A Novel Tomato-Soy Juice Induces a Dose-Response Increase in Urinary and Plasma Phytochemical Biomarkers in Men with Prostate Cancer

A Novel Tomato-Soy Juice Induces a Dose-Response Increase in Urinary and Plasma Phytochemical... ABSTRACT Background Tomato and soy intake is associated with reduced prostate cancer risk or severity in epidemiologic and experimental studies. Objective On the basis of the principle that multiple bioactives in tomato and soy may act on diverse anticancer pathways, we developed and characterized a tomato-soy juice for clinical trials. In this phase 2 dose-escalating study, we examined plasma, prostate, and urine biomarkers of carotenoid and isoflavone exposure. Methods Men scheduled for prostatectomy were recruited to consume 0, 1, or 2 cans of tomato-soy juice/d before surgery (mean ± SD duration: 24 ± 4.6 d). The juice provided 20.6 mg lycopene and 66 mg isoflavone aglycone equivalents/177-mL can. Plasma carotenoids and urinary isoflavone metabolites were quantified by HPLC–photometric diode array and prostate carotenoids and isoflavones by HPLC–tandem mass spectrometry. Results We documented significant dose-response increases (P < 0.05) in plasma concentrations of tomato carotenoids. Plasma concentrations were 1.86-, 1.69-, 1.73-, and 1.69-fold higher for lycopene, β-carotene, phytoene, and phytofluene, respectively, for the 1-can/d group and 2.34-, 3.43-, 2.54-, and 2.29-fold higher, respectively, for the 2-cans/d group compared with 0 cans/d. Urinary isoflavones daidzein, genistein, and glycitein increased in a dose-dependent manner. Prostate carotenoid and isoflavone concentrations were not dose-dependent in this short intervention; yet, correlations between plasma carotenoid and urinary isoflavones with respective prostate concentrations were documented (R2 = 0.78 for lycopene, P < 0.001; R2 = 0.59 for dihydrodaidzein, P < 0.001). Secondary clustering analyses showed urinary isoflavone metabolite phenotypes. To our knowledge, this is the first demonstration of the phytoene and phytofluene in prostate tissue after a dietary intervention. Secondary analysis showed that the 2-cans/d group experienced a nonsignificant decrease in prostate-specific antigen slope compared with 0 cans/d (P = 0.078). Conclusion These findings provide the foundation for evaluating a well-characterized tomato-soy juice in human clinical trials to define the impact on human prostate carcinogenesis. This trial is registered at clinicaltrials.gov as NCT01009736. carotenoids, lycopene, isoflavones, tomato, soy, prostate cancer Introduction Prostate cancer remains an enormous health burden, and although diagnosis and treatment options have improved over the past 2 decades, specific strategies to prevent prostate cancer remain elusive. Geographic variation in risk around the globe as well as changing risk with migration and, over time, within nations strongly implicate dietary and lifestyle patterns as contributing factors (1). Although a few large-population studies have identified foods and dietary patterns that may reduce prostate cancer risk (2–5), analytic epidemiology has struggled to provide a foundation of data upon which to develop and test dietary prevention strategies (6–11). Several nutrients, phytochemicals, and dietary patterns, with support from in vitro mechanistic studies, including components found in tomatoes and soy foods, affect experimental prostate carcinogenesis (12–15). Although a causal relation between foods, such as tomatoes or soy, and prostate cancer risk reduction is not clearly established, several lines of evidence suggest that these foods have components that inhibit prostate carcinogenesis (14–26). Decades of research on pharmacologic cancer therapies have established the paradigm that combinations of active agents with multiple mechanisms of action and nonoverlapping toxicity provide the greatest efficacy in curing advanced malignancies (27, 28). We hypothesize that a similar strategy can form a foundation for the design of functional foods or dietary patterns for prostate cancer prevention (29–31). To this end, we propose a combination of tomato and soy. Previous work showed that feeding a 10% tomato powder plus 2% soy germ–containing diet resulted in significantly reduced prostate tumor incidence in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model compared with mice fed a control diet or a diet containing tomato powder or soy germ alone (tumor incidence in tomato powder plus soy = 45% compared with 100% in controls, 61% in the tomato powder group, and 66% in the soy germ group) (30). To extend our findings, it is first necessary to develop and fully classify the bioactive components of a novel food product that can be applied to human studies. To optimize incorporation into a daily diet and promote compliance, we previously developed a tomato-soy juice product that provides consistent phytochemical exposure (32). We targeted tomato lycopene and soy isoflavone content from two 177-mL cans/d to achieve physiologic exposure mimicking the dose range associated with lower cancer risk in human studies (Table 1) (2, 33, 34). The tomato-soy juice was previously developed, subjected to sensory evaluation, and analyzed for phytochemical content and stability. In an 8-wk phase 1 study, we showed its compliance and safety in healthy humans (32). The objective of the current study is to define both the compliance and safety of 2 different doses of tomato-soy juice, as well as carotenoid and isoflavone biodistribution and metabolism, in men with prostate cancer. TABLE 1 Nutrient and phytochemical content of each 177-mL (6-fluid-ounce) can of tomato-soy juice Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 View Large TABLE 1 Nutrient and phytochemical content of each 177-mL (6-fluid-ounce) can of tomato-soy juice Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 View Large Methods Materials HPLC-grade methyl-tert butyl ether (MTBE), methanol (MeOH), acetonitrile, and water and reagent-grade hexane, ethanol, acetone, and toluene were purchased from Fisher Scientific. Lycopene (>95%) was isolated and purified as previously reported (35). β-Carotene and lutein standards (≥95% purity) were purchased from Sigma Aldrich (35). Phytoene and phytofluene standards (≥95% purity) were purchased from Carotenature (35). Subject recruitment Men with biopsy-proven carcinoma of the prostate who had chosen a radical prostatectomy for treatment were recruited from the James Cancer Hospital and The Ohio State University Medical Center (Columbus, OH). There were no age restrictions, and subjects were required to have an Eastern Cooperative Oncology Group performance status of 0–1 and have no clinical abnormalities in kidney, liver, or hematopoietic function, as determined by preoperative assessment. Subjects were asked to discontinue all nutritional supplements, including lycopene and soy. A multivitamin-mineral (CVS Brand) was provided for the study duration. Patients were excluded if they were receiving neoadjuvant hormonal (thyroid, adrenocorticotropic, or growth hormone) or chemotherapy or if they had an active malignancy other than prostate cancer requiring therapy, a history of castration, or other endocrine disorders requiring hormone administration, with the exceptions of diabetes and osteoporosis. Patients with a history of malabsorptive disorders or disorders requiring special diet recommendations (e.g., low-sodium diet for hypertension), severe constipation, or a recent history of anemia or iron deficiency were also excluded. Patients were excluded if they were currently taking finasteride, other agents for benign prostatic hypertrophy, or medications for urinary outlet obstruction. Study design All of the procedures were approved by the Ohio State University Institutional Review Board (clinicaltrials.gov: NCT01009736). Eligible subjects completed informed consent. Upon enrollment, subjects completed a baseline tomato/high-lycopene food-intake questionnaire (Supplemental Data 1), which queried about serving size and frequency of consumption of fresh tomatoes, tomato juice, soup, pizza, pasta sauce, watermelon, pink grapefruit (including juice), salsa, ketchup, and barbeque sauce. In addition, participants answered questions about soy food intake and documented intakes of other soy not listed (Supplemental Data 2). Participants were assigned to an intervention group using a dose-escalation design. The planned intervention consisted of consuming 0, 1, 2, or 3 cans/d of the tomato-soy juice developed by the Department of Food Science and Technology at The Ohio State University (formulation: OSU-TSJ-001) (32) for a 3- to 5-wk duration, which varied on the basis of surgery scheduling. Toxicity to OSU-TSJ-001 was assessed using the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 3.0, and the study physician (SKC) was notified immediately of any adverse events. Each study group was fully enrolled before enrollment in the next dose level began. The study proceeded to the next dose level only if there were <2 episodes of grade I/II toxicity and no episodes of grade III or IV toxicity attributed to the food product after evaluation of each cohort. Ultimately, however, the 3-cans/d group was not enrolled, because 2 of the 30 men (7%) in the 2-cans/d group experienced grade I adverse events. In addition, men in the 2-cans/d group indicated potential difficulties with consuming 3 cans/d. Instead, we expanded the 2-cans/d cohort. Tomato-soy juice intervention product The tomato juice was prepared from tomatoes grown by the Department of Horticulture and Crop Science at Ohio State University. A high-carotenoid tomato variety (FG99-218), which has excellent juice-producing properties, grows successfully in Ohio, and is homozygous for the high pigment-2 dark-green allele (hp-2dg) and for the old gold crimson allele (og c), was selected. Tomato juice was initially produced using a hot-break treatment and preserved in no. 10 cans, as previously published (36), to capture the tomato flavor and carotenoid profile at the peak of ripeness. To produce the tomato-soy juice, the already preserved tomato juice was pooled in an agitated, steam-jacketed kettle and combined with salt, 1% extra-virgin olive oil (Bertolli) added to enhance taste and palatability, and 0.10% soybean-isoflavone–rich extract (wt:wt; Solgen 40S; Solbar Plant Extracts, Ltd.) on a weight basis and heated to 95°C. The juice was then hot-filled into 177-mL (6 fluid ounces) cans and retorted at 100°C for 15 min. All constituents used for juice production originated from single batches of ingredients. After sealing and cooling, this product was stored at room temperature. The juice nutrient and phytochemical content is detailed in Table 1. Blood, urine, and prostate sample collection Venous blood samples (17 mL) were collected upon enrollment (nonfasting) and surgery (fasting) from subjects into 1 serum separator Vacutainer tube and 2 EDTA Vacutainer tubes (BD). Tubes were light-protected with foil, maintained on ice, and centrifuged at 1100 rotations per minute or 500 relative centrifugal force (× g) for 20 min at 4°C. The serum and plasma were divided into aliquots, placed into vials, and stored at −80°C for analyses. A 10-mL spot urine sample was collected upon enrollment, and a complete 24-h urine collection was begun 1 d before surgery. Throughout collection, the urine containers were kept refrigerated, and samples were divided into aliquots and stored at −80°C until analysis. Prostate tissue samples were obtained at surgical resection, and a piece of prostate tissue (noncancerous by gross examination) was immediately frozen in liquid nitrogen and stored at −80°C for phytochemical analysis. A separate tissue specimen was transported to the Ohio State University Surgical Pathology Department–Comprehensive Cancer Center Tissue Procurement Center for pathologic examination by a board-certified pathologist. Compliance monitoring All participants were instructed to follow a “controlled lycopene diet.” We restricted consumption of high-lycopene foods and limited lower-lycopene tomato foods to provide ≤5 mg lycopene/d. Participants were also instructed to avoid foods containing soy protein and soy isoflavones. All men completed a daily log (Supplemental Data 1 and 2) to document consumption of the study product (for the intervention groups) and adherence to or deviations from the controlled lycopene diet. Biochemical measurements Prostate-specific antigen and lipid profiles Plasma prostate-specific antigen (PSA) and serum TG, LDL-, HDL-, and total-cholesterol concentrations were measured at baseline and at the end of study using standard clinical procedures by the Ohio State Center for Clinical and Translational Science Laboratory and the Ohio State University Medical Center Clinical Laboratory. PSA was analyzed by the Medical Center Clinical Laboratory on a Centaur XP (Siemens Medical Diagnostics) using sandwich chemiluminescent immunological reaction with paramagnetic particles as the solid phase and acridinium ester as the chemiluminescent label. Lipids were analyzed using the Dimension Xpand Clinical Chemistry System (Siemens Medical Diagnostics). The analytical sensitivity was 3.0 mg/dL for HDL cholesterol, 5 mg/dL for LDL cholesterol, 50 mg/dL for total cholesterol, 15 mg/dL for TGs, and 0.01ng/mL for PSA. Plasma and prostate tissue carotenoid extraction and analysis Carotenoid concentrations of the plasma and juice samples were analyzed using reverse-phase HPLC coupled with photometric diode array using