Thermally Abused Frying Oil Potentiates Metastasis to Lung in a Murine Model of Late-Stage Breast Cancer

Fats account for approximately 35% of the average U.S. American's caloric intake, which creates the salient task of understanding the relationships between lipid consumption and human health. The seventh international symposium on deep-frying emphasized understanding of the health effects of oxidized triacylglyerides and identification of toxic and carcinogenic compounds formed during frying as important research goals (1). Dietary oil consumed from deep fried food, a major method used in food preparation, is of specific interest because chemical alterations of lipids occur when oils are heated to deep frying temperatures (175–192°C; refs. 2, 3). The deep-frying process, particularly after repeated frying or reusing oil, produces hydrolyzed, polymerized, and oxidized lipid degradation products including polymeric acylglycerides, oxidized acylglycerides and fatty acids, and cyclic monomers. As these molecules accumulate throughout repeated frying cycles, fresh frying oil progresses to thermally abused frying oil (TAFO). The new matrix of chemicals in TAFO raises both nutritional and toxicologic concerns, causing numerous countries worldwide to implement regulations governing oil quality (3–5). Under these legislations restaurant operations are permitted between 24% and 27% total polar material in frying oil (1, 4, 6, 7). In frying oils, polar materials are triglycerides and fatty acids whose chemical compositions are altered by the introduction of heat, air, and moisture during frying. Polarity is described by a peroxide value (POV), a measurement of the amount of hydroperoxides in oil which interact with a known concentration of iodide ions. No regulations for these polar compounds exist in the United States, although total polar content in severely abused oils can approach 25%.

The consumption of thermal degradation products in TAFOs can be altogether avoided through oil disposal after each instance of heating. However, this practice is unreasonable for the budgets of families and businesses, and reused oils contain desirable flavor and aroma compounds (2, 8). Therefore, regular dietary exposure to polar materials in TAFOs will likely continue to be a widespread phenomenon. Food lipid oxidation products such as lipid peroxides have been reported to promote atherosclerosis and coronary heart disease (9, 10). However, concrete and accepted demonstrations of relationships between dietary lipids and tumorigenesis have remained elusive [American Institute for Cancer Research (AICR), 1997]. This indicates a critical need to understand how lipid consumption and cancer progression are conflated. Many plausible mechanisms for dietary lipid-induced carcinogenesis have been proposed, including the promotion of oxidative stress and immune response, two favorable characteristics of tumorigenic environments (11–13). Fully elucidating the mechanisms behind dietary lipid driven tumorigenicity can inform preclinical questions and translate to clinical practices, including new dietary recommendations for patients and new therapeutic targets. However, much work toward this end-goal is necessary.

Although early research on the impact of food processing has implicated thermally abused oils as drivers of undesirable pathologies, these studies have been scrutinized and dismissed due to impractical temperatures used to abuse frying oils or irrelevant procedures, including intravenous injection, to administer abused oils to animals (4, 14). Further, validated epidemiological studies rely on diet histories which cannot precisely measure dietary fat intake, long-term dietary fat exposure, where the fat comes from, or if this exposure promotes cancer progression (12). Due to those experimental shortcomings, the accepted and un-criticized notion for over 20 years has been that good practices mitigate the risk of dietary oil being abused to the point of health problems (4, 14). We will reassess the nutritional and toxicologic risks of thermally abused oils incorporated into a low-fat diet.

This study investigates the relationships between thermally induced chemical alterations of dietary oil and breast cancer metastasis at operationally relevant temperatures (180°C ± 5°C). Metastatic breast cancer increases the likelihood of fatality by two-fold and severely depreciates patients “quality of life” (15). Because metastasis plays a fatal role in the etiology of breast cancer and the national intake of fried foods is high, this research focuses on understanding if the dissemination and progression of breast cancer cells from a primary tumor to a distal site is facilitated by the consumption of TAFO. The general preclinical and clinical understanding is that a low calorie, low-fat diet is ideal to minimize risk of invasive breast cancer or recurrence (16–18). Therefore, we incorporated TAFO into a low-fat diet (10% w/w) to test the notion that dietary oil composition contributes to breast cancer progression independent of oil quantity.

