Editor's Highlight: Application of Gene Set Enrichment Analysis for Identification of Chemically Induced, Biologically Relevant Transcriptomic Networks and Potential Utilization in Human Health Risk Assessment.

The rate of new chemical development in commerce combined with a paucity of toxicity data for legacy chemicals presents a unique challenge for human health risk assessment. There is a clear need to develop new technologies and incorporate novel data streams to more efficiently inform derivation of toxicity values. One avenue of exploitation lies in the field of transcriptomics and the application of gene expression analysis to characterize biological responses to chemical exposures. In this context, gene set enrichment analysis (GSEA) was employed to evaluate tissue-specific, dose-response gene expression data generated following exposure to multiple chemicals for various durations. Patterns of transcriptional enrichment were evident across time and with increasing dose, and coordinated enrichment plausibly linked to the etiology of the biological responses was observed. GSEA was able to capture both transient and sustained transcriptional enrichment events facilitating differentiation between adaptive versus longer term molecular responses. When combined with benchmark dose (BMD) modeling of gene expression data from key drivers of biological enrichment, GSEA facilitated characterization of dose ranges required for enrichment of biologically relevant molecular signaling pathways, and promoted comparison of the activation dose ranges required for individual pathways. Median transcriptional BMD values were calculated for the most sensitive enriched pathway as well as the overall median BMD value for key gene members of significantly enriched pathways, and both were observed to be good estimates of the most sensitive apical endpoint BMD value. Together, these efforts support the application of GSEA to qualitative and quantitative human health risk assessment.

A sensitivity analysis using alternative toxic equivalency factors to estimate U.S. dietary exposures to dioxin-like compounds.

EPA recommends sensitivity analyses when applying the toxic equivalency factor (TEF) method to evaluate exposures to dioxin-like compounds (DLCs). Applying the World Health Organization's (WHO) 2005 TEF values and estimating average U.S. daily dietary intakes of 25 DLCs from eight food categories, we estimate a toxic equivalency (TEQ) intake of 23 pg/day. Among DLCs, PCB 126 (26%) and 1,2,3,7,8-PeCDD (23%) dominate TEQ intakes. Among food categories, milk (14%), other dairy (28%), beef (25%), and seafood (18%) most influenced TEQ intakes. We develop two approaches to estimate alternative TEF values. Based on WHO's assumption regarding TEF uncertainty, Approach1 estimates upper and lower TEFs for each DLC by multiplying and dividing, respectively, its individual TEF by ± half a log. Based on compiled empirical ranges of relative potency estimates, Approach2 uses percentile values for individual TEFs. Total TEQ intake estimates using the lower and upper TEFs based on Approach1 were 8 and 68 pg TEQ/day, respectively. The 25th and 75th percentile TEFs from Approach2 yielded 12 and 28 pg TEQ/day, respectively. The influential DLCs and food categories remained consistent across alternative TEFs, except at the 90th percentile using Approach2. We highlight the need for developing underlying TEF probability distributions.

Inhibition of insulin synthesis by cyproheptadine: effects on translation.

The antihistaminic, antiserotonergic drug cyproheptadine (CPH) is known to inhibit insulin synthesis in vivo and in vitro. This inhibition of insulin synthesis occurs without a commensurate decrease in preproinsulin mRNA (PPImRNA) levels, suggesting a post-transcriptional mechanism of action. The goal of the present study was to investigate the direct effects of CPH on translation of PPImRNA in RINm5F cells. Results produced using a subcellular fractionation technique followed by real-time RT-PCR indicated that a 2-h 10 microM CPH treatment resulted in a decrease in the percentage of cellular PPImRNA associated with endoplasmic reticulum (ER) bound polysomes and increases in the percentages of translationally uninitiated and monoribosome-associated PPImRNA. These alterations in PPImRNA distribution were found to be concentration-dependent, chemical structure-specific, and reversible with a time course consistent with a previously reported CPH-induced inhibition of insulin synthesis. Further investigations to examine the possible effect of CPH on translation initiation were then undertaken by examining the phosphorylation state of the translation initiation factors eIF2alpha, eIF4E, and 4E-BP1 after CPH treatment. CPH (10 microM) treatment resulted in increased phosphorylation of eIF2alpha, and decreased phosphorylation of both eIF4E and 4E-BP1. These changes are all consistent with decreased initiation of translation. Taken together, these results suggest that the inhibition of insulin synthesis known to be elicited by CPH treatment of RINm5F cells and intact animals involves alterations of initiation factor phosphorylation leading to a decrease in insulin synthesis and of stored insulin in insulin-producing cells.