our previously published method (37). The tomato-soy juice carotenoids were extracted following a previously described method (35) and analyzed using the HPLC method cited above. Plasma lycopene, β-carotene, α-carotene, zeaxanthin, lutein, and β-cryptoxanthin were quantified using external standard curves. Plasma and tomato-soy juice phytoene and phytofluene were quantitated by comparing the molar extinction coefficient of lycopene with those of phytoene and phytofluene and by adjusting the standard curve slope of lycopene appropriately for phytoene and phytofluene. Prostate tissues (20–200 mg) were pulverized to fine particles with an anvil-in-cup apparatus by repeated striking with a rubberized hammer. Pulverized material was weighed into an 11-mL glass vial, water was added (1 mL), the suspension probe sonicated (5 s, default energy; Sonic Dismembrator Model 150E; Fisher Scientific), ethanol added [1 mL with 1% (wt:vol) butylated hydroxytoluene], probe sonicated, and extracted twice with 5 mL HEAT (10 hexane:6 ethanol:7 acetone:7 toluene; vol:vol:vol:vol). The upper organic layers from both were combined into a 40-mL glass vial and dried under nitrogen, then redissolved in 200 µL 1:1 MTBE:MeOH, and finally filtered through 0.2-µm nylon syringe filters. Reconstituted extracts were injected (40 µL) on the QTrap5500 LCMS system (AB Sciex) (38). Lycopene, β-carotene, phytoene, and phytofluene concentrations were quantified against an external standard curve. Urine and tissue isoflavone analysis Urine isoflavones were extracted as described previously (24). Isoflavone metabolites were extracted from 20–200 mg prostate tissue by suspending in 100 µL water, probe sonicating (5 s; default energy), and adding 200 µL acetonitrile. This solvent composition liberates isoflavone metabolites while precipitating proteins and other sample matrix components. Samples were centrifuged for 5 min at 21,130 × g, and supernatant was collected. The pellet was resuspended in 300 µL of 2:1 acetonitrile:water, probe sonicated, and centrifuged again. The 2 supernatants were pooled and dried under nitrogen. The residues were resuspended in 1 mL of 1 M sodium acetate buffer, pH 5.5, and 10 µL of 54 mg glucuronidase-sulfatase/mL in 0.2% NaCl was added to deconjugate over 2 h at 37°C, and extracted twice with 3 mL ether each. Aglycone extracts were dried under nitrogen and reconstituted in 150 µL methanol with bath sonication and filtered through 0.2-µm nylon syringe filters. LC-MS analyses of daidzein, genistein, equol, dihydrodaidzein (dhDaidzein), dihydrogenistein (dhGenistein), 6-hydroxy-O-desmethylangolensin (6-OH-ODMA), and O-desmethylangolensin (ODMA) in prostate tissue and glycitein in urine were conducted as described in Ahn-Jarvis et al. (24). Statistical analyses Baseline characteristics were summarized using means and SDs. Differences between groups were determined using ANOVA, with the exception of the baseline habitual phytochemical intakes, for which the Kruskal-Wallis nonparametric test was used due to multiple observations of no soy intake (39). In the statistical analyses of the plasma and prostate outcomes, the primary goal was to estimate the effect of the intervention. As such, covariates were included in the statistical models if they modified the effect of the intervention by >15% (40). Interactions with the intervention were included at the 0.05 significance level. For plasma, the preintervention measure of the outcome was always included in the model, consistent with an ANCOVA approach. Pearson correlations were calculated between plasma and tissue carotenoid concentrations. When observations for plasma or tissue were below the detection limit, a Wilcoxon Rank Sum test, as opposed to a parametric test, was used to compare urinary and tissue isoflavone concentrations between groups (39). Similarly, for correlation analyses, a Kendall's τ-b nonparametric correlation was calculated between urinary and tissue isoflavone concentrations due to the presence of nondetects. Change in PSA was evaluated using ANCOVA. In all parametric analyses, concentrations were log-transformed before analysis to improve normality and homoscedasticity. Where applicable, the stepdown Bonferroni (Holms) procedure was used to adjust P values for multiplicity. Data included those for each participant who completed ≥2 wk of the intervention. Statistical analyses were performed in SAS version 9.4 (SAS Institute) or Stata version 13.1 (StataCorp). Clustering of the urine isoflavones was conducted using hierarchical agglomerative methods. Proportions of the 3 components of the daidzein family of metabolites, equol, ODMA, and dhDaidzein and daidzein (combined) were computed over the daidzein family total. These proportions were transformed using the arcsin square root to stabilize their variance and improve normality. Clusters were formed using Euclidean distance measure and average linkage. Final determination of the number of clusters present was conducted by visual inspection of the dendrogram. Results Sixty men consented to participate. Three men withdrew for the following reasons: 1 opted for surgery at another hospital, 1 did not want to complete the questionnaires, and 1 postponed surgery indefinitely. In addition, 1 subject did not complete baseline sample collection, and another only consumed 20% of the targeted intervention goal. All 5 of these men were excluded from statistical analyses. Three men were excluded from tissue isoflavone analysis only, and an additional 3 men were excluded from both tissue carotenoid and tissue isoflavone analyses due to inadequate prostate tissue procurement. Finally, men who did not drink the juice for the 2 d before surgery (confusion with preoperative instructions) or who did not complete a full 24-h urine collection were excluded from the urinary isoflavone analysis (n = 8). The prestudy habitual daily tomato or soy intakes reported at baseline are shown in Table 2. Subjects reported usual consumption of 10.7 ± 8.2 (mean ± SD) servings of lycopene-rich foods/wk, with the most frequently consumed category being ketchup or barbeque sauce, with 3.8 ± 5.1 servings/wk contributing 6.05 ± 8.17 mg lycopene/wk to the diet. The greatest sources of lycopene in the diet were pasta sauce (27% of lycopene intake; 15.6 ± 12.16 mg/wk), followed by tomato juice (20%; 11.5 ± 35.2 mg/wk), pizza (13%; 7.4 ± 8.4 mg/wk), and ketchup (11%). Subjects reported negligible baseline consumption of any soy-based foods. With the exception of age, other baseline characteristics of the 55 men who completed all study interventions were not significantly different between groups and are summarized in Table 1. The mean ± SD number of days receiving the intervention was 24 ± 4.6. TABLE 2 Baseline characteristics and reported habitual phytochemical intake of patients with prostate cancer participating in the OSU tomato-soy juice study1 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 1 Values are means ± SDs unless otherwise indicated; n = 55 for all measures. OSU, Ohio State University. 2 Mean ± SD ages of the 0-, 1-, and 2-cans/d groups were 54.7 ± 8, 60.2 ± 7, and 63.2 ± 7 y, respectively. The men consuming 2 cans/d were significantly younger than men consuming 0 cans/d (P < 0.05). 3 Prestudy carotenoid intake is based on subject responses to a baseline questionnaire querying the usual number of weekly servings consumed of the following high-lycopene foods: raw tomato, tomato juice, tomato-based mixed-vegetable juice, tomato soup, pizza, tomato-based pasta sauce (marinara), watermelon, grapefruit, tomato salsa, ketchup, and barbeque sauce. Prestudy soy isoflavone intake is based on subject responses to a baseline questionnaire querying weekly intake of soymilk, soy burgers, soy nuts, and other soy products (open response). 4 Value includes 40% of men who were taking a statin medication for cholesterol management. View Large TABLE 2 Baseline characteristics and reported habitual phytochemical intake of patients with prostate cancer participating in the OSU tomato-soy juice study1 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 1 Values are means ± SDs unless otherwise indicated; n = 55 for all measures. OSU, Ohio State University. 2 Mean ± SD ages of the 0-, 1-, and 2-cans/d groups were 54.7 ± 8, 60.2 ± 7, and 63.2 ± 7 y, respectively. The men consuming 2 cans/d were significantly younger than men consuming 0 cans/d (P < 0.05). 3 Prestudy carotenoid intake is based on subject responses to a baseline questionnaire querying the usual number of weekly servings consumed of the following high-lycopene foods: raw tomato, tomato juice, tomato-based mixed-vegetable juice, tomato soup, pizza, tomato-based pasta sauce (marinara), watermelon, grapefruit, tomato salsa, ketchup, and barbeque sauce. Prestudy soy isoflavone intake is based on subject responses to a baseline questionnaire querying weekly intake of soymilk, soy burgers, soy nuts, and other soy products (open response). 4 Value includes 40% of men who were taking a statin medication for cholesterol management. View Large Subject compliance and toxicity Men in all 3 groups (0, 1, and 2 cans/d) were compliant with the “controlled lycopene diet” and maintained a diet of ≤5 mg lycopene on 97% (1272 of 1310) of study days (mean lycopene ± SD = 1.1 ± 0.29 mg/d, 0.54 ± 0.2 mg/d, and 0.47 ± 0.10 mg/d, respectively). Men in both the 1-can/d and 2-cans/d groups, who were also compliant with the intervention product, consumed 97% and 91% of the targeted dose, respectively. In the 2-cans/d group, there were 2 grade I adverse events. One man experienced grade I diarrhea and discontinued the juice after 7 d, at which time his symptoms resolved; because of the short intervention duration, he was excluded from data analysis. A second man discontinued the juice after 16 d due to hypertension, which did not resolve after discontinuation of the study product. Because isoflavones are quickly metabolized and excreted, as marked by a plasma half-life of 7 h (41), we did not include this subject in isoflavone analysis. However, lycopene has a plasma half-life of 6 d and a tissue half-life of 12 d (42); therefore, this participant was included in both blood and tissue carotenoid analyses. Clinical measures of toxicity were within normal limits at both baseline and at the end of study for all men. Plasma carotenoids Plasma lycopene, β-carotene, phytofluene, and phytoene concentrations at baseline and at the end of the intervention are shown in Table 3. There was a dose-dependent and significant increase for each of the tomato carotenoids analyzed, with the exception of phytoene in the 1-can/d group. In addition, the model-adjusted analysis for men consuming 2 cans/d showed plasma concentrations that were 2.34-fold (95% CI: 1.95-, 2.81-fold), 3.43-fold (95% CI: 2.53-, 4.66-fold), 2.54-fold (95% CI: 1.86-, 3.45-fold), and 2.29-fold (95% CI: 1.78, 2.94-fold) higher than the 0-cans/d group for lycopene, β-carotene, phytoene, and phytofluene, respectively. The men consuming 1 can/d had plasma concentrations that were 1.86-fold (95% CI: 1.49-, 2.31-fold), 1.69-fold (95% CI: 1.2-, 2.37-fold), 1.73-fold (95% CI: 1.22-, 2.46-fold), 1.69-fold (95% CI: 1.27-, 2.23-fold) higher than the 0-cans/d group, respectively. For lycopene, the interaction between intervention group and duration was significant (P = 0.0099), such that a longer intervention before scheduled prostatectomy led to greater plasma lycopene changes within the intervention group. Therefore, intervention duration and baseline plasma lycopene concentration were included in the analysis of the effect of treatment group on final plasma lycopene concentration. Other covariates are listed in Table 3. TABLE 3 Plasma carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* 1 Values are means ± SDs. Labeled means in a column without a common superscript letter differ, P < 0.05. *Different from baseline, P < 0.05. The interactions between the length of intervention and intervention group and baseline lycopene were included in the model for lycopene. Age, BMI, and baseline values were included for β-carotene and phytofluene. Other covariates tested (plasma volume, blood lipoproteins, usual reported carotenoid intake) did not affect outcomes. There were no significant differences between the 3 groups for baseline carotenoid concentrations. View Large TABLE 3 Plasma carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* 1 Values are means ± SDs. Labeled means in a column without a common superscript letter differ, P < 0.05. *Different from baseline, P < 0.05. The interactions between the length of intervention and intervention group and baseline lycopene were included in the model for lycopene. Age, BMI, and baseline values were included for β-carotene and phytofluene. Other covariates tested (plasma volume, blood lipoproteins, usual reported carotenoid intake) did not affect outcomes. There were no significant differences between the 3 groups for baseline carotenoid concentrations. View Large Prostate carotenoids Prostate carotenoid concentrations were assessed at prostatectomy only and are shown in Table 4. There were no significant differences in prostate carotenoids between the 3 groups for this short-term intervention (Table 4) TABLE 4 Prostate carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 1 Values are means ± SDs. Group effects were statistically compared using linear models, and prostate carotenoid concentrations were not significantly different by treatment group. View Large TABLE 4 Prostate carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 1 Values are means ± SDs. Group effects were statistically compared using linear models, and prostate carotenoid concentrations were not significantly different by treatment group. View Large Correlation between plasma and prostate carotenoid concentrations Baseline and end-of-study plasma lycopene concentrations were significantly correlated with prostatic lycopene, as were baseline and end-of-study plasma β-carotene with prostatic β-carotene concentrations. However, plasma measures of carotenoids of lower concentrations, phytoene or phytofluene, were not significantly correlated with respective prostatic concentrations. Correlation plots for plasma and prostatic lycopene and β-carotene are shown in Figure 1. FIGURE 1 View largeDownload slide (A–D) Correlations between baseline or end-of-study plasma carotenoid concentrations with prostate carotenoid concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Pearson correlation coefficient. Plasma and prostate carotenoid concentrations were log-transformed before calculating correlations to improve normality, and untransformed data are presented here for ease of interpretation. FIGURE 1 View largeDownload slide (A–D) Correlations between baseline or end-of-study plasma carotenoid concentrations with prostate carotenoid concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Pearson correlation coefficient. Plasma and prostate carotenoid concentrations were log-transformed before calculating correlations to improve normality, and untransformed data are presented here for ease of interpretation. Urine isoflavones Parent isoflavones (genistein, daidzein, and glycitein) and their metabolites (ODMA, 6-OH-ODMA, dhDaidzein, dhGenistein, and equol) were below the minimal detection limits in urine from men in the 0-cans/d group. Total urinary isoflavone excretion increased with increasing tomato-soy juice dose (Table 5). A significant dose response was observed for each individual parent isoflavone comparing 1 can of tomato-soy juice/d with 2 cans/d (Wilcoxon's P = 0.0009, 0.0131, and 0.0193 for daidzein, glycitein, and genistein, respectively). There was no difference between groups for urinary isoflavone metabolite concentration. TABLE 5 Urinary isoflavone concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) 1 Values are medians (25th–75th percentiles). Labeled medians in a column without a common superscript letter differ, P < 0.05. Group responses were compared pairwise by the Wilcoxon Rank Sum test. The urinary isoflavone output was below the minimal detection limit in 100% of the subjects for all isoflavones in the 0-cans/d group. MDL (nmol/L) urine analysis: daidzein, 1.08; ODMA, 0.42; genistein, 3.8; glycitein, 2.44; dhDaidzein, 0.18; dhGenistein, 0.24, equol, 0.28. dhDiadzein, dihydrodaidzein; dhGenistein, dihydrogenistein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin. View Large TABLE 5 Urinary isoflavone concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) 1 Values are medians (25th–75th percentiles). Labeled medians in a column without a common superscript letter differ, P < 0.05. Group responses were compared pairwise by the Wilcoxon Rank Sum test. The urinary isoflavone output was below the minimal detection limit in 100% of the subjects for all isoflavones in the 0-cans/d group. MDL (nmol/L) urine analysis: daidzein, 1.08; ODMA, 0.42; genistein, 3.8; glycitein, 2.44; dhDaidzein, 0.18; dhGenistein, 0.24, equol, 0.28. dhDiadzein, dihydrodaidzein; dhGenistein, dihydrogenistein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin. View Large Prostate isoflavones Prostate concentrations of the isoflavones and metabolites are presented in Table 6. Daidzein and genistein were quantifiable in all subjects across all groups. The genistein metabolite 6-OH-ODMA and the daidzein metabolite equol were only detected in one patient, so neither are shown in Table 6. dhDaidzein was detectable in 15 patients in the juice-consuming groups but in no subjects consuming 0 cans/d. ODMA was detected in 24 study participants in the tomato-soy juice groups but not in any of the 0-cans/d group. Both dhDaidzein and ODMA concentrations were greater in the 2-cans/d group than in the 0-cans/d group (Table 6). TABLE 6 Prostate isoflavone and isoflavone metabolite concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 1 Values are means ± SDs unless otherwise indicated. Only one patient, respectively, had measurable tissue concentrations of 6-OH-ODMA or equol; therefore, those data were not analyzable. Group effects for isoflavones (daidzein and genistein) were present in the majority of subjects and were compared by ANOVA. Isoflavone metabolites (dhDaidzein and ODMA) were below the MDL for many participants and therefore were compared using the Wilcoxon Rank Sum test (39). MDL (nmol/g) for tissue analysis: daidzein, 0.01; dhDaidzein, 0.001; genistein, 0.02; ODMA, 0.002. Medians in a column without a common superscript letter differ, P < 0.05. dhDaidzein, dihydrodaidzein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin; 6-OH-ODMA, 6-hydroxy-O-desmethylangolensin. 2 All subjects had detectable prostate daidzein and genistein. View Large TABLE 6 Prostate isoflavone and isoflavone metabolite concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 1 Values are means ± SDs unless otherwise indicated. Only one patient, respectively, had measurable tissue concentrations of 6-OH-ODMA or equol; therefore, those data were not analyzable. Group effects for isoflavones (daidzein and genistein) were present in the majority of subjects and were compared by ANOVA. Isoflavone metabolites (dhDaidzein and ODMA) were below the MDL for many participants and therefore were compared using the Wilcoxon Rank Sum test (39). MDL (nmol/g) for tissue analysis: daidzein, 0.01; dhDaidzein, 0.001; genistein, 0.02; ODMA, 0.002. Medians in a column without a common superscript letter differ, P < 0.05. dhDaidzein, dihydrodaidzein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin; 6-OH-ODMA, 6-hydroxy-O-desmethylangolensin. 2 All subjects had detectable prostate daidzein and genistein. View Large Correlations between urinary and prostate isoflavone concentrations Prostate and urinary daidzein at the time of surgery were not correlated (r = −0.14, P = 0.28) and neither were tissue and urinary genistein concentrations (r = −0.07, P = 0.60). Tissue and urinary dhDaidzein concentrations significantly correlated, as were dhGenistein and ODMA tissue and urinary concentrations (Figure 2). FIGURE 2 View largeDownload slide (A–C) Correlations between 24-h urinary isoflavone metabolite output with prostate isoflavone metabolite concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Kendall's τ-b correlation coefficient, which allows for inclusion of samples below the minimal detection limit in analysis by rank-order analysis. Urinary and prostate isoflavone concentrations were log10-transformed for correlation analysis to improve normality assumptions; however, untransformed data are presented here for ease of interpretation. dhDaidzein, dihydrodaidzein; dhGenistein, dihydrogenistein; ODMA, O-desmethylangolensin. FIGURE 2 View largeDownload slide (A–C) Correlations between 24-h urinary isoflavone metabolite output with prostate isoflavone metabolite concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Kendall's τ-b correlation coefficient, which allows for inclusion of samples below the minimal detection limit in analysis by rank-order analysis. Urinary and prostate isoflavone concentrations were log10-transformed for correlation analysis to improve normality assumptions; however, untransformed data are presented here for ease of interpretation. dhDaidzein, dihydrodaidzein; dhGenistein, dihydrogenistein; ODMA, O-desmethylangolensin. Isoflavone metabolism phenotype clusters Based on the dendogram, subjects clustered into 5 distinct isoflavone metabolic phenotype groups on the basis of urinary isoflavone excretion (Figure 3). The 2 largest groups consisted of individuals with either similar proportions of daidzein and dhDaidzein and ODMA (cluster 1) or a greater proportion of daidzein and dhDaidzein than ODMA (cluster 2) in the urine. The third largest group was similar to the second but also excreted equol in the urine (cluster 3), whereas the fourth group had no daidzein metabolites (equol, dhDaidzein, or ODMA) (cluster 4) in the urine. The final and smallest group (cluster 5), with only one member, excreted large amounts of equol with no ODMA. Cluster groups 1, 2, and 3 contained subjects from the 1- and 2-cans/d intervention groups, whereas cluster 4 contained only men consuming 2 cans tomato-soy juice/d, and the one man in cluster 5 consumed 1 can/d. FIGURE 3 View largeDownload slide Clustered daidzein urinary metabolic phenotypes in men with prostate cancer consuming either 1 or 2 cans tomato-soy juice/d. The proportions of daidzein and daidzein metabolites were analyzed using agglomerative clustering with the Euclidean distance dissimilarity measure and the average linkage metric. Brackets with numbers along the x axis indicate of which supplemented group each subject was a member. Relative abundance data were arcsin square root transformed for greater detection of dissimilarity in cluster analysis. dhD, dihydrodaidzein; ODMA, O-desmethylangolensin. FIGURE 3 View largeDownload slide Clustered daidzein urinary metabolic phenotypes in men with prostate cancer consuming either 1 or 2 cans tomato-soy juice/d. The proportions of daidzein and daidzein metabolites were analyzed using agglomerative clustering with the Euclidean distance dissimilarity measure and the average linkage metric. Brackets with numbers along the x axis indicate of which supplemented group each subject was a member. Relative abundance data were arcsin square root transformed for greater detection of dissimilarity in cluster analysis. dhD, dihydrodaidzein; ODMA, O-desmethylangolensin. PSA kinetics PSA ranged from 0.11 to 12.8 ng/mL at enrollment, which is typical for most prostatectomy cohorts of men screened by digital rectal examination and PSA. Due to the heterogeneity and modest study size, no differences were detected between groups at baseline or the end of study. Calculated mean ± SD PSA velocities were 6.3 ± 16.8, 7.7 ± 21.5, and 1.7 ± 22.7 ng ⋅ mL−1 ⋅ y−1 for the 0-, 1-, and 2-cans/d groups, respectively; these values were not significantly different. The change in PSA from study start to end by duration of the intervention was evaluated using an ANCOVA approach. For the control group, little change was observed regarding duration. For the 1- and 2-cans/d groups, a decrease in PSA was detected and was greater for those who were receiving the intervention longer. The difference in the slopes between the 0-cans/d group and the 2-cans/d group was not significant (P = 0.078; Figure 4). Gleason grade from the surgical sample was not significantly different between groups. FIGURE 4 View largeDownload slide Change in PSA over ∼3 wk of a dietary intervention with either 0, 1, or 2 cans of tomato-soy juice/d in men with prostate cancer. The lines indicate the linear slope for each group from baseline to the end of the intervention. PSA, prostate-specific antigen. FIGURE 4 View largeDownload slide Change in PSA over ∼3 wk of a dietary intervention with either 0, 1, or 2 cans of tomato-soy juice/d in men with prostate cancer. The lines indicate the linear slope for each group from baseline to the end of the intervention. PSA, prostate-specific antigen. Discussion Historically, cancer chemoprevention strategies have utilized a pharmacologic model with drugs (43), nutrients (44, 45), or pure phytochemicals (46). In contrast, multifactor interventions focusing on dietary patterns representing a diverse array of variables have been examined (47). A third strategy, engaging foods of defined compositions conducive to inhibition of carcinogenesis, has undergone less clinical investigation. Plant-based foods can be studied in preclinical mechanistic models to define bioactive profiles with additive or synergistic anticancer activity and nonoverlapping toxicity. Plant genetics, horticultural conditions, and food processing can be engineered to enhance ingredient bioactive profiles, lengthen shelf-life, and improve patient compliance through optimized sensory and packaging characteristics for ease of use (32). This dose-escalating phase I/II trial evaluated a tomato-soy juice assessed short-term (∼3 wk) safety and compliance, as well as phytochemical exposure and metabolism in a cohort of men undergoing prostatectomy for clinically localized prostate cancer. Compliance with the tomato-soy juice was excellent. No grade II/III or IV toxicities or clinical laboratory abnormalities were observed. However, with 2 cans/d we observed one case of grade I gastrointestinal complaint and one case of grade I hypertension. We have observed similar dose-dependent complaints in past studies with soy products (24, 32). Thus, to focus our study on tolerable and achievable doses, we eliminated the 3-cans/d group and expanded the 2-cans/d group. Our past intervention studies (19, 24, 31, 32, 48) document profound human heterogeneity in absorption and metabolism of food bioactives. This heterogeneity is likely due to intrinsic factors, such as genetics and the microbiome (24), as well as extrinsic variables, such as medications and diet composition (49). We controlled background dietary variability of tomato and soy foods to better isolate effects of the intervention. All 4 monitored plasma