We previously developed a preclinical model of late-stage of breast cancer that recapitulates a micro-metastatic tumor in bone marrow that has regained proliferative and migratory activity after long-term dormancy, a common event in late-stage breast cancer (19, 20). This model was used to test the hypothesis that TAFO consumption enhances breast cancer metastasis. 4T1 tumor cells, which spontaneously metastasize to the lung, liver, and lymph nodes, were inoculated into the tibia of ovariectomized Balb/c mice which were fed either a 10% (w/w) fresh soybean oil (SBO) or TAFO diet. 4T1 cells expressed the luciferase enzyme, allowing bioluminescent monitoring of metastasis and tumor progression throughout the time course.

DMEM was purchased from the Cell Media Facility (School of Chemical Sciences, University of Illinois at Urbana-Champaign, Urbana, Illinois). Heat-inactivated FBS was purchased from Atlanta Biological. Streptomycin/penicillin and sodium pyruvate were purchased from Cellgro. Matrigel was purchased from BD Biosciences. d-luciferin potassium was purchased from Regis Technologies. Cleaved caspase-3 rabbit polyclonal antibody and Ki-67 rabbit monoclonal antibody were purchased from Biocare Medical. Liquid soybean frying oil (Harvest Value) and raw frozen and breaded catfish (Bluewater) were purchased through U.S. Foods Inc.

TAFO was produced under controlled conditions in the Food Science and Human Nutrition Pilot Processing Plant (University of Illinois at Urbana-Champaign). Catfish was fried using 7 L of industry-standard soybean oil in each fryer. A Cayenne 15 lb. electric counter top fryer (The Vollrath Company, L.L.C.) was used to fry 55 consecutive and continuous cycles (400 g food/cycle) over the course of 16 hours at 180 ± 5°C. The catfish entered the frying cycle while frozen per the manufacturer's instructions and fried 5 minutes, to ensure the safe minimum internal temperature mandated by the USDA was reached. Aliquots of oil were reserved after every five frying cycles to monitor the degradation of the oil throughout frying. The TAFO samples produced after 16 hours of frying, were sent to Harlan Laboratories, Inc., to be incorporated into AIN93G-based mouse diets.

The triple negative and luciferase-expressing murine 4T1 mammary carcinoma cell line was provided by Dr. David Piwnica-Worms from Washington University (St. Louis, MO) in 2003 with a negative test for infectious agents, including mycoplasma. Diagnostic tests were conducted in the Washington University Department of Comparative Biomedicine in 2003. Upon receipt, cells were thawed, expanded to create a stock, and passaged less than 10 times before use in this study. No short tandem repeat assays are currently available to authenticate murine cell lines; therefore, no authentication was completed in our lab. 4T1 cells were cultured in DMEM supplemented with 10% HI-FBS, 100 unit/mL penicillin, and 100 μg/mL streptomycin at 37°C in 5% CO₂. Cells were harvested at 70% confluence and 1,000 cells were seeded suspended in Matrigel for inoculation into mice.

