Lignans

In vitro cytotoxicity and clonogenicity assays were performed using the sulforhodamine B (SRB) (MP Biomedicals, LLC) protein staining assay [40 ,41 (link),42 (link),43 ], using the cell lines described before. Briefly, cells were seeded in 384- or 96-well microtiter plates at a density of 1 × 10⁴ cells/mL, and placed in an incubator (5% CO₂ and 37 °C) for 8 h. Afterward, different concentrations (0.08, 0.4, 2, and 10 µg/mL) of pure compounds and positive controls (POD and VP-16) were added in triplicate and incubated for 72 h.
Afterward, cells were fixed with cold trichloroacetic acid (30% in water) and stained with SRB (0.4% in a 1% of acetic acid solution). Cells were washed with 1% of acetic acid solution. Finally, the bound colorant was solubilized with Tris-base to obtain optical density (OD_sample). The bound colorant was proportional with either total protein or cells amount. DMSO (final concentration of 0.5%) was used as vehicle and blank (OD_blank). The total protein concentration in a single plate with cells at the beginning of the assay was considered as zero (OD_zero). Microtiter plates were incubated for 72 h after which the total protein concentration was determined with Equation (1). This assay measures the respective absorbance at 490 nm using a spectrophotometer (Molecular Devices, SPECTRA max plus 384).

To determine the recovery percentage, an in vitro clonogenicity assay was performed. Cells were seeded, incubated, and treated in microtiter plates under the previously described conditions. After 72 h of treatment, the culture media of each well was removed, followed by two washes with phosphate buffered saline (PBS), before warm media supplemented with FBS was added to the cells and incubated for an additional 72 h in the same conditions. The percentage of proliferation was then determined using Equation (1).
We have defined percentage of recovery as the percentage of survival at 72 h of treatment added to the percentage of proliferation calculated 72 h post-treatment. This is outlined in Equation (2) and Figure 4:

The half inhibitory concentrations of lignans and controls were calculated from six concentrations (0.0032, 0.016, 0.08, 0.4, 2, and 10 µg/mL). Data analysis was performed using the relationship between the % of survival cells (total protein, Equation (1)) and the relative optical density vs. concentration as a logarithm expression. The IC₅₀ values were reported as a mean of three independent experiments ± standard deviation (S.D.).

We used the same procedures in the NJ Ovarian Cancer Study and the EDGE Study to standardize data collection in cases and controls. Interviewers were trained using the same procedures and same training manual. Interviews, conducted by telephone for most respondents, covered established and suspected risk factors for ovarian cancer. In addition to the interview, participants were mailed a package with instructions for providing buccal specimens and waist and hip circumference measurements and the Block 98.2 food frequency questionnaire (FFQ). Participants were instructed to report their usual intake of the food items in the questionnaire during the six months before diagnosis (for cases) or the date of the interview (for controls). Two hundred and five (88%) cases and 398 controls (85%) returned the FFQ. The participants who returned the FFQ tended to be older, but there were no significant differences in education, oral contraceptive use, hormone replacement therapy use, tubal ligation or family history of ovarian cancer (data not shown). Eight of the controls were excluded because both of their ovaries had been removed, placing them at negligible risk of developing ovarian cancer, resulting in 390 controls being included in analyses.
The Block 98.2 FFQ (NutritionQuest, Berkeley, CA) includes 110 food items and was developed using the NHANES (National Health and Nutrition Examination Survey) III dietary recall data. It also includes questions on portion size for each food, and pictures are provided to facilitate estimation. The questionnaire includes a variety of foods containing phytoestrogens, such as several kinds of beans, tofu, soymilk, canned tuna fish, meat substitutes (e.g., veggie burgers, veggie chicken), and whole wheat bread. To supplement the list of foods, we added one page with 21 additional food items, based on the LACE questionnaire [28 (link)] and including other food items that have been identified as important sources of phytoestrogens [29 (link)]. The additional foods in the supplemental page that we added, and that are not included in the Block 98.2 questionnaire, are listed in Appendix 1. We also asked about the use of phytoestrogen/soy supplements including frequency and duration of use. NutritionQuest provided nutrient calculations using the USDA Nutrient Database for Standard Reference. For phytoestrogen calculations we used a Canadian database with detailed analyses of phytoestrogen content of foods, including detailed values for lignans [23 (link)]. Given the global food trading, we do not expect major differences in lignan composition between foods available in the United States and Canada.

The levels of isoflavone metabolites, daidzein (ng/mL), equol (ng/mL), genistein (ng/mL), and O-desmethylangolensin (ODMA, ng/mL) as well as lignan metabolites, enterodiol (ng/mL) and enterolactone (ng/mL) in the urine were measured by high-performance liquid chromatography-atmospheric pressure photoionization-tandem mass spectrometry. The association between urinary phytoestrogen levels and isoflavone intake in the years 2007–2010 was analyzed using the Pearson correlation method.

A semi-quantitative FFQ, previously validated with 136 food records, was used to evaluate dietary intake at both study inclusion and at 10-year of follow-up [25 (link),26 (link),27 (link)]. For each food item, 9 options of intake were provided in the FFQ (>6 times/day; 4–6 times/day; 2–3 times/day; once/day; 5–6 times/week; 2–4 times/week; once/week; 1–3 times/month and never or almost never). To calculate the daily intake of nutrients and energy, the standard servings were multiplied by the frequency of consumption of each food and by the nutritional content or kilocalories of energy specified in Spanish food composition tables [28 ,29 ]. Other eating habits such as snacking between meals or following a special diet were also gathered at baseline. Compliance with the Mediterranean diet was measured with the score (0–9) of Trichopoulou et al. [30 (link)].
The Phenol-Explorer database version 3.6 (www.phenol-explorer.eu (accessed on 15 December 2022), whose methodology has been published elsewhere, was used to calculate the PC content of each food [31 (link),32 (link)]. The most common extraction method used for estimating PC intake was high-performance liquid chromatography (HPLC), which simultaneously quantifies phenolic acid esters and phenol glycosides together with aglycones and free phenolic acids, and normal-phase HPLC to estimate the proanthocyanidins content. Data from HPLC after hydrolysis method were used, if available, to calculate the aglycone in phenolic acids and lignans in some foods such as olives, beans and grains, since this analytical method uses hydrolysis to release certain compounds which cannot be solubilized without hydrolysis. Foods with only traces of PC were excluded. A weighted average was calculated according to the average dietary consumption of the Spanish adult population when the FFQ items contained more than one food [33 ], and for processed foods and recipes PC content was calculated according to their ingredients. Finally, the PC intake from each food was calculated by multiplying the content of each PC by the daily consumption of each food. The total PC intake (milligrams per day) was determined as the sum of all PC of each food item reported in the FFQ, and was also calculated by class intake. Additionally, we calculated the contribution of each specific food or food group to the total PC intake and expressed it as percentage.