carotenoids significantly increased in a dose-response manner. End-of-study plasma lycopene was 2.3-fold greater in the 2-cans/d group compared with the 0-cans/d groups. The final plasma lycopene concentrations reported here for both intervention groups are greater than those previously achieved with a similar amount of lycopene from tomato products (mean ± SEM: 0.78 ± 0.1 to 1.14 ± 0.1 µmol/L) (48) or from a combination of tomato products (0.91 µmol/L) (50); this may be due to greater baseline plasma lycopene concentrations (1.17 ± 0.50 µmol/L) in the current study than in previous studies (range: 0.45–0.67 µmol/L) (48, 50). To our knowledge, this is the first report of the dose-response impact of a tomato food-product intervention in patients with prostate cancer on plasma phytoene and phytofluene. Although plasma phytoene-to-lycopene ratios mirrored that in the juice (1:10), plasma phytofluene:lycopene was 1:5, differing from the juice ratios of 1:20, which may suggest differing pharmacokinetics of phytofluene and phytoene. The dose-response effect of this product on plasma carotenoids provides a foundation for use in larger clinical trials. There are several limitations to our study. First, based on the prostate carotenoid data, the duration of feeding may have been too brief to change prostate carotenoid concentrations in these subjects with elevated baseline carotenoid status. Wait times at our clinic for curative cancer surgery limited the intervention period to ∼3 wk. Secondary analyses of prostate carotenoid outcomes provided insights. First, according to the Plateau Principle, it takes 3.3 half-lives to reach 90% of an expected steady-state concentration (51). The estimated half-life of lycopene in slow-turnover tissues, such as the prostate, is estimated to be 12 d (42); therefore, it may require ∼41 d to reach 90% of the steady-state prostate tissue lycopene concentration. The longer time to reach tissue than plasma carotenoid steady-state concentrations may also explain why baseline plasma lycopene and β-carotene concentrations were more strongly correlated with corresponding prostate carotenoid concentrations than were end-of-study plasma carotenoid concentrations. Second, the lack of a washout in the current study design may have affected some of our study results. We previously found that a similar amount (25–35 mg/d) of daily lycopene from tomato products provided for ∼23 d increased prostate lycopene by 2- to 3-fold compared with controls (48); that study engaged a 1-wk lycopene washout before a 3-wk intervention, and thus subjects had lower baseline lycopene status than in the current study. Indeed, prostate lycopene in the current control group (mean ± SD: 0.584 ± 0.096 nmol/g) was greater than that in the previous control group (mean ± SEM: 0.16 ± 0.06 nmol/g) (48). In the current study, in which subjects began with a higher lycopene status, we found that baseline plasma lycopene and β-carotene concentrations were more strongly correlated with end-of-study prostate carotenoid concentrations than were their end-of-study plasma lycopene and β-carotene concentrations. This suggests that, if at a high baseline carotenoid status, previous steady-state conditions may be more influential on prostate carotenoids than a short intervention. In addition, plasma carotenoid responses may have been repressed in this study due to an interaction with soy components of the drink. Zuniga and Erdman (52) previously found that soy germ consumed with tomato led to reduced serum and prostate lycopene compared with tomato feeding alone in rodents. Even so, Zuniga et al. (30) later found that a tomato and soy-germ combination was more effective than either component alone in reducing prostate cancer incidence in TRAMP mice. Furthermore, our studies (42, 48, 53) indicate that longer-term carotenoid interventions are required to change prostate carotenoid concentrations in those with a higher baseline carotenoid status. Finally, we were not able to procure malignant prostate tissue samples due to pathologic evaluation after surgery. Our previous study (54) showed that cancerous regions accumulate greater lycopene and all-trans β-carotene, but not α-carotene, β-cryptoxanthin, or lutein and zeaxanthin. Future studies to define the differential accumulation of phytoene, phytofluene, and isoflavones in cancerous compared with noncancerous regions would be of interest. To our knowledge, this is the first report of prostate phytoene and phytofluene concentrations (0.2–0.3 nmol/g) in response to a tomato product intervention. Although these are minor carotenoids in the juice, they accumulate to nearly half the level of lycopene in the prostate, consistent with previous kinetic studies in rodent models (53, 55, 56) and in humans, suggesting that phytoene is more bioavailable and more rapidly taken up by slow-turnover tissue pools than lycopene (42, 57). Urinary isoflavones are a marker of soy food intake (58), but, to our knowledge, the relation between urinary isoflavones and tissue concentrations has not been explored. Urinary isoflavone metabolite (dhDaidzein, dhGenistein, ODMA) concentrations correlated with their respective tissue concentrations, whereas the parent isoflavones (genistein and daidzein) did not. In particular, urinary ODMA may be a useful biomarker of prostatic ODMA, because it was excreted by most subjects. Finally, the patterns of parent isoflavone and metabolite urinary excretion clustered into groups similar to our previous report (24). We postulate that these metabolic phenotypes represent the combination of host genetics and microbiome determinants of isoflavone metabolism. Critically, the application of statistical and bioinformatic techniques to isoflavone metabolites may provide insight into how these various phenotypes relate to health outcomes. This study is the first, to our knowledge, to report prostatic isoflavone concentrations after a food-based soy intervention study. We observed a nonsignificant (P > 0.1) dose-response effect for genistein and daidzein, which is unsurprising given the person-to-person heterogeneity observed. Tissue isoflavone concentrations in the current study are lower than in previous reports with botanical dietary supplements resulting in genistein and daidzein concentrations of ∼1–2.4 nmol/g (59–61). However, these studies were of shorter duration (3–14 d), which could also affect isoflavone metabolic rate by the host or colonic microbiota. In sum, isoflavones do accumulate in prostatic tissue, although with significant individual variability, as is characteristic of phytochemicals. Although not a primary outcome, we measured PSA kinetics as a marker of prostate cancer status and found a trend supporting a dietary impact for the 2-cans/d dose in reducing PSA (Figure 4). We observed a prolongation of PSA doubling time in a previous short-term study (24), and others have reported favorable changes in PSA after a tomato-based intervention (50, 62). To conclude, an optimized tomato-soy juice reliably increases plasma carotenoids and urinary markers of soy isoflavone intake with minimal grade I toxicity events. Complementary genetic and microbiome studies can define the key predictors of plasma and tissue exposure responses. Future studies may utilize this characterized product for longer-term interventions to determine efficacy for slowing disease development or progression in high-risk individuals or progression in patients with low-risk prostate cancer on active surveillance. Acknowledgments We thank Caryn Fasko, Anna Maria Bittoni, Ashley Schmitz, and Christina Simpson for assistance in editing and entering patient compliance, 3-d diet records, and anthropometric data, and Jennifer Ahn-Jarvis for analyzing the juice isoflavone content. The authors’ responsibilities were as follows—SKC, SJS, and EMG: designed the study; SKC, SJS, KMR, REK, EMG, and NEM: formulated the research questions; SKC, RRB and RA: recruited the subjects; DMF: developed, grew, and harvested the FG99-218 tomatoes; SKC, EMG, and RA: carried out the clinical study; EMG, REK, LW, and KMR: analyzed the samples; GSY, EMG, KMR, NEM, JT-A, SJS, and SKC: analyzed the data; EMG, NEM, and SKC: drafted the manuscript; and all authors: read and approved the final manuscript. Notes Supported by the National Cancer Institute of the NIH by R01CA112632 (principal investigator: SKC). Additional resources have been provided through the National Cancer Institute P30CA01605-supported Molecular Carcinogenesis and Chemoprevention Program and the Nutrient and Phytochemical Analytic Shared Resource and the National Center for Advancing Translational Sciences by the award UL1TR001070-supported Clinical Research Center. NEM was funded by the National Center for Complementary and Integrative Health and Office of Dietary Supplements under award K99/R00 AT008576 and by the USDA–Agricultural Research Service under CRIS 3092-51000-056-03S. Additional support was provided by The Ohio State University's Food Innovation Center, The Center for Advancement of Functional Foods Research and Entrepreneurship, and The Ohio State University James Development funds [Prostate Cancer Prevention and Treatment Fund (302024), Bionutrition and Cancer Prevention Fund (310684), and Hammond Cancer Research Fund (262914)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the USDA. Author disclosures: EMG, NEM, DMF, SJS, LW, JT-A, REK, KMR, GSY, RA, RRB, and SKC, no conflicts of interest. Supplemental Data 1 and 2 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/. Present address for REK: Department of Human Sciences, 1787 Neil Avenue, 067 Campbell Hall, Columbus, OH 43210. Present address for RA: OhioHealth Robotic Urologic Surgeons, Dublin Methodist Hospital, 7450 Hospital Drive, Suite 300, Dublin, OH 43016. Abbreviations used: dhDaidzein, dihydrodaidzein; dhGenistein, dihydrogenistein; ODMA, O-desmethylangolensin; PSA, prostate-specific antigen; 6-OH-ODMA, 6-hydroxy-O-desmethylangolensin. References 1. Grainger E , Clinton SK , Giovannucci E . Diet and nutrition in the etiology and prevention of cancer . In: Bast RC , Croce CM , Hait WN , Hong WK , Kufe DW , Piccart-Gebhart M , Pollock R , Weichselbaum RR , Want H , Holland JF , editors. Cancer medicine . Hoboken (NJ) : John Wiley and Sons, Inc. ; 2017 : 415 – 31 . 2. Giovannucci E , Ascherio A , Rimm EB , Stampfer MJ , Colditz GA , Willett WC . Intake of carotenoids and retinol in relation to risk of prostate cancer . J Natl Cancer Inst 1995 ; 87 ( 23 ): 1767 – 76 . Google Scholar Crossref Search ADS PubMed 3. Graff RE , Pettersson A , Lis RT , Ahearn TU , Markt SC , Wilson KM , Rider JR , Fiorentino M , Finn S , Kenfield SA et al. Dietary lycopene intake and risk of prostate cancer defined by ERG protein expression . Am J Clin Nutr 2016 ; 103 ( 3 ): 851 – 60 . Google Scholar Crossref Search ADS PubMed 4. Zu K , Mucci L , Rosner BA , Clinton SK , Loda M , Stampfer MJ , Giovannucci E . Dietary lycopene, angiogenesis, and prostate cancer: a prospective study in the prostate-specific antigen era . J Natl Cancer Inst 2014 ; 106 ( 2 ): djt430 . Google Scholar Crossref Search ADS PubMed 5. Key TJ , Appleby PN , Travis RC , Albanes D , Alberg AJ , Barricarte A , Black A , Boeing H , Bueno-de-Mesquita HB , Chan JM et al. Carotenoids, retinol, tocopherols, and prostate cancer risk: pooled analysis of 15 studies . Am J Clin Nutr 2015 ; 102 ( 5 ): 1142 – 57 . Google Scholar Crossref Search ADS PubMed 6. World Cancer Research Fund/American Institute for Cancer Research . Food, nutrition, physical activity and the prevention of cancer: a global perspective . Washington (DC) : American Institute for Cancer Research; 2007 . 7. World Cancer Research Fund International/American Institute for Cancer Research . Diet, nutrition, physical activity, and prostate cancer. Continuous Update Project Expert Report . 20 14 . 8. Cohen JH , Kristal AR , Stanford JL . Fruit and vegetable intakes and prostate cancer risk . J Natl Cancer Inst 2000 ; 92 ( 1 ): 61 – 8 . Google Scholar Crossref Search ADS PubMed 9. Giovannucci E . Does prostate-specific antigen screening influence the results of studies of tomatoes, lycopene, and prostate cancer risk? J Natl Cancer Inst 2007 ; 99 ( 14 ): 1060 – 2 . Google Scholar Crossref Search ADS PubMed 10. International Agency for Research on Cancer; WHO; International Association of Cancer Registries . Cancer incidence in five continents . Lyon (France) : IARC Press ; 2005 . 