The mice were maintained in accordance with the NIH Guide for the Care and the Use of Laboratory Animals and the experimental protocol was approved by the Institutional Animal Care and Use Committee (IACUC) at the University of Illinois - protocol #13315, approved September 2013. No patient studies are included in this investigation, eliminating a need for compliance with human subjects consent and ethical guidelines or IRB approval. Four-week-old ovariectomized female Balb/c mice were purchased and housed using procedures and protocols as described previously (20). Ovariectimizing mice eliminate complex effects of endogenous estradiol (21, 22). Further, the strain of mouse in this study was obesity resistant. Obesity in Balb/c mice results in systemic modifications of immune and metabolic profiles (23, 24), two biological factors that modulate breast cancer progression (25, 26). Like obesity, dietary lipid compounds can also facilitate immune mediated metastasis (27) and alter markers of dietary lipid metabolism in Balb/c mice (24). Therefore, the use of obesity resistant animals uncouples effects of obesity-induced changes from those induced by the treatment diet. Once received, the animals were acclimated to standard AIN-93G purified diets for two weeks followed by one additional week of transition to a low-fat AIN-93G control diet before receiving the TAFO treatment diet. At 7 weeks of age, animals received a standard AIN93 diet containing either 10% by weight (23.4% kcal) unheated fresh soybean frying (n = 7) oil or 10% fish TAFO (n = 9) for 16 weeks. Additional constituents of the diet include 57.4% (w/w) carbohydrates and 17.7% (w/w) protein, levels previously reported for optimal rodent health (28).

A Hunter colorimeter (HunterLab) was used to measure the color in L*a*b* mode in triplicate. L* is on the lightness axis (0 is black, 100 is white, and 50 is middle gray). a* is on the red-green axis (positive values are red, negative values are green and 0 is neutral). b* is on the blue-yellow axis (positive values are yellow, negative values are blue and 0 is neutral). Color changes of oil samples were further analyzed by the total color difference, which is denoted as ΔE*, which is calculated as follows:

POVs and p-anisidine values, measures of primary and secondary lipid peroxidation products, were determined according to AOCS Official Method Cd 18-90 (29). The amount of free fatty acid (FFA) in fats and oils can be used to indicate the extent of TAFO deterioration due to hydrolysis of triglyceride. FFA value was determined by AOCS Official Method Ca 5a-40 (29).

Intra-tibial inoculation of mice with 4T1 BC cells was conducted using a procedure described previously (20). After an acclimation period of 3 weeks and feeding period of 16 weeks, 4T1 metastatic mammary gland tumor cells (1 × 10³) expressing luciferase were transplanted intra-tibially. Therefore, our study of metastatic progression began in animals at 23 weeks (5.75 months) of age. Tumor progression was monitored by BLI every 4 days. The mice remained on their treatment diets for an additional 23 days postinjection prior to sacrifice.

Bioluminescence imaging (BLI) was conducted to monitor the growth and progression of the tumors using a procedure described previously (20). Every 4 days, mice were injected intraperitoneally with luciferin (150 mg luciferin per kilogram mouse) and imaged via BLI following an incubation period of 3 minutes. Imaging occurred on days 4, 8, 12, 16, and 20. The relative intensity of BLI output in bone and lung were measured by NIH ImageJ software (version 1.51k, public domain). An area that fully encompassed lung and bone metastasis was selected. For each animal, pixel quantities were measured, and background pixilation was subtracted within the predetermined area.

After euthanasia, blood was collected from the posterior vena cava and preserved at −80°C. Lungs, liver, spleen, and tibiae of each animal were fixed in 10% neutral buffered formalin or frozen at −80°C. Tissue trimming was standardized across all animals. Histologic slide preparation and IHC of lung was conducted by the Veterinary Diagnostic Laboratory (University of Illinois at Urbana-Champaign) according to SOPs. Tissues were embedded using Leica Formula R paraffin, sectioned at 4 μm, and stained with hematoxylin and eosin (H&E). Cell proliferation in tumors was determined using IHC analysis (antibody and reagents from Biocare Medical) Ki-67 (RM, 1:300 dilution) was applied to consecutive sections using an automated stainer and coverslipper (Sakura Finetek). The expression and area sums (μm²) of the raw images were quantified with AxioVision Rel 4.8 (Carl Zeiss AG).

Gross quantification of metastatic nodules in formalin-fixed lungs was conducted in a blind fashion without the knowledge of treatment groups in mice showing metastatic growth (n_SBO = 7, n_TAFO = 9). The independent counts from three trained observers were utilized for the final calculation. Histopathologic evaluation of metastatic lung tumors was conducted and regions of hypercellularity were examined at up to 20× magnification. Tissues were imaged and evaluated using NanoZoomer Digital Pathology System and NDP.view 2 software (Hamamatsu).

