Evidence is accumulating that xenoestrogens significantly affect gene expression in mammalian cells at doses far lower than their commonly considered toxic doses. Animal experiments suggest that in vivo effects of xenoestrogens follow non-monotonous dose response curves. However, whether such non-linearity is observed at the level of individual cells is unknown. To address this question, we developed a comprehensive database of dose-dependent transcriptomal effects of representative xenoestrogens in MCF-7 cells, which strongly express estrogen receptor a but not estrogen receptor β. For each xenoestrogen [estradiol, diethylstilbestrol, ethynylestradiol, Bisphenol A (BPA), genistein, daidzein, p-nonylphenol], transcriptomal snapshots were determined for 6 to 12 different doses in a single experiment using Affymetrix HG-U133-2+ microarray, and experiments were repeated at least five times. Databases were constructed to handle these ~480 snapshots (30~90 snapshots for each chemical). Exposure time was fixed to 48 hours, which was selected for best reproducibility of the snapshots. Exposure to correct doses was confirmed by qPCR determination of mRNA expression for WISP2 (estrogen-inducible marker) and BIK (estrogen-suppressible marker). The dose-specific snapshot data were integrated into the transcriptomal profile vectors for each chemical. Each element of the profile vector was a summary of multiple snapshots determined for a single concentration. The 50% effective doses (AC50) with 95% confidence intervals were calculated from the profile vectors for each of the 500 genes showing the best scores of curve fitting. By applying the B-spline curve fitting algorithm, mathematical models of the dose-dependent transcriptomal changes were produced form the profile vectors for each gene. These models generated estimations of transcriptomal profiles for unlimited numbers of concentrations within the concentrations ranges covered by the snapshot data. High-density virtual 3-D heatmaps drawn using these models revealed that mRNA expression of BPA-upregulated genes showed a small but significant peak at 10 pM~100 pM, once decreased at 1 nM~10 nM, and then increased again at 100 nM~10μM. This “second peak” of mRNA induction very low doses was specific to BPA as the virtual 3-D heatmaps of estradiol or other xenoestrogens did not show such peaks. In contrast, however, genes down-regulated by genistein showed significantly biphasic responses whereas genes up-regulated by the same ligand did not. Analyses on the AC50 distribution curves revealed that BPA-suppressible genes tend to be more sensitive than genes inducible by the same ligand with 2.3-fold differences in the average AC50, suggesting that relatively low concentrations of BPA may selectively suppress gene expression whereas its effects on gene induction are still weak. On the other hand, the AC50 distributions of estradiol-inducible and suppressible genes mostly overlapped, suggesting that the gene induction and suppression by this ligand occur simultaneously at all ranges of ligand concentrations. Thus, our comprehensive analyses on the dose-response characteristics of xenoestrogen-regulated gene expression show significant ligand species-dependent differences at low doses, suggesting that the low dose effects may occur even at the level of individual cells. This study was supported by Susan G. Komen for Cure grants FAS0703860 and KG090515 to TS.
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