11. Miyanaga N , Akaza H , Hinotsu S , Fujioka T , Naito S , Namiki M , Takahashi S , Hirao Y , Horie S , Tsukamoto T et al. Prostate cancer chemoprevention study: an investigative randomized control study using purified isoflavones in men with rising prostate-specific antigen . Cancer Sci 2012 ; 103 ( 1 ): 125 – 30 . Google Scholar Crossref Search ADS PubMed 12. Boileau TW , Liao Z , Kim S , Lemeshow S , Erdman JW Jr. , Clinton SK . Prostate carcinogenesis in N-methyl-N-nitrosourea (NMU)-testosterone-treated rats fed tomato powder, lycopene, or energy-restricted diets . J Natl Cancer Inst 2003 ; 95 ( 21 ): 1578 – 86 . Google Scholar Crossref Search ADS PubMed 13. Liu AG , Juvik JA , Jeffery EH , Berman-Booty LD , Clinton SK , Erdman JW Jr . Enhancement of broccoli indole glucosinolates by methyl jasmonate treatment and effects on prostate carcinogenesis . J Med Food 2014 ; 17 ( 11 ): 1177 – 82 . Google Scholar Crossref Search ADS PubMed 14. Tan HL , Thomas-Ahner JM , Moran NE , Cooperstone JL , Erdman JW Jr. , Young GS , Clinton SK . beta-Carotene 9′,10′ oxygenase modulates the anticancer activity of dietary tomato or lycopene on prostate carcinogenesis in the TRAMP model . Cancer Prev Res 2017 ; 10 ( 2 ): 161 – 9 . Google Scholar Crossref Search ADS 15. Zhou JR , Gugger ET , Tanaka T , Guo Y , Blackburn GL , Clinton SK . Soybean phytochemicals inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice . J Nutr 1999 ; 129 ( 9 ): 1628 – 35 . Google Scholar Crossref Search ADS PubMed 16. Mein JR , Lian F , Wang XD . Biological activity of lycopene metabolites: implications for cancer prevention . Nutr Rev 2008 ; 66 ( 12 ): 667 – 83 . Google Scholar Crossref Search ADS PubMed 17. Sies H , Stahl W . Lycopene: antioxidant and biological effects and its bioavailability in the human . Proc Soc Exp Biol Med 1998 ; 218 ( 2 ): 121 – 4 . Google Scholar Crossref Search ADS PubMed 18. Wan L , Tan HL , Thomas-Ahner JM , Pearl DK , Erdman JW Jr. , Moran NE , Clinton SK . Dietary tomato and lycopene impact androgen signaling- and carcinogenesis-related gene expression during early TRAMP prostate carcinogenesis . Cancer Prev Res 2014 ; 7 ( 12 ): 1228 – 39 . Google Scholar Crossref Search ADS 19. Lesinski GB , Reville PK , Mace TA , Young GS , Ahn-Jarvis J , Thomas-Ahner J , Vodovotz Y , Ameen Z , Grainger E , Riedl K et al. Consumption of soy isoflavone enriched bread in men with prostate cancer is associated with reduced proinflammatory cytokines and immunosuppressive cells . Cancer Prev Res 2015 ; 8 ( 11 ): 1036 – 44 . Google Scholar Crossref Search ADS 20. Singh AV , Franke AA , Blackburn GL , Zhou JR . Soy phytochemicals prevent orthotopic growth and metastasis of bladder cancer in mice by alterations of cancer cell proliferation and apoptosis and tumor angiogenesis . Cancer Res 2006 ; 66 ( 3 ): 1851 – 8 . Google Scholar Crossref Search ADS PubMed 21. Wang S , DeGroff VL , Clinton SK . Tomato and soy polyphenols reduce insulin-like growth factor-I-stimulated rat prostate cancer cell proliferation and apoptotic resistance in vitro via inhibition of intracellular signaling pathways involving tyrosine kinase . J Nutr 2003 ; 133 ( 7 ): 2367 – 76 . Google Scholar Crossref Search ADS PubMed 22. Sargeant AM , Klein RD , Rengel RC , Clinton SK , Kulp SK , Kashida Y , Yamaguchi M , Wang X , Chen CS . Chemopreventive and bioenergetic signaling effects of PDK1/Akt pathway inhibition in a transgenic mouse model of prostate cancer . Toxicol Pathol 2007 ; 35 ( 4 ): 549 – 61 . Google Scholar Crossref Search ADS PubMed 23. Zhou JR , Yu L , Zhong Y , Blackburn GL . Soy phytochemicals and tea bioactive components synergistically inhibit androgen-sensitive human prostate tumors in mice . J Nutr 2003 ; 133 ( 2 ): 516 – 21 . Google Scholar Crossref Search ADS PubMed 24. Ahn-Jarvis JH , Clinton SK , Grainger EM , Riedl KM , Schwartz SJ , Lee ML , Cruz-Cano R , Young GS , Lesinski GB , Vodovotz Y . Isoflavone pharmacokinetics and metabolism after consumption of a standardized soy and soy-almond bread in men with asymptomatic prostate cancer . Cancer Prev Res 2015 ; 8 ( 11 ): 1045 – 54 . Google Scholar Crossref Search ADS 25. Varinska L , Gal P , Mojzisova G , Mirossay L , Mojzis J . Soy and breast cancer: focus on angiogenesis . Int J Mol Sci 2015 ; 16 ( 5 ): 11728 – 49 . Google Scholar Crossref Search ADS PubMed 26. Karsli-Ceppioglu S , Ngollo M , Adjakly M , Dagdemir A , Judes G , Lebert A , Boiteux JP , Penault LF , Bignon YJ , Guy L et al. Genome-wide DNA methylation modified by soy phytoestrogens: role for epigenetic therapeutics in prostate cancer? Omics 2015 ; 19 ( 4 ): 209 – 19 . Google Scholar Crossref Search ADS PubMed 27. DeVita VT , Hellman S , Rosenberg SA . Cancer, principles & practice of oncology . Philadelphia : Lippincott, Williams, & Wilkins ; 2005 . 28. Hong WK ; American Association for Cancer Research . Holland Frei cancer medicine 8 . Shelton (CT) : People's Medical Pub. House ; 2010 . 29. Canene-Adams K , Lindshield BL , Wang S , Jeffery EH , Clinton SK , Erdman JW Jr . Combinations of tomato and broccoli enhance antitumor activity in Dunning r3327-h prostate adenocarcinomas . Cancer Res 2007 ; 67 ( 2 ): 836 – 43 . Google Scholar Crossref Search ADS PubMed 30. Zuniga KE , Clinton SK , Erdman JW Jr . The interactions of dietary tomato powder and soy germ on prostate carcinogenesis in the TRAMP model . Cancer Prev Res 2013 ; 6 ( 6 ): 548 – 57 . Google Scholar Crossref Search ADS 31. Grainger EM , Schwartz SJ , Wang S , Unlu NZ , Boileau TW , Ferketich AK , Monk JP , Gong MC , Bahnson RR , DeGroff VL et al. A combination of tomato and soy products for men with recurring prostate cancer and rising prostate specific antigen . Nutr Cancer 2008 ; 60 ( 2 ): 145 – 54 . Google Scholar Crossref Search ADS PubMed 32. Bohn T , Blackwood M , Francis D , Tian Q , Schwartz SJ , Clinton SK . Bioavailability of phytochemical constituents from a novel soy fortified lycopene rich tomato juice developed for targeted cancer prevention trials . Nutr Cancer 2013 ; 65 ( 6 ): 919 – 29 . Google Scholar Crossref Search ADS PubMed 33. Giovannucci E , Rimm EB , Liu Y , Stampfer MJ , Willett WC . A prospective study of tomato products, lycopene, and prostate cancer risk . J Natl Cancer Inst 2002 ; 94 ( 5 ): 391 – 8 . Google Scholar Crossref Search ADS PubMed 34. Shu XO , Zheng Y , Cai H , Gu K , Chen Z , Zheng W , Lu W . Soy food intake and breast cancer survival . JAMA 2009 ; 302 ( 22 ): 2437 – 43 . Google Scholar Crossref Search ADS PubMed 35. Kopec RE , Riedl KM , Harrison EH , Curley RW Jr. , Hruszkewycz DP , Clinton SK , Schwartz SJ . Identification and quantification of apo-lycopenals in fruits, vegetables, and human plasma . J Agric Food Chem 2010 ; 58 ( 6 ): 3290 – 6 . Google Scholar Crossref Search ADS PubMed 36. Xu SY , Shoemaker CF , Luh BS . Effect of break temperature on rheological properties and microstructure of tomato juices and pastes . J Food Sci 1986 ; 51 ( 2 ): 399 . Google Scholar Crossref Search ADS 37. Barona J , Jones JJ , Kopec RE , Comperatore M , Andersen C , Schwartz SJ , Lerman RH , Fernandez ML . A Mediterranean-style low-glycemic-load diet increases plasma carotenoids and decreases LDL oxidation in women with metabolic syndrome . J Nutr Biochem 2012 ; 23 ( 6 ): 609 – 15 . Google Scholar Crossref Search ADS PubMed 38. Cooperstone JL , Ralston RA , Riedl KM , Haufe TC , Schweiggert RM , King SA , Timmers CD , Francis DM , Lesinski GB , Clinton SK et al. Enhanced bioavailability of lycopene when consumed as cis-isomers from tangerine compared to red tomato juice, a randomized, cross-over clinical trial . Mol Nutr Food Res 2015 ; 59 ( 4 ): 658 – 69 . Google Scholar Crossref Search ADS PubMed 39. Zhang D , Fan C , Zhang J , Zhang CH . Nonparametric methods for measurements below detection limit . Stat Med 2009 ; 28 ( 4 ): 700 – 15 . Google Scholar Crossref Search ADS PubMed 40. Mickey RM , Greenland S . The impact of confounder selection criteria on effect estimation . Am J Epidemiol 1989 ; 129 ( 1 ): 125 – 37 . Google Scholar Crossref Search ADS PubMed 41. Anupongsanugool E , Teekachunhatean S , Rojanasthien N , Pongsatha S , Sangdee C . Pharmacokinetics of isoflavones, daidzein and genistein, after ingestion of soy beverage compared with soy extract capsules in postmenopausal Thai women . BMC Clin Pharmacol 2005 ; 5 : 2 . Google Scholar Crossref Search ADS PubMed 42. Moran NE , Cichon MJ , Riedl KM , Grainger EM , Schwartz SJ , Novotny JA , Erdman JW Jr. , Clinton SK . Compartmental and noncompartmental modeling of 13C-lycopene absorption, isomerization, and distribution kinetics in healthy adults . Am J Clin Nutr 2015 ; 102 ( 6 ): 1436 – 49 . Google Scholar Crossref Search ADS PubMed 43. Fisher B , Redmond C , Brown A , Wolmark N , Wittliff J , Fisher ER , Plotkin D , Bowman D , Sachs S , Wolter J et al. Treatment of primary breast cancer with chemotherapy and tamoxifen . N Engl J Med 1981 ; 305 ( 1 ): 1 – 6 . Google Scholar Crossref Search ADS PubMed 44. Albanes D , Heinonen OP , Taylor PR , Virtamo J , Edwards BK , Rautalahti M , Hartman AM , Palmgren J , Freedman LS , Haapakoski J et al. Alpha-Tocopherol and beta-carotene supplements and lung cancer incidence in the alpha-tocopherol, beta-carotene cancer prevention study: effects of base-line characteristics and study compliance . J Natl Cancer Inst 1996 ; 88 ( 21 ): 1560 – 70 . Google Scholar Crossref Search ADS PubMed 45. Klein EA , Thompson IM Jr. , Tangen CM , Crowley JJ , Lucia MS , Goodman PJ , Minasian LM , Ford LG , Parnes HL , Gaziano JM et al. Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT) . JAMA 2011 ; 306 ( 14 ): 1549 – 56 . Google Scholar Crossref Search ADS PubMed 46. Lazarevic B , Hammarstrom C , Yang J , Ramberg H , Diep LM , Karlsen SJ , Kucuk O , Saatcioglu F , Tasken KA , Svindland A . The effects of short-term genistein intervention on prostate biomarker expression in patients with localised prostate cancer before radical prostatectomy . Br J Nutr 2012 ; 108 ( 12 ): 2138 – 47 . Google Scholar Crossref Search ADS PubMed 47. Pierce JP , Faerber S , Wright FA , Rock CL , Newman V , Flatt SW , Kealey S , Jones VE , Caan BJ , Gold EB et al. A randomized trial of the effect of a plant-based dietary pattern on additional breast cancer events and survival: the Women's Healthy Eating and Living (WHEL) Study . Control Clin Trials 2002 ; 23 ( 6 ): 728 – 56 . Google Scholar Crossref Search ADS PubMed 48. Grainger EM , Hadley CW , Moran NE , Riedl KM , Gong MC , Pohar K , Schwartz SJ , Clinton SK . A comparison of plasma and prostate lycopene in response to typical servings of tomato soup, sauce or juice in men before prostatectomy . Br J Nutr 2015 ; 114 ( 4 ): 1 – 12 . Google Scholar Crossref Search ADS PubMed 49. Williams AW , Boileau TW , Erdman JW Jr . Factors influencing the uptake and absorption of carotenoids . Proc Soc Exp Biol Med 1998 ; 218 ( 2 ): 106 – 8 . Google Scholar Crossref Search ADS PubMed 50. Paur I , Lilleby W , Bohn SK , Hulander E , Klein W , Vlatkovic L , Axcrona K , Bolstad N , Bjoro T , Laake P et al. Tomato-based randomized controlled trial in prostate cancer patients: effect on PSA . Clin Nutr 2017 ; 36 ( 3 ): 672 – 9 . Google Scholar Crossref Search ADS PubMed 51. Atkinson AJ Jr. , Huang S-M , Lertora JJL , Markey SP . Principles of clinical pharmacology . 3 rd ed . San Diego (CA) : Elsevier ; 2012 . 52. Zuniga KE , Erdman JW Jr . Combined consumption of soy germ and tomato powders results in altered isoflavone and carotenoid bioavailability in rats . J Agric Food Chem 2011 ; 59 ( 10 ): 5335 – 41 . Google Scholar Crossref Search ADS PubMed 53. Moran NE , Clinton SK , Erdman JW Jr . Differential bioavailability, clearance, and tissue distribution of the acyclic tomato carotenoids lycopene and phytoene in Mongolian gerbils . J Nutr 2013 ; 143 ( 12 ): 1920 – 6 . Google Scholar Crossref Search ADS PubMed 54. Clinton SK , Emenhiser C , Schwartz SJ , Bostwick DG , Williams AW , Moore BJ , Erdman JWJ . cis-trans Lycopene isomers, carotenoids, and retinol in the human prostate . Cancer Epidemiol Biomarkers Prev 1996 ; 5 ( 10 ): 823 – 33 . Google Scholar PubMed 55. Conlon LE , King RD , Moran NE , Erdman JW Jr . Coconut oil enhances tomato carotenoid tissue accumulation compared to safflower oil in the Mongolian gerbil (Meriones unguiculatus) . J Agric Food Chem 2012 ; 60 ( 34 ): 8386 – 94 . Google Scholar Crossref Search ADS PubMed 56. Campbell JK , Engelmann NJ , Lila MA , Erdman JW Jr . Phytoene, phytofluene, and lycopene from tomato powder differentially accumulate in tissues of male Fisher 344 rats . Nutr Res 2007 ; 27 ( 12 ): 794 – 801 . Google Scholar Crossref Search ADS PubMed 57. Moran NE , Novotny JA , Cichon MJ , Riedl KM , Rogers RB , Grainger EM , Schwartz SJ , Erdman JW Jr. , Clinton SK . Absorption and distribution kinetics of the 13C-labeled tomato carotenoid phytoene in healthy adults . J Nutr 2015 ; 146 ( 2 ): 368 – 76 . Google Scholar Crossref Search ADS PubMed 58. Morimoto Y , Beckford F , Franke AA , Maskarinec G . Urinary isoflavonoid excretion as a biomarker of dietary soy intake during two randomized soy trials . Asia Pac J Clin Nutr 2014 ; 23 ( 2 ): 205 – 9 . Google Scholar PubMed 59. Gardner CD , Oelrich B , Liu JP , Feldman D , Franke AA , Brooks JD . Prostatic soy isoflavone concentrations exceed serum levels after dietary supplementation . Prostate 2009 ; 69 ( 7 ): 719 – 26 . Google Scholar Crossref Search ADS PubMed 60. Rannikko A , Petas A , Raivio T , Janne OA , Rannikko S , Adlercreutz H . The effects of short-term oral phytoestrogen supplementation on the hypothalamic-pituitary-testicular axis in prostate cancer patients . Prostate 2006 ; 66 ( 10 ): 1086 – 91 . Google Scholar Crossref Search ADS PubMed 61. Guy L , Vedrine N , Urpi-Sarda M , Gil-Izquierdo A , Al-Maharik N , Boiteux JP , Scalbert A , Remesy C , Botting NP , Manach C . Orally administered isoflavones are present as glucuronides in the human prostate . Nutr Cancer 2008 ; 60 ( 4 ): 461 – 8 . Google Scholar Crossref Search ADS PubMed 62. Bowen P , Chen L , Stacewicz-Sapuntzakis M , Duncan C , Sharifi R , Ghosh L , Kim HS , Christov-Tzelkov K , van Breemen R . Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis . Exp Biol Med 2002 ; 227 ( 10 ): 886 – 93 . Google Scholar Crossref Search ADS © 2018 American Society for Nutrition. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Nutrition Oxford University Press

A Novel Tomato-Soy Juice Induces a Dose-Response Increase in Urinary and Plasma Phytochemical Biomarkers in Men with Prostate Cancer

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Oxford University Press
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© 2018 American Society for Nutrition.
