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A dose-ranging study is performed using young estrogen-depleted rats to determine whether dietary isoliquiritigenin (ILQ) alters bone metabolism and if the effects are associated with estrogen receptor signaling.Six-week-old rats (ovariectomized at 4 weeks of age) are fed diets containing 0, 100, 250, or 750 ppm ILQ (n = 5/treatment) for 7 days. Gene expression in femur and uterus, blood markers of bone turnover, body composition, and uterine weight and epithelial cell height are determined. Because ILQ lowers bone resorption, the effect of ILQ on in vitro differentiation of osteoclasts from bone marrow of mice is assessed. Treatment resulted in a dose-dependent increases in serum ILQ but no changes in serum osteocalcin, a marker of global bone formation. Contrastingly, ILQ administration results in reduced serum CTX-1, a marker of global bone resorption, and reduces tartrate resistant acid phosphatase expression in osteoclast culture. ILQ treatment and endogenous estrogen production had limited overlap on gene expression in femur and uterus. However, uterine epithelial cell hyperplasia is observed in two of five animals treated with 750 ppm.In conclusion, dietary ILQ reduces bone resorption in vivo and osteoclast differentiation in vitro, by mechanisms likely differing from actions of ovarian hormones.

OBJECTIVES: Widely consumed foods such as chicken, fish, and potatoes are regularly prepared by deep frying. The frying process involves temperatures exceeding 180 degrees Celsius and repeated frying cycles that result in thermally induced chemical changes of the oil's lipid structures. One such chemical change is an accumulation of polar compounds, including secondary lipid oxidation products, which are associated with several disease pathologies. Many European countries adhere to strict limits of less than 30% polar compounds within recycled fryer oils. There are no such regulations in the United States. Using a murine model of late-stage breast cancer (BC), we previously demonstrated an increased metastatic burden in mice consuming a diet of thermally abused frying oil (TAFO) compared with mice consuming fresh vegetable oil. To further understand this observation, we assessed 1) the amount of polar material in oil recycled for 300 minutes of deep frying, and 2) the effect the fractionated polar material from TAFO has on in vitro migration of 4T1 murine cancer cells. METHODS: We used silica column chromatography to separate the TAFO into polar and non-polar fractions. The polar fraction of TAFO (TAFO-PF) was retained from oil used to fry fish nuggets for a duration of 300 minutes. In vitro wound healing migration assays were conducted in the presence or absence of TAFO-PF in a concentration dependent manner. We assessed the wound closure rates (motility) of the highly metastatic 4T1 murine mammary carcinoma cell line. Live images were captured every hour for 24 hours to measure cell migration using brightfield microscopy. RESULTS: We found that after 300 minutes of frying, oil contained 74 ± 7.8 μg/mL of polar compound. 4T1 cells incubated over a period of 24 hours with diluted TAFO-PF achieved faster wound closure rates compared with cells incubated in growth media alone. CONCLUSIONS: Our results suggest that TAFO-PF increases motility as an indicator of the metastatic potential of BC cells. Ongoing work is being focused on conducting in vitro invasion assays on both 4T1 and human BC MDA-MB-231 cell lines in order to further understand the potential mechanisms and effects TAFO-PF has on cancer metastatic progression. FUNDING SOURCES: NIEHS Training (T32) Grant ES007326 Fellowship to JRH and ABO; UIUC Campus Research Board Beckman Grant and UIUC Hatch 1011659_ILLU-698-357.

For the past four decades, dietary fat intake has composed a significant portion of the typical Western diet, at 33-35% of total kilocalories. Approximately 8% of this dietary fat is from consumption of deep fat fried foods. Oil has been demonstrated to be absorbed by food during frying and contributes between 8 to 40% of the food’s final weight. As oils are used and reused for frying, complex chemical reactions take place creating deleterious polar compounds (e.g., acrylamide, sterol derivatives, or other reactive oxidative species.) Although these compounds are associated with health risks, i.e., heart disease, diabetes mellitus, and cancer, there is no regulation in the United States for upper limits of polar material in frying oil. This raises an important question as to whether thermally abused fryer oil (TAFO) can alter metastatic breast cancer progression. We initially conducted an in vivo study in which a modest amount of dietary fat, 25% of kilocalories, came from fresh fryer oil or TAFO. Ovariectomized BALB/C mice fed the diet containing TAFO for 16 weeks and inoculated with luciferase-expressing 4T1 murine mammary cancer cells had significantly more metastasis to lungs than control-fed animals (p < 0.05, n=21). Serum metabolic analysis in these animals revealed significantly elevated levels of fumarate, a known onco-metabolite, in TAFO-fed compared with control-fed animals (p < 0.05, n=9). To confirm that fumarate was mechanistically involved in metastatic progression, we conducted in vitro studies. 4T1 cells exposed to 500 nM to 10 µM monomethyl fumarate (MMF), a cell-permeable form of fumarate, trended toward an increase in the healing rate over 24 hours in a wound healing (scratch) assay compared to control (n =2). This preliminary evidence supports the hypothesis that fumarate accumulation creates a protumorigenic and metastatic environment and needs confirmation in subsequent investigation. This information can help elucidate mechanistic details of the effects of TAFO on breast cancer metastasis. Further work will assess rates of 4T1 cellular proliferation and invasion induced by MMF. In summary, dietary intake of TAFO increased metastasis of tumor cells to lungs, and dysregulation of the metabolite fumarate may be mechanistically involved. Citation Format: Jennifer R. Hughes, Kaitlyn M. Joyce, Anthony Cam, William G. Helferich, Zeynep Madak-Erdogan. Thermally abused frying oil enhances metastatic progression in vivo and in vitro: A link to elevated fumarate levels [abstract]. In: Proceedings of the AACR Special Conference: Advances in Breast Cancer Research; 2017 Oct 7-10; Hollywood, CA. Philadelphia (PA): AACR; Mol Cancer Res 2018;16(8_Suppl):Abstract nr B26.