Total RNA from the liver samples was isolated from four randomly selected liver from each treatment group using a Direct-zol RNA MiniPrep Kit (Zymo Research). For RNA-seq, the TruSeq RNA Sample Prep Kit was used according to the manufacturer's guidelines (Illumina Inc.). Agilent Technologies Human UHR total RNA was used as a positive control sample. The library was constructed according to a standard protocol provided by Illumina Inc. and pooled in equimolar concentrations, quantitated by qPCR, and sequenced on three lanes for 101 cycles with a HiSeq2500 sequencing system in the High-Throughput Sequencing and Genotyping Unit by the Roy J. Carver Biotechnology Center (University of Illinois at Urbana-Champaign). After removal of adapter sequences, Illumina sequences, DNA contamination, and reads less than 80 base pairs, Fastq files were generated, demultiplexed with the bcl2fastq v1.8.4 Conversion Software (Illumina). Reads were aligned with the Mus musculus reference genome obtained from the Ensembl website using the TopHat alignment software. Feature counts were generated. The R statistical analysis software was used to identify differentially expressed genes (DEG).

Bone response to tumor was evaluated as described (30). In brief, 4T1 cell-inoculated tibiae were scanned using a μCT40 scanner (Scanco Medical AG). The bone was evaluated using semiquantitative (pathology score) and quantitative (measurement of bone) assays.

Tibiae were imaged at a threshold of 245 (scale of 0–1,000) and the reconstructed three-dimensional images were used for assessment of pathologic bone response to tumor. Bone response was characterized by periosteal bone proliferation (woven bone extending from the periosteum) and osteolysis (pathologic bone resorption) and was scored from 0 (normal bone architecture) to 5 (extensive woven bone/osteolysis).

To quantify the extent of periosteal woven bone, μCT image stacks of 500 slices (8 mm of bone) proximal to tibiofibular junction were analyzed. Woven bone was distinguished from cortical bone based on μCT voxel threshold. All voxels residing within 64 μm of the apparent periosteal surface were excluded from analysis to remove partial volume effects. Pathological woven bone was defined by voxels with thresholds values ranging from 175 to 423.

Quantitative data describing metastatic effects of TAFO were analyzed with a one-way ANOVA, Student t test, Mann–Whitney or PROC GENMOD using Statistical Analysis System software version 9.3 (SAS Institute, Cary, NC). Significant differences are reported at P < 0.05. Mean values and standard error of the mean are reported throughout this paper.

The TAFO showed many changes throughout the frying period, including shifts in (i) color, (ii) peroxidation levels, and (iii) concentrations of free fatty acids. Color changes were indicated by measures of lightness (l*), redness (a*), and yellowness (b*) and consolidated into the total color change measurement, ΔE*. ΔE* shifted from 0 to 23.0 ± 0.87. Peroxidation in TAFO was also shifted significantly as indicated by a change in peroxide value from 0.3 meq/kg in SBO to 18.1 ± 6.6 meq/kg in TAFO and a change in p-anisidine value from 0.2 to 22.7 ± 0.7. The percent free fatty acids increased from 0.1% to 32.4%, indicating high levels of hydrolytic de-esterification of triglycerides in the TAFO. Finally, the percent of polar material in oil increased from 3.84% to 15.2%. Together, these data indicate dramatic chemical changes between the fresh oil and the TAFO.

