7)-guanine (4-OHE2-N(7)-guanine) adduct from the reaction of estradiol-3,4-quinone with DNA and its detection in vivo has been established. With the ultimate goal of exploring estrogen-DNA adducts as biomarkers in experimental and human investigations, the 4-OHE2-N(7)-guanine was synthesized, and preliminary studies demonstrated that this adduct was detectable in all 10 female human urine samples examined. Therefore, more extensive investigations were conducted to study this compound's chemical-physical properties and to examine the stability of 4-OHE2-N(7)-guanine under a range of pH conditions that might influence biomarker measurement. Under neutral to alkaline conditions, 4-OHE2-N(7)-guanine could completely oxidize to an 8-oxo-guanine derivative. This derivative was isolated by HPLC, and mass spectrometry confirmed the oxidized compound by demonstrating the formation of an m/ z 168 fragment, generated by oxygen addition to guanine. Furthermore, investigation of the 4-OHE2-N(7)-2'-deoxyguanosine nucleoside adduct showed that under alkaline conditions a formamidopyrimidine analogue was produced. The formamidopyrimidine derivative forms from ring opening of the guanine imidazole ring following C-8 oxidation in the N(7), N(9) disubstituted guanine. Formation of both of these oxidized estrogen-guanine DNA adducts has precedent with other chemical agents that covalently bind to the N(7) position in guanine. Therefore, the development and application of methods to measure estrogen-guanine adducts will need to also consider these new adducts, and the biological implications of these compounds will need to be explored to determine their contribution to estrogen toxicology.