Selecting the optimum diet for endocrine disruptor (ED) research and testing studies in rodents is critical because the diet may determine the sensitivity to detect or properly evaluate an ED compound. Dietary estrogens can profoundly influence many molecular and cellular event actions on estrogen receptors and estrogen-sensitive genes. The source, concentration, relative potency, and significance of dietary estrogens in rodent diets are reviewed, including dietary factors that focus specifically on total metabolizable energy and phytoestrogen content, which potentially affect ED studies in rodents. Research efforts to determine dietary factors in commercially available rodent diets that affect uterotrophic assays and the time of vaginal opening in immature CD-1 mice are summarized. A checklist is provided of important factors to consider when selecting diets for ED research and testing studies in rodents. Specific metabolizable energy levels are recommended for particular bioassays. Discussions include the between-batch variation in content of the phytoestrogens daidzein and genistein, the effects of total metabolizable energy and phytoestrogens on the timing (i.e., acceleration) of vaginal opening, and increased uterine weight in immature CD-1 mice. It is concluded that rodent diets differ significantly in estrogenic activity primarily due to the large variations in phytoestrogen content; therefore animal diets used in all ED studies should ideally be free of endocrine-modulating compounds.

BackgroundThe optimum test diet and rodent species/strain for evaluating endocrine-disrupting compounds (EDCs) are critical.ObjectivesWe conducted studies to evaluate rodent species sensitivity and the effects of diets varying in phytoestrogen content on the time of vaginal opening (VO) in CD-1 mice, Fischer 344 (F344) rats, and CD Sprague-Dawley (S-D) rats.MethodsMice were weaned on postnatal day (PND) 15 and rats on PND19 and randomly assigned to control or test diets. Body weights, food consumption, and time of VO were recorded.ResultsThe time of VO was significantly advanced in F344 rats fed diets containing daidzein and genistein, whereas these same diets did not advance VO in S-D rats. When animals were fed the AIN-76A diet spiked with genistein, time of VO was significantly advanced at all doses in CD-1 mice, at the two highest doses in F344 rats, and at the highest dose in S-D rats. The time of VO in F344 rats was more highly correlated with the phytoestrogen content than with the total metabolizable energy (ME) of 12 diets.ConclusionsThe S-D rat is less sensitive to dietary phytoestrogens compared with the F344 rat or the CD-1 mouse, suggesting that the S-D rat is not the ideal model for evaluating estrogenic activity of EDCs. The profound effects of dietary phytoestrogens on the time of VO, an estrogen-sensitive marker, indicate that a standardized open-formula phytoestrogen-free diet containing a low ME level should be used to optimize the sensitivity of estrogenic bioassays.

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e14614 Background: Assessing tumor-derived somatic variants from blood plasma provides minimally invasive tumor profiling, decreased cost relative to traditional tissue biopsy and rapid turn-around-time. We describe here the analytical validation of the Oncomine Pan-Cancer Cell-Free assay in a CAP-accredited, CLIA-certified laboratory. The assay is designed to detect somatic DNA single-nucleotide variants (SNV), insertions/deletions (INDEL), copy number variants (CNV), and gene fusions in cell-free total nucleic acid (cfTNA) across 52 genes. For research use only. Not for use in diagnostic procedures. Methods: We assessed the sensitivity, specificity, accuracy, and precision of the assay. Pre-characterized reference materials with alterations at known allelic frequencies (AF) were used to establish performance characteristics for each variant class followed by verification on clinical specimens. Whole blood (n = 73) from healthy (10), breast cancer (9), colorectal cancer (32), and lung cancer (22) donors were collected in K2EDTA tubes and plasma was separated within 8 hours of collection. cfTNA was isolated using the MagMAX Cell-Free Total Nucleic Acid Isolation Kit on the KingFisher Flex. Libraries were prepared following kit instructions. Templating and sequencing were performed using the Ion 550 Kit on the Ion Chef and S5 XL systems. Alignment to hg19 and variant calling were performed using Torrent Suite and Ion Reporter software. Results: We observed a sensitivity of 80% at 0.1% AF and > 99.9% at 0.5% AF for SNV/INDEL. Sensitivity was > 99.9% at 1.34 fold-change for CNV, and > 99.9% at 0.4% fusion fraction. Specificity and accuracy were > 99% for all variant classes. Precision was 98% for SNV/INDEL and > 99.9% for CNV/fusions. Analysis of donor plasma demonstrated clinical feasibility; on average, we detected 1 hotspot SNV/INDEL (range 0-4) across the 3 cancer types. For SNV/INDEL in plasma where matched tissue genotyping was available (n = 17), we observed > 99% concordance (AF range 0.09%-52%). Conclusions: We have demonstrated that this assay is a sensitive method for evaluating tumor-derived somatic variants in blood plasma. It may provide an alternative and minimally invasive method for mutation profiling.