Metastatic lung colonization was assayed by BLI of luciferase-transduced cells, ex vivo examination of the lungs, and histologic analysis. At day 4 post-inoculation, BLI showed similar tumor colonization at tibial sites in both TAFO and SBO treatment groups, but no metastasis was observed (Fig. 1). By day 20, mice consuming the SBO diet had minimal metastatic growth detectable by BLI whereas the signal intensity in animals consuming the TAFO diet was markedly increased; the primary tibial tumor continued growing in situ in both treatment groups. Metastatic growth in the TAFO treatment group showed a four-fold increase (P < 0.05) in bioluminescent output by day 20, whereas there was no statistically significant difference between the bone tumor output when treatment and control groups were compared at day 20.

Histologic examination of lung sections revealed an approximately 2.5-fold increase in the number of metastases per animal in the TAFO group as compared with those consuming the fresh oil treatment diet. The metastatic tumor colonies had several distinct phenotypes, categorized descriptively as (i) solid tumors—well-differentiated solid tumors protruding from the lung surface (Fig. 2A and B) or within the parenchyma, with some tumors demonstrating a perivascular location; (ii) intravascular colonization—solid but poorly differentiated tumors (foci of cells with spindeloid morphology and high mitotic index) that colonized and expanded the vasculature, primarily the pulmonary veins (Fig. 2C); (iii) micro-metastases—small circumscribed foci of 2 to 100 cells, presumably within capillaries (Fig. 2D); and (iv) dispersed tumor—neoplastic cells were located free in alveoli or infiltrating alveolar walls. Because continuous migration of neoplastic cells from the primary tibial tumor site is expected, some of these phenotypes could simply be a manifestation of the different stages of metastases. However, although the TAFO group showed an increase in the number of each tumor phenotype, there was a striking increase in the dispersed tumor phenotype, with all (7/7) TAFO animals having multiple (up to 17) dispersed tumors while one dispersed tumor was observed in a single control animal (1/9). Intravascular colonization was also increased in six out of seven TAFO-treated animals as compared with only three of nine animals in the SBO group, likely indicating a decrease in tumor migration in SBO group. Dispersed tumors often involved an extensive area so they could not be adequately counted. In several animals, this tumor phenotype was associated with alveolar edema and hemorrhage.

Next, we examined the effects of the TAFO diet on metastatic tumor proliferation. Ki-67 reactivity was quantified using AxioVision Rel 4.8 measuring Ki-67 reactivity in solid non-necrotic cells from well-differentiated metastatic tumors that did not contain areas of necrosis. This phenotype was chosen because it provided a large discrete tumor area; however, cell proliferation was likely underestimated because this phenotype was the most differentiated. TAFO diet increased Ki-67 reactivity by 40.2% when compared with the SBO diet (Fig. 3), suggesting that mitogenic activity is promoted by TAFO consumption.

Liver is the principal location of lipid metabolism and the first organ TAFOs encounter after absorption from the gastrointestinal tract. Therefore, we conducted transcriptome analyses of liver to determine response to TAFO consumption when compared with SBO consumption in the animals. A significant increase was observed in liver weight relative to total animal weight (P < 0.05) in the TAFO group as compared with SBO (Fig. 4). RNA-sequencing revealed 1,411 differentially expressed genes in the liver (FDR < 0.05). Four hundred and fifty-five genes showed at least two-fold higher or lower expression in TAFO liver compared with SBO (Fig. 5A). Cluster analysis indicates alterations in lipid metabolism (Fig. 5B) and xenobiotic metabolic pathways (Fig. 5C).

To examine the influence of TAFO on primary tumor growth, tibial tumors were examined. The effect of treatment on tibial response to 4T1 cell inoculation is shown in Fig. 6. Significant differences in total tibia pathology score (Fig. 6A) or periosteal woven bone (Fig. 6B) were not detected between mice consuming SBO and mice consuming TAFO. Notably, pathology due to inoculation of tumor was extensive in all animals in both treatment groups. The woven bone in response to treatment can be readily appreciated in Fig. 6C.