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0022-3166
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1541-6100
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10.1093/jn/nxy232
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Abstract

ABSTRACT Background Tomato and soy intake is associated with reduced prostate cancer risk or severity in epidemiologic and experimental studies. Objective On the basis of the principle that multiple bioactives in tomato and soy may act on diverse anticancer pathways, we developed and characterized a tomato-soy juice for clinical trials. In this phase 2 dose-escalating study, we examined plasma, prostate, and urine biomarkers of carotenoid and isoflavone exposure. Methods Men scheduled for prostatectomy were recruited to consume 0, 1, or 2 cans of tomato-soy juice/d before surgery (mean ± SD duration: 24 ± 4.6 d). The juice provided 20.6 mg lycopene and 66 mg isoflavone aglycone equivalents/177-mL can. Plasma carotenoids and urinary isoflavone metabolites were quantified by HPLC–photometric diode array and prostate carotenoids and isoflavones by HPLC–tandem mass spectrometry. Results We documented significant dose-response increases (P < 0.05) in plasma concentrations of tomato carotenoids. Plasma concentrations were 1.86-, 1.69-, 1.73-, and 1.69-fold higher for lycopene, β-carotene, phytoene, and phytofluene, respectively, for the 1-can/d group and 2.34-, 3.43-, 2.54-, and 2.29-fold higher, respectively, for the 2-cans/d group compared with 0 cans/d. Urinary isoflavones daidzein, genistein, and glycitein increased in a dose-dependent manner. Prostate carotenoid and isoflavone concentrations were not dose-dependent in this short intervention; yet, correlations between plasma carotenoid and urinary isoflavones with respective prostate concentrations were documented (R2 = 0.78 for lycopene, P < 0.001; R2 = 0.59 for dihydrodaidzein, P < 0.001). Secondary clustering analyses showed urinary isoflavone metabolite phenotypes. To our knowledge, this is the first demonstration of the phytoene and phytofluene in prostate tissue after a dietary intervention. Secondary analysis showed that the 2-cans/d group experienced a nonsignificant decrease in prostate-specific antigen slope compared with 0 cans/d (P = 0.078). Conclusion These findings provide the foundation for evaluating a well-characterized tomato-soy juice in human clinical trials to define the impact on human prostate carcinogenesis. This trial is registered at clinicaltrials.gov as NCT01009736. carotenoids, lycopene, isoflavones, tomato, soy, prostate cancer Introduction Prostate cancer remains an enormous health burden, and although diagnosis and treatment options have improved over the past 2 decades, specific strategies to prevent prostate cancer remain elusive. Geographic variation in risk around the globe as well as changing risk with migration and, over time, within nations strongly implicate dietary and lifestyle patterns as contributing factors (1). Although a few large-population studies have identified foods and dietary patterns that may reduce prostate cancer risk (2–5), analytic epidemiology has struggled to provide a foundation of data upon which to develop and test dietary prevention strategies (6–11). Several nutrients, phytochemicals, and dietary patterns, with support from in vitro mechanistic studies, including components found in tomatoes and soy foods, affect experimental prostate carcinogenesis (12–15). Although a causal relation between foods, such as tomatoes or soy, and prostate cancer risk reduction is not clearly established, several lines of evidence suggest that these foods have components that inhibit prostate carcinogenesis (14–26). Decades of research on pharmacologic cancer therapies have established the paradigm that combinations of active agents with multiple mechanisms of action and nonoverlapping toxicity provide the greatest efficacy in curing advanced malignancies (27, 28). We hypothesize that a similar strategy can form a foundation for the design of functional foods or dietary patterns for prostate cancer prevention (29–31). To this end, we propose a combination of tomato and soy. Previous work showed that feeding a 10% tomato powder plus 2% soy germ–containing diet resulted in significantly reduced prostate tumor incidence in a transgenic adenocarcinoma of the mouse prostate (TRAMP) model compared with mice fed a control diet or a diet containing tomato powder or soy germ alone (tumor incidence in tomato powder plus soy = 45% compared with 100% in controls, 61% in the tomato powder group, and 66% in the soy germ group) (30). To extend our findings, it is first necessary to develop and fully classify the bioactive components of a novel food product that can be applied to human studies. To optimize incorporation into a daily diet and promote compliance, we previously developed a tomato-soy juice product that provides consistent phytochemical exposure (32). We targeted tomato lycopene and soy isoflavone content from two 177-mL cans/d to achieve physiologic exposure mimicking the dose range associated with lower cancer risk in human studies (Table 1) (2, 33, 34). The tomato-soy juice was previously developed, subjected to sensory evaluation, and analyzed for phytochemical content and stability. In an 8-wk phase 1 study, we showed its compliance and safety in healthy humans (32). The objective of the current study is to define both the compliance and safety of 2 different doses of tomato-soy juice, as well as carotenoid and isoflavone biodistribution and metabolism, in men with prostate cancer. TABLE 1 Nutrient and phytochemical content of each 177-mL (6-fluid-ounce) can of tomato-soy juice Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 View Large TABLE 1 Nutrient and phytochemical content of each 177-mL (6-fluid-ounce) can of tomato-soy juice Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 Nutrient or phytochemical Value Energy, kcal 43 Fat, g 2 Cholesterol, mg 0 Sodium, mg 447 Total carbohydrate, g 7  Dietary fiber 1  Sugars 6 Protein, g 1 Carotenoids, mg  Total lycopene 20.6   All-trans lycopene 18   cis-Lycopene 2.6  β-Carotene 1.15  Phytoene 2.4  Phytofluene 1.1 Isoflavones (total aglycone equivalents), mg 65  Genistein 42.3  Daidzein 20.7  Glycitein 2.0 View Large Methods Materials HPLC-grade methyl-tert butyl ether (MTBE), methanol (MeOH), acetonitrile, and water and reagent-grade hexane, ethanol, acetone, and toluene were purchased from Fisher Scientific. Lycopene (>95%) was isolated and purified as previously reported (35). β-Carotene and lutein standards (≥95% purity) were purchased from Sigma Aldrich (35). Phytoene and phytofluene standards (≥95% purity) were purchased from Carotenature (35). Subject recruitment Men with biopsy-proven carcinoma of the prostate who had chosen a radical prostatectomy for treatment were recruited from the James Cancer Hospital and The Ohio State University Medical Center (Columbus, OH). There were no age restrictions, and subjects were required to have an Eastern Cooperative Oncology Group performance status of 0–1 and have no clinical abnormalities in kidney, liver, or hematopoietic function, as determined by preoperative assessment. Subjects were asked to discontinue all nutritional supplements, including lycopene and soy. A multivitamin-mineral (CVS Brand) was provided for the study duration. Patients were excluded if they were receiving neoadjuvant hormonal (thyroid, adrenocorticotropic, or growth hormone) or chemotherapy or if they had an active malignancy other than prostate cancer requiring therapy, a history of castration, or other endocrine disorders requiring hormone administration, with the exceptions of diabetes and osteoporosis. Patients with a history of malabsorptive disorders or disorders requiring special diet recommendations (e.g., low-sodium diet for hypertension), severe constipation, or a recent history of anemia or iron deficiency were also excluded. Patients were excluded if they were currently taking finasteride, other agents for benign prostatic hypertrophy, or medications for urinary outlet obstruction. Study design All of the procedures were approved by the Ohio State University Institutional Review Board (clinicaltrials.gov: NCT01009736). Eligible subjects completed informed consent. Upon enrollment, subjects completed a baseline tomato/high-lycopene food-intake questionnaire (Supplemental Data 1), which queried about serving size and frequency of consumption of fresh tomatoes, tomato juice, soup, pizza, pasta sauce, watermelon, pink grapefruit (including juice), salsa, ketchup, and barbeque sauce. In addition, participants answered questions about soy food intake and documented intakes of other soy not listed (Supplemental Data 2). Participants were assigned to an intervention group using a dose-escalation design. The planned intervention consisted of consuming 0, 1, 2, or 3 cans/d of the tomato-soy juice developed by the Department of Food Science and Technology at The Ohio State University (formulation: OSU-TSJ-001) (32) for a 3- to 5-wk duration, which varied on the basis of surgery scheduling. Toxicity to OSU-TSJ-001 was assessed using the National Cancer Institute's Common Terminology Criteria for Adverse Events, version 3.0, and the study physician (SKC) was notified immediately of any adverse events. Each study group was fully enrolled before enrollment in the next dose level began. The study proceeded to the next dose level only if there were <2 episodes of grade I/II toxicity and no episodes of grade III or IV toxicity attributed to the food product after evaluation of each cohort. Ultimately, however, the 3-cans/d group was not enrolled, because 2 of the 30 men (7%) in the 2-cans/d group experienced grade I adverse events. In addition, men in the 2-cans/d group indicated potential difficulties with consuming 3 cans/d. Instead, we expanded the 2-cans/d cohort. Tomato-soy juice intervention product The tomato juice was prepared from tomatoes grown by the Department of Horticulture and Crop Science at Ohio State University. A high-carotenoid tomato variety (FG99-218), which has excellent juice-producing properties, grows successfully in Ohio, and is homozygous for the high pigment-2 dark-green allele (hp-2dg) and for the old gold crimson allele (og c), was selected. Tomato juice was initially produced using a hot-break treatment and preserved in no. 10 cans, as previously published (36), to capture the tomato flavor and carotenoid profile at the peak of ripeness. To produce the tomato-soy juice, the already preserved tomato juice was pooled in an agitated, steam-jacketed kettle and combined with salt, 1% extra-virgin olive oil (Bertolli) added to enhance taste and palatability, and 0.10% soybean-isoflavone–rich extract (wt:wt; Solgen 40S; Solbar Plant Extracts, Ltd.) on a weight basis and heated to 95°C. The juice was then hot-filled into 177-mL (6 fluid ounces) cans and retorted at 100°C for 15 min. All constituents used for juice production originated from single batches of ingredients. After sealing and cooling, this product was stored at room temperature. The juice nutrient and phytochemical content is detailed in Table 1. Blood, urine, and prostate sample collection Venous blood samples (17 mL) were collected upon enrollment (nonfasting) and surgery (fasting) from subjects into 1 serum separator Vacutainer tube and 2 EDTA Vacutainer tubes (BD). Tubes were light-protected with foil, maintained on ice, and centrifuged at 1100 rotations per minute or 500 relative centrifugal force (× g) for 20 min at 4°C. The serum and plasma were divided into aliquots, placed into vials, and stored at −80°C for analyses. A 10-mL spot urine sample was collected upon enrollment, and a complete 24-h urine collection was begun 1 d before surgery. Throughout collection, the urine containers were kept refrigerated, and samples were divided into aliquots and stored at −80°C until analysis. Prostate tissue samples were obtained at surgical resection, and a piece of prostate tissue (noncancerous by gross examination) was immediately frozen in liquid nitrogen and stored at −80°C for phytochemical analysis. A separate tissue specimen was transported to the Ohio State University Surgical Pathology Department–Comprehensive Cancer Center Tissue Procurement Center for pathologic examination by a board-certified pathologist. Compliance monitoring All participants were instructed to follow a “controlled lycopene diet.” We restricted consumption of high-lycopene foods and limited lower-lycopene tomato foods to provide ≤5 mg lycopene/d. Participants were also instructed to avoid foods containing soy protein and soy isoflavones. All men completed a daily log (Supplemental Data 1 and 2) to document consumption of the study product (for the intervention groups) and adherence to or deviations from the controlled lycopene diet. Biochemical measurements Prostate-specific antigen and lipid profiles Plasma prostate-specific antigen (PSA) and serum TG, LDL-, HDL-, and total-cholesterol concentrations were measured at baseline and at the end of study using standard clinical procedures by the Ohio State Center for Clinical and Translational Science Laboratory and the Ohio State University Medical Center Clinical Laboratory. PSA was analyzed by the Medical Center Clinical Laboratory on a Centaur XP (Siemens Medical Diagnostics) using sandwich chemiluminescent immunological reaction with paramagnetic particles as the solid phase and acridinium ester as the chemiluminescent label. Lipids were analyzed using the Dimension Xpand Clinical Chemistry System (Siemens Medical Diagnostics). The analytical sensitivity was 3.0 mg/dL for HDL cholesterol, 5 mg/dL for LDL cholesterol, 50 mg/dL for total cholesterol, 15 mg/dL for TGs, and 0.01ng/mL for PSA. Plasma and prostate tissue carotenoid extraction and analysis Carotenoid concentrations of the plasma and juice samples were analyzed using reverse-phase HPLC coupled with photometric diode array using our previously published method (37). The tomato-soy juice carotenoids were extracted following a previously described method (35) and analyzed using the HPLC method cited above. Plasma lycopene, β-carotene, α-carotene, zeaxanthin, lutein, and β-cryptoxanthin were quantified using external standard curves. Plasma and tomato-soy juice phytoene and phytofluene were quantitated by comparing the molar extinction coefficient of lycopene with those of phytoene and phytofluene and by adjusting the standard curve slope of lycopene appropriately for phytoene and phytofluene. Prostate tissues (20–200 mg) were pulverized to fine particles with an anvil-in-cup apparatus by repeated striking