To our best knowledge, we show for the first time that consumption of a low-fat diet with deteriorated oil heightens the frequency of metastatic tumor formation in a late-stage breast cancer model. We also observed phenotypic changes in the metastatic tumors that suggest TAFO consumption can lead to less differentiated metastatic tumors that are more difficult to treat. Spindeloid epithelial cells in some tumors suggested mesenchymal transformation. These findings add to recent evidence demonstrating the potential therapeutic efficacy of monitoring chemical compositions of dietary lipid intake (31–34) and emphasize a need to better understand the interplay between breast cancer metastasis and dietary fats. We propose that further investigation of the chemical structures accumulating in TAFOs and the biological responses to consumption of those altered lipid products is necessary. Specifically, the mechanistic drivers behind the influence of thermally altered dietary lipids on breast cancer metastasis should be thoroughly studied.

The literature strongly supports lipid peroxidation as a toxicologic concern due to oxidative cascades initiated by secondary peroxides; meanwhile, primary peroxides from ingested dietary lipids are considered of less toxicologic relevance (4, 9, 35). We can infer from the high rates of metastasis to lung after 20 days in animals consuming TAFO that the primary lipid source, the TAFO, could in fact be toxicologically relevant in addition to the secondary peroxides it produces (35). However, performing a dose response would be necessary to confirm TAFOs toxicologic outcomes, an endeavor outside the scope of this study. Further, the propensity of the metastatic neoplastic cells to attach to the vascular endothelia is shown by frequent instances of intravascular tumor colonization in the lung of TAFO fed animals (Fig. 2). This supports a model of metastasis whereby tumors metastasize to the lungs via proliferation of endothelium-attached cells, rather than extravasation of disseminated tumor cells (36). However, marked extravasation of neoplastic cells was also present in TAFO-treated animals with neoplastic cells free in alveoli in some cases.

The association between a low-fat diet of TAFO and metastatic progression we observed raises the question “Should breast cancer patients and survivors be advised to consider not only the quantity (percentage) of dietary lipids in their diets, but also the quality of them?” The general preclinical and clinical understanding is that a low calorie, low fat diet is ideal to minimize risk of invasive breast cancer or reoccurrence (17, 18). Fresh oil in this study referred to unheated oil and was characterized by free fatty acid content. The measured free fatty acid content of the fresh soybean oil used in our study was 0.073%, lower than the 0.1% cutoff that indicates freshness (37). In our model, in addition to enhancing metastasis, the diet composed of 10% by weight TAFO induced a xenobiotic response and altered lipid metabolism in the liver when compared with fresh oil. The xenobiotic response in liver suggests that TAFO is a toxicologic trigger that prompts the liver's detoxification response. We hypothesize that lipid structures in TAFO prompt xenobiotic metabolism in liver in lieu of or addition to canonical lipid catabolic enzymes. This xenobiotic response leads us to believe that patients with breast cancer and survivors may benefit from considering the quality of oil being consumed.

As expected (30), intra-tibial inoculation of mice with 1 × 10³ 4T1 cells resulted in periosteal woven bone formation and cortical bone osteolysis. We did not detect a treatment effect on the magnitude of bone damage assessed visually or on quantity of periosteal woven bone in response to 4T1 cells; the response was very severe in all mice. We cannot rule out the possibility that an effect of consuming TAFO may have been observed had the study been terminated earlier. However, doing so would have compromised detection of lung metastasis, the primary goal of the study. Bone tumor burden was not measured in this study. However, we previously demonstrated a strong temporal association between tumor cell infiltration of bone marrow and bone destruction (30). We also showed that increased tumor burden and bone destruction associated with feeding mice a low calcium diet did not alter metastasis to lung. In contrast, reduced estrogen levels increased 4T1 cell-induced bone damage while decreasing tumor metastasis from bone to lung (38, 39). Taken together, these findings suggest that dietary and/or endocrine factors can alter the likelihood of metastasis independent of tumor burden.

It is important to note that this study presents the findings of abused oil produced from frying a single food source, battered catfish. Varying food sources require different frying times to meet USDA safety guidelines and appropriate internal temperatures. This fact, coupled with vastly different chemical compositions among food sources results in many possible variations of oxidation, hydrolysis, isomerization, and polymerization products during frying (2, 40). Differences among the resulting lipid profiles mean the physiologic influence by TAFO consumption will vary by food source. This is consistent with literature in food chemistry regarding Maillard reactions, hydrocarbon behavior at high temperatures, and the established reaction mechanisms of lipid hydrolysis, oxidation, and polymerization (2, 8, 41). Other studies have mitigated these variations by frying multiple food sources in the production of a single TAFO (42). However, in our studies this multiple food source approach would weaken our precision when investigating mechanistic details of TAFO induced breast cancer metastasis in future work. The data presented here cannot be extrapolated to other food sources but remains a reliable demonstration that thermal alterations of lipids during frying can promote breast cancer metastasis.

Finally, there is some biochemical and nutritional discrepancy between consuming TAFOs rather than consuming fried food. As we work toward a more complete understanding of the complex chemistry that occurs between food and oil, a model that wholly reflects eating fried food can be developed. The observed relationship between a 10% diet of TAFO and the progression of breast cancer from tibia to lung remains strong because as much as 40% of a fried food's weight can consist of oil absorbed during the frying process (43). Therefore, consumers of fried foods are consumers of TAFOs.

We conclude that breast cancer metastasis in this model is at least in part promoted by chemical alterations arising from heating dietary oils to frying temperatures. The data generated from this study can prompt biological, nutritional, and biomedical researchers and clinicians to consider how diseases, especially metastatic breast cancer, and organ systems are impacted by TAFO consumption. This understanding may offer more comprehensive strategies for breast cancer survivors to reduce risk of recurrence through diet modification. Further, these results can act as a precursor for development of food policies and regulation bodies. In the United States, the current approach to regulating the reuse of frying oils in industrial settings is more relaxed than that of many comparable developed countries. Countries including Austria, Belgium, The Netherlands, Finland, France, Japan, and Spain have implemented restrictions or guidelines that prohibit the use of dietary oils exceeding a polar content of 25% to 27% or falling beneath a smoke-point of 170°C (6, 10). Based on this study's observation of an adverse biological response to the consumption of degraded lipids, the following questions must be addressed in the United States: (i) Should the United States establish regulations on the reuse of deep-frying oils and (ii) Should these regulations match or exceed the stringency of comparable nations? More evidence-based information must be generated to answer these questions. Therefore, this research raises many questions for nutritionists of varying specialties, including medicine, toxicology, food policy and regulation, nutritional education, and epidemiology.

No potential conflicts of interest were disclosed.

Conception and design: A. Cam, A.B. Oyirifi, W.M. Haschek, W.G. Helferich

Development of methodology: A. Cam, Y. Liu, W.G. Helferich

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc.): A.B. Oyirifi, Y. Liu, W.M. Haschek, U.T. Iwaniec, N.J. Engeseth, W.G. Helferich

Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): A. Cam, A.B. Oyirifi, Y. Liu, W.M. Haschek, U.T. Iwaniec, R.T. Turner, W.G. Helferich

Writing, review, and/or revision of the manuscript: A. Cam, A.B. Oyirifi, Y. Liu, W.M. Haschek, U.T. Iwaniec, R.T. Turner, N.J. Engeseth, W.G. Helferich

Administrative, technical, or material support (i.e., reporting or organizing data, constructing databases): A. Cam, A.B. Oyirifi, W.G. Helferich

Study supervision: A. Cam, W.G. Helferich

This work was supported by the NIH [P50AT006268] (to W.G. Helferich) from the National Center for Complementary and Integrative Health (NCCIH), the Office of Dietary Supplements (ODS), the National Cancer Institute (NCI), and National Institute of Environmental Health Sciences Grant T32 ES007326 (to A.B. Oyirifi). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCCIH, ODS, NCI, or the NIH.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.