with a rubberized hammer. Pulverized material was weighed into an 11-mL glass vial, water was added (1 mL), the suspension probe sonicated (5 s, default energy; Sonic Dismembrator Model 150E; Fisher Scientific), ethanol added [1 mL with 1% (wt:vol) butylated hydroxytoluene], probe sonicated, and extracted twice with 5 mL HEAT (10 hexane:6 ethanol:7 acetone:7 toluene; vol:vol:vol:vol). The upper organic layers from both were combined into a 40-mL glass vial and dried under nitrogen, then redissolved in 200 µL 1:1 MTBE:MeOH, and finally filtered through 0.2-µm nylon syringe filters. Reconstituted extracts were injected (40 µL) on the QTrap5500 LCMS system (AB Sciex) (38). Lycopene, β-carotene, phytoene, and phytofluene concentrations were quantified against an external standard curve. Urine and tissue isoflavone analysis Urine isoflavones were extracted as described previously (24). Isoflavone metabolites were extracted from 20–200 mg prostate tissue by suspending in 100 µL water, probe sonicating (5 s; default energy), and adding 200 µL acetonitrile. This solvent composition liberates isoflavone metabolites while precipitating proteins and other sample matrix components. Samples were centrifuged for 5 min at 21,130 × g, and supernatant was collected. The pellet was resuspended in 300 µL of 2:1 acetonitrile:water, probe sonicated, and centrifuged again. The 2 supernatants were pooled and dried under nitrogen. The residues were resuspended in 1 mL of 1 M sodium acetate buffer, pH 5.5, and 10 µL of 54 mg glucuronidase-sulfatase/mL in 0.2% NaCl was added to deconjugate over 2 h at 37°C, and extracted twice with 3 mL ether each. Aglycone extracts were dried under nitrogen and reconstituted in 150 µL methanol with bath sonication and filtered through 0.2-µm nylon syringe filters. LC-MS analyses of daidzein, genistein, equol, dihydrodaidzein (dhDaidzein), dihydrogenistein (dhGenistein), 6-hydroxy-O-desmethylangolensin (6-OH-ODMA), and O-desmethylangolensin (ODMA) in prostate tissue and glycitein in urine were conducted as described in Ahn-Jarvis et al. (24). Statistical analyses Baseline characteristics were summarized using means and SDs. Differences between groups were determined using ANOVA, with the exception of the baseline habitual phytochemical intakes, for which the Kruskal-Wallis nonparametric test was used due to multiple observations of no soy intake (39). In the statistical analyses of the plasma and prostate outcomes, the primary goal was to estimate the effect of the intervention. As such, covariates were included in the statistical models if they modified the effect of the intervention by >15% (40). Interactions with the intervention were included at the 0.05 significance level. For plasma, the preintervention measure of the outcome was always included in the model, consistent with an ANCOVA approach. Pearson correlations were calculated between plasma and tissue carotenoid concentrations. When observations for plasma or tissue were below the detection limit, a Wilcoxon Rank Sum test, as opposed to a parametric test, was used to compare urinary and tissue isoflavone concentrations between groups (39). Similarly, for correlation analyses, a Kendall's τ-b nonparametric correlation was calculated between urinary and tissue isoflavone concentrations due to the presence of nondetects. Change in PSA was evaluated using ANCOVA. In all parametric analyses, concentrations were log-transformed before analysis to improve normality and homoscedasticity. Where applicable, the stepdown Bonferroni (Holms) procedure was used to adjust P values for multiplicity. Data included those for each participant who completed ≥2 wk of the intervention. Statistical analyses were performed in SAS version 9.4 (SAS Institute) or Stata version 13.1 (StataCorp). Clustering of the urine isoflavones was conducted using hierarchical agglomerative methods. Proportions of the 3 components of the daidzein family of metabolites, equol, ODMA, and dhDaidzein and daidzein (combined) were computed over the daidzein family total. These proportions were transformed using the arcsin square root to stabilize their variance and improve normality. Clusters were formed using Euclidean distance measure and average linkage. Final determination of the number of clusters present was conducted by visual inspection of the dendrogram. Results Sixty men consented to participate. Three men withdrew for the following reasons: 1 opted for surgery at another hospital, 1 did not want to complete the questionnaires, and 1 postponed surgery indefinitely. In addition, 1 subject did not complete baseline sample collection, and another only consumed 20% of the targeted intervention goal. All 5 of these men were excluded from statistical analyses. Three men were excluded from tissue isoflavone analysis only, and an additional 3 men were excluded from both tissue carotenoid and tissue isoflavone analyses due to inadequate prostate tissue procurement. Finally, men who did not drink the juice for the 2 d before surgery (confusion with preoperative instructions) or who did not complete a full 24-h urine collection were excluded from the urinary isoflavone analysis (n = 8). The prestudy habitual daily tomato or soy intakes reported at baseline are shown in Table 2. Subjects reported usual consumption of 10.7 ± 8.2 (mean ± SD) servings of lycopene-rich foods/wk, with the most frequently consumed category being ketchup or barbeque sauce, with 3.8 ± 5.1 servings/wk contributing 6.05 ± 8.17 mg lycopene/wk to the diet. The greatest sources of lycopene in the diet were pasta sauce (27% of lycopene intake; 15.6 ± 12.16 mg/wk), followed by tomato juice (20%; 11.5 ± 35.2 mg/wk), pizza (13%; 7.4 ± 8.4 mg/wk), and ketchup (11%). Subjects reported negligible baseline consumption of any soy-based foods. With the exception of age, other baseline characteristics of the 55 men who completed all study interventions were not significantly different between groups and are summarized in Table 1. The mean ± SD number of days receiving the intervention was 24 ± 4.6. TABLE 2 Baseline characteristics and reported habitual phytochemical intake of patients with prostate cancer participating in the OSU tomato-soy juice study1 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 1 Values are means ± SDs unless otherwise indicated; n = 55 for all measures. OSU, Ohio State University. 2 Mean ± SD ages of the 0-, 1-, and 2-cans/d groups were 54.7 ± 8, 60.2 ± 7, and 63.2 ± 7 y, respectively. The men consuming 2 cans/d were significantly younger than men consuming 0 cans/d (P < 0.05). 3 Prestudy carotenoid intake is based on subject responses to a baseline questionnaire querying the usual number of weekly servings consumed of the following high-lycopene foods: raw tomato, tomato juice, tomato-based mixed-vegetable juice, tomato soup, pizza, tomato-based pasta sauce (marinara), watermelon, grapefruit, tomato salsa, ketchup, and barbeque sauce. Prestudy soy isoflavone intake is based on subject responses to a baseline questionnaire querying weekly intake of soymilk, soy burgers, soy nuts, and other soy products (open response). 4 Value includes 40% of men who were taking a statin medication for cholesterol management. View Large TABLE 2 Baseline characteristics and reported habitual phytochemical intake of patients with prostate cancer participating in the OSU tomato-soy juice study1 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 Baseline characteristics Value n 55 Age,2 y 60.5 ± 7.9 Race, n (%)  White 54 (98)  Black 1 (2) Body mass, kg 100 ± 19 BMI, kg/m2 31.4 ± 5.5 Prestudy estimated tomato carotenoid and soy isoflavone intake,3 mg/d  Lycopene 8.12 ± 7.20  Phytoene 1.43 ± 1.38  Phytofluene 0.97 ± 0.69  Daidzein 0.33 ± 1.17  Genistein 0.45 ± 1.72  Glycitein 0.02 ± 0.09 Plasma lipids, mg/dL  Total cholesterol4 173 ± 41  LDL cholesterol 110 ± 34  HDL cholesterol 42 ± 11  TGs 189 ± 116 1 Values are means ± SDs unless otherwise indicated; n = 55 for all measures. OSU, Ohio State University. 2 Mean ± SD ages of the 0-, 1-, and 2-cans/d groups were 54.7 ± 8, 60.2 ± 7, and 63.2 ± 7 y, respectively. The men consuming 2 cans/d were significantly younger than men consuming 0 cans/d (P < 0.05). 3 Prestudy carotenoid intake is based on subject responses to a baseline questionnaire querying the usual number of weekly servings consumed of the following high-lycopene foods: raw tomato, tomato juice, tomato-based mixed-vegetable juice, tomato soup, pizza, tomato-based pasta sauce (marinara), watermelon, grapefruit, tomato salsa, ketchup, and barbeque sauce. Prestudy soy isoflavone intake is based on subject responses to a baseline questionnaire querying weekly intake of soymilk, soy burgers, soy nuts, and other soy products (open response). 4 Value includes 40% of men who were taking a statin medication for cholesterol management. View Large Subject compliance and toxicity Men in all 3 groups (0, 1, and 2 cans/d) were compliant with the “controlled lycopene diet” and maintained a diet of ≤5 mg lycopene on 97% (1272 of 1310) of study days (mean lycopene ± SD = 1.1 ± 0.29 mg/d, 0.54 ± 0.2 mg/d, and 0.47 ± 0.10 mg/d, respectively). Men in both the 1-can/d and 2-cans/d groups, who were also compliant with the intervention product, consumed 97% and 91% of the targeted dose, respectively. In the 2-cans/d group, there were 2 grade I adverse events. One man experienced grade I diarrhea and discontinued the juice after 7 d, at which time his symptoms resolved; because of the short intervention duration, he was excluded from data analysis. A second man discontinued the juice after 16 d due to hypertension, which did not resolve after discontinuation of the study product. Because isoflavones are quickly metabolized and excreted, as marked by a plasma half-life of 7 h (41), we did not include this subject in isoflavone analysis. However, lycopene has a plasma half-life of 6 d and a tissue half-life of 12 d (42); therefore, this participant was included in both blood and tissue carotenoid analyses. Clinical measures of toxicity were within normal limits at both baseline and at the end of study for all men. Plasma carotenoids Plasma lycopene, β-carotene, phytofluene, and phytoene concentrations at baseline and at the end of the intervention are shown in Table 3. There was a dose-dependent and significant increase for each of the tomato carotenoids analyzed, with the exception of phytoene in the 1-can/d group. In addition, the model-adjusted analysis for men consuming 2 cans/d showed plasma concentrations that were 2.34-fold (95% CI: 1.95-, 2.81-fold), 3.43-fold (95% CI: 2.53-, 4.66-fold), 2.54-fold (95% CI: 1.86-, 3.45-fold), and 2.29-fold (95% CI: 1.78, 2.94-fold) higher than the 0-cans/d group for lycopene, β-carotene, phytoene, and phytofluene, respectively. The men consuming 1 can/d had plasma concentrations that were 1.86-fold (95% CI: 1.49-, 2.31-fold), 1.69-fold (95% CI: 1.2-, 2.37-fold), 1.73-fold (95% CI: 1.22-, 2.46-fold), 1.69-fold (95% CI: 1.27-, 2.23-fold) higher than the 0-cans/d group, respectively. For lycopene, the interaction between intervention group and duration was significant (P = 0.0099), such that a longer intervention before scheduled prostatectomy led to greater plasma lycopene changes within the intervention group. Therefore, intervention duration and baseline plasma lycopene concentration were included in the analysis of the effect of treatment group on final plasma lycopene concentration. Other covariates are listed in Table 3. TABLE 3 Plasma carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* 1 Values are means ± SDs. Labeled means in a column without a common superscript letter differ, P < 0.05. *Different from baseline, P < 0.05. The interactions between the length of intervention and intervention group and baseline lycopene were included in the model for lycopene. Age, BMI, and baseline values were included for β-carotene and phytofluene. Other covariates tested (plasma volume, blood lipoproteins, usual reported carotenoid intake) did not affect outcomes. There were no significant differences between the 3 groups for baseline carotenoid concentrations. View Large TABLE 3 Plasma carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* Lycopene β-Carotene Phytoene Phytofluene Group Baseline End of study Baseline End of study Baseline End of study Baseline End of study 0 cans/d (n = 13), µmol/L 1.32 ± 0.45 0.99 ± 0.36c* 0.47 ± 0.66 0.36 ± 0.40c 0.12 ± 0.06 0.09 ± 0.05c 0.20 ± 0.11 0.17 ± 0.08c 1 can/d (n = 15), µmol/L 1.17 ± 0.64 1.48 ± 0.62b* 0.50 ± 0.60 0.60 ± 0.47b* 0.17 ± 0.13 0.20 ± 0.12b 0.23 ± 0.14 0.33 ± 0.22b* 2 cans/d (n = 27), µmol/L 1.1 ± 0.42 1.93 ± 0.50a* 0.33 ± 0.25 0.86 ± 0.42a* 0.13 ± 0.05 0.33 ± 0.10a* 0.21 ± 0.11 0.41 ± 0.17a* 1 Values are means ± SDs. Labeled means in a column without a common superscript letter differ, P < 0.05. *Different from baseline, P < 0.05. The interactions between the length of intervention and intervention group and baseline lycopene were included in the model for lycopene. Age, BMI, and baseline values were included for β-carotene and phytofluene. Other covariates tested (plasma volume, blood lipoproteins, usual reported carotenoid intake) did not affect outcomes. There were no significant differences between the 3 groups for baseline carotenoid concentrations. View Large Prostate carotenoids Prostate carotenoid concentrations were assessed at prostatectomy only and are shown in Table 4. There were no significant differences in prostate carotenoids between the 3 groups for this short-term intervention (Table 4) TABLE 4 Prostate carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 1 Values are means ± SDs. Group effects were statistically compared using linear models, and prostate carotenoid concentrations were not significantly different by treatment group. View Large TABLE 4 Prostate carotenoid concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 Group Lycopene β-Carotene Phytoene Phytofluene 0 cans/d (n = 13), nmol/g 0.58 ± 0.10 0.16 ± 0.03 0.23 ± 0.23 0.28 ± 0.17 1 can/d (n = 15), nmol/g 0.48 ± 0.076 0.22 ± 0.09 0.22 ± 0.22 0.30 ± 0.24 2 cans/d (n = 24), nmol/g 0.67 ± 0.06 0.21 ± 0.04 0.21 ± 0.13 0.28 ± 0.18 1 Values are means ± SDs. Group effects were statistically compared using linear models, and prostate carotenoid concentrations were not significantly different by treatment group. View Large Correlation between plasma and prostate carotenoid concentrations Baseline and end-of-study plasma lycopene concentrations were significantly correlated with prostatic lycopene, as were baseline and end-of-study plasma β-carotene with prostatic β-carotene concentrations. However, plasma measures of carotenoids of lower concentrations, phytoene or phytofluene, were not significantly correlated with respective prostatic concentrations. Correlation plots for plasma and prostatic lycopene and β-carotene are shown in Figure 1. FIGURE 1 View largeDownload slide (A–D) Correlations between baseline or end-of-study plasma carotenoid concentrations with prostate carotenoid concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Pearson correlation coefficient. Plasma and prostate carotenoid concentrations were log-transformed before calculating correlations to improve normality, and untransformed data are presented here for ease of interpretation. FIGURE 1 View largeDownload slide (A–D) Correlations between baseline or end-of-study plasma carotenoid concentrations with prostate carotenoid concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Pearson correlation coefficient. Plasma and prostate carotenoid concentrations were log-transformed before calculating correlations to improve normality, and untransformed data are presented here for ease of interpretation. Urine isoflavones Parent isoflavones (genistein, daidzein, and glycitein) and their metabolites (ODMA, 6-OH-ODMA, dhDaidzein, dhGenistein, and equol) were below the minimal detection limits in urine from men in the 0-cans/d group. Total urinary isoflavone excretion increased with increasing tomato-soy juice dose (Table 5). A significant dose response was observed for each individual parent isoflavone comparing 1 can of tomato-soy juice/d with 2 cans/d (Wilcoxon's P = 0.0009, 0.0131, and 0.0193 for daidzein, glycitein, and genistein, respectively). There was no difference between groups for urinary isoflavone metabolite concentration. TABLE 5 Urinary isoflavone concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) 1 Values are medians (25th–75th percentiles). Labeled medians in a column without a common superscript letter differ, P < 0.05. Group responses were compared pairwise by the Wilcoxon Rank Sum test. The urinary isoflavone output was below the minimal detection limit in 100% of the subjects for all isoflavones in the 0-cans/d group. MDL (nmol/L) urine analysis: daidzein, 1.08; ODMA, 0.42; genistein, 3.8; glycitein, 2.44; dhDaidzein, 0.18; dhGenistein, 0.24, equol, 0.28. dhDiadzein, dihydrodaidzein; dhGenistein, dihydrogenistein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin. View Large TABLE 5 Urinary isoflavone concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) Group Total isoflavones Daidzein ODMA Genistein Glycitein dhDaidzein dhGenistein Equol 0 cans/d (n = 13), µmol/24 h <MDLc <MDLc <MDLb <MDLc <MDLc <MDLb <MDLb <MDLb 1 can/d (n = 13), µmol/24 h 49 (22.2–71)b 16.7 (9.5–27.9)b 14.1 (3.6–21.2)a 3.8 (2.4–6.2)b 2.2 (1.1–3.9)b 2.5 (1.0–6.1)a 0.4 (<MDL–1.9)a <MDLa (<MDL–2.3) 2 cans/d (n = 21), µmol/24 h 92.8 (69.4–118)a 39.4 (28–56.6)a 15.7 (4.7–30.7)a 7.1 (5.0 –14.9)a 4.4 (2.4 –7.2)a 5.6 (3.0–14.8)a 1.1 (0.3–3.2)a <MDLa,b (<MDL–<MDL) 1 Values are medians (25th–75th percentiles). Labeled medians in a column without a common superscript letter differ, P < 0.05. Group responses were compared pairwise by the Wilcoxon Rank Sum test. The urinary isoflavone output was below the minimal detection limit in 100% of the subjects for all isoflavones in the 0-cans/d group. MDL (nmol/L) urine analysis: daidzein, 1.08; ODMA, 0.42; genistein, 3.8; glycitein, 2.44; dhDaidzein, 0.18; dhGenistein, 0.24, equol, 0.28. dhDiadzein, dihydrodaidzein; dhGenistein, dihydrogenistein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin. View Large Prostate isoflavones Prostate concentrations of the isoflavones and metabolites are presented in Table 6. Daidzein and genistein were quantifiable in all subjects across all groups. The genistein metabolite 6-OH-ODMA and the daidzein metabolite equol were only detected in one patient, so neither are shown in Table 6. dhDaidzein was detectable in 15 patients in the juice-consuming groups but in no subjects consuming 0 cans/d. ODMA was detected in 24 study participants in the tomato-soy juice groups but not in any of the 0-cans/d group. Both dhDaidzein and ODMA concentrations were greater in the 2-cans/d group than in the 0-cans/d group (Table 6). TABLE 6 Prostate isoflavone and isoflavone metabolite concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 1 Values are means ± SDs unless otherwise indicated. Only one patient, respectively, had measurable tissue concentrations of 6-OH-ODMA or equol; therefore, those data were not analyzable. Group effects for isoflavones (daidzein and genistein) were present in the majority of subjects and were compared by ANOVA. Isoflavone metabolites (dhDaidzein and ODMA) were below the MDL for many participants and therefore were compared using the Wilcoxon Rank Sum test (39). MDL (nmol/g) for tissue analysis: daidzein, 0.01; dhDaidzein, 0.001; genistein, 0.02; ODMA, 0.002. Medians in a column without a common superscript letter differ, P < 0.05. dhDaidzein, dihydrodaidzein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin; 6-OH-ODMA, 6-hydroxy-O-desmethylangolensin. 2 All subjects had detectable prostate daidzein and genistein. View Large TABLE 6 Prostate isoflavone and isoflavone metabolite concentrations in men with prostate cancer after a 3-wk intervention with 0, 1, or 2 cans of tomato-soy juice/d1 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 dhDaidzein ODMA Group Daidzein2 Median (25th, 75th percentile) <MDL, % Genistein2 Median (25th, 75th percentile) <MDL, % 0 cans/d (n = 13), nmol/g 0.22 ± 0.11 <MDLb 100 0.54 ± 0.30 <MDLb 100 1 can/d (n = 14), nmol/g 0.28 ± 0.12 <MDL (<MDL, 0.003)a,b 71.4 0.60 ± 0.27 0.02 (<MDL, 0.03)a 35.7 2 cans/d (n = 22), nmol/g 0.33 ± 0.20 0.50 (<MDL, 0.02)a 50 0.75 ± 0.64 0.025 (<MDL, 0.06)a 27.3 1 Values are means ± SDs unless otherwise indicated. Only one patient, respectively, had measurable tissue concentrations of 6-OH-ODMA or equol; therefore, those data were not analyzable. Group effects for isoflavones (daidzein and genistein) were present in the majority of subjects and were compared by ANOVA. Isoflavone metabolites (dhDaidzein and ODMA) were below the MDL for many participants and therefore were compared using the Wilcoxon Rank Sum test (39). MDL (nmol/g) for tissue analysis: daidzein, 0.01; dhDaidzein, 0.001; genistein, 0.02; ODMA, 0.002. Medians in a column without a common superscript letter differ, P < 0.05. dhDaidzein, dihydrodaidzein; MDL, minimal detectable level; ODMA, O-desmethylangolgensin; 6-OH-ODMA, 6-hydroxy-O-desmethylangolensin. 2 All subjects had detectable prostate daidzein and genistein. View Large Correlations between urinary and prostate isoflavone concentrations Prostate and urinary daidzein at the time of surgery were not correlated (r = −0.14, P = 0.28) and neither were tissue and urinary genistein concentrations (r = −0.07, P = 0.60). Tissue and urinary dhDaidzein concentrations significantly correlated, as were dhGenistein and ODMA tissue and urinary concentrations (Figure 2). FIGURE 2 View largeDownload slide (A–C) Correlations between 24-h urinary isoflavone metabolite output with prostate isoflavone metabolite concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Kendall's τ-b correlation coefficient, which allows for inclusion of samples below the minimal detection limit in analysis by rank-order analysis. Urinary and prostate isoflavone concentrations were log10-transformed for correlation analysis to improve normality assumptions; however, untransformed data are presented here for ease of interpretation. dhDaidzein, dihydrodaidzein; dhGenistein, dihydrogenistein; ODMA, O-desmethylangolensin. FIGURE 2 View largeDownload slide (A–C) Correlations between 24-h urinary isoflavone metabolite output with prostate isoflavone metabolite concentrations in men with prostate cancer consuming 0, 1, or 2 cans tomato-soy juice/d. R2 represents the Kendall's τ-b correlation coefficient, which allows for inclusion of samples below the minimal detection limit in analysis by rank-order analysis. Urinary and prostate isoflavone concentrations were log10-transformed for correlation analysis to improve normality assumptions; however, untransformed data are presented here for ease of interpretation. dhDaidzein, dihydrodaidzein; dhGenistein, dihydrogenistein; ODMA, O-desmethylangolensin. Isoflavone metabolism phenotype clusters Based on the dendogram, subjects clustered into 5 distinct isoflavone metabolic phenotype groups on the basis of urinary isoflavone excretion (Figure 3). The 2 largest groups consisted of individuals with either similar proportions of daidzein and dhDaidzein and ODMA (cluster 1) or a greater proportion of daidzein and dhDaidzein than ODMA (cluster 2) in the urine. The third largest group was similar to the second but also excreted equol in the urine (cluster 3), whereas the fourth group had no daidzein metabolites (equol, dhDaidzein, or ODMA) (cluster 4) in the urine. The final and smallest group (cluster 5), with only one member, excreted large amounts of equol with no ODMA. Cluster groups 1, 2, and 3 contained subjects from the 1- and 2-cans/d intervention groups, whereas cluster 4 contained only men consuming 2 cans tomato-soy juice/d, and the one man in cluster 5 consumed 1 can/d. FIGURE 3 View largeDownload slide Clustered daidzein urinary metabolic phenotypes in men with prostate cancer consuming either 1 or 2 cans tomato-soy juice/d. The proportions of daidzein and daidzein metabolites were analyzed using agglomerative clustering with the Euclidean distance dissimilarity measure and the average linkage metric. Brackets with numbers along the x axis indicate of which supplemented group each subject was a member. Relative abundance data were arcsin square root transformed for greater detection of dissimilarity in cluster analysis. dhD, dihydrodaidzein; ODMA, O-desmethylangolensin. FIGURE 3 View largeDownload slide Clustered daidzein urinary metabolic phenotypes in men with prostate cancer consuming either 1 or 2 cans tomato-soy juice/d. The proportions of daidzein and daidzein metabolites were analyzed using agglomerative clustering with the Euclidean distance dissimilarity measure and the average linkage metric. Brackets with numbers along the x axis indicate of which supplemented group each subject was a member. Relative abundance data were arcsin square root transformed for greater detection of dissimilarity in cluster analysis. dhD, dihydrodaidzein; ODMA, O-desmethylangolensin. PSA kinetics PSA ranged from 0.11 to 12.8 ng/mL at enrollment, which is typical for most prostatectomy cohorts of men screened by digital rectal examination and PSA. Due to the heterogeneity and modest study size, no differences were detected between groups at baseline or the end of study. Calculated mean ± SD PSA velocities were 6.3 ± 16.8, 7.7 ± 21.5, and 1.7 ± 22.7 ng ⋅ mL−1 ⋅ y−1 for the 0-, 1-, and 2-cans/d groups, respectively; these values were not significantly different. The change in PSA from study start to end by duration of the intervention was evaluated using an ANCOVA approach. For the control group, little change was observed regarding duration. For the 1- and 2-cans/d groups, a decrease in PSA was detected and was greater for those who were receiving the intervention longer. The difference in the slopes between the 0-cans/d group and the 2-cans/d group was not significant (P = 0.078; Figure 4). Gleason grade from the surgical sample was not significantly different between groups. FIGURE 4 View largeDownload slide Change in PSA over ∼3 wk of a dietary intervention with either 0, 1, or 2 cans of tomato-soy juice/d in men with prostate cancer. The lines indicate the linear slope for each group from baseline to the end of the intervention. PSA, prostate-specific antigen. FIGURE 4 View largeDownload slide Change in PSA over ∼3 wk of a dietary intervention with either 0, 1, or 2 cans of tomato-soy juice/d in men with prostate cancer. The lines indicate the linear slope for each group from baseline to the end of the intervention. PSA, prostate-specific antigen. Discussion Historically, cancer chemoprevention strategies have utilized a pharmacologic model with drugs (43), nutrients (44, 45), or pure phytochemicals (46). In contrast, multifactor interventions focusing on dietary patterns representing a diverse array of variables have been examined (47). A third strategy, engaging foods of defined compositions conducive to inhibition of carcinogenesis, has undergone less clinical investigation. Plant-based foods can be studied in preclinical mechanistic models to define bioactive profiles with additive or synergistic anticancer activity and nonoverlapping toxicity. Plant genetics, horticultural conditions, and food processing can be engineered to enhance ingredient bioactive profiles, lengthen shelf-life, and improve patient compliance through optimized sensory and packaging characteristics for ease of use (32). This dose-escalating phase I/II trial evaluated a tomato-soy juice assessed short-term (∼3 wk) safety and compliance, as well as phytochemical exposure and metabolism in a cohort of men undergoing prostatectomy for clinically localized prostate cancer. Compliance with the tomato-soy juice was excellent. No grade II/III or IV toxicities or clinical laboratory abnormalities were observed. However, with 2 cans/d we observed one case of grade I gastrointestinal complaint and one case of grade I hypertension. We have observed similar dose-dependent complaints in past studies with soy products (24, 32). Thus, to focus our study on tolerable and achievable doses, we eliminated the 3-cans/d group and expanded the 2-cans/d group. Our past intervention studies (19, 24, 31, 32, 48) document profound human heterogeneity in absorption and metabolism of food bioactives. This heterogeneity is likely due to intrinsic factors, such as genetics and the microbiome (24), as well as extrinsic variables, such as medications and diet composition (49). We controlled background dietary variability of tomato and soy foods to better isolate effects of the intervention. All 4 monitored plasma carotenoids significantly increased in a dose-response manner. End-of-study plasma lycopene was 2.3-fold greater in the 2-cans/d group compared with the 0-cans/d groups. The final plasma lycopene concentrations reported here for both intervention groups are greater than those previously achieved with a similar amount of lycopene from tomato products (mean ± SEM: 0.78 ± 0.1 to 1.14 ± 0.1 µmol/L) (48) or from a combination of tomato products (0.91 µmol/L) (50); this may be due to greater baseline plasma lycopene concentrations (1.17 ± 0.50 µmol/L) in the current study than in previous studies (range: 0.45–0.67 µmol/L) (48, 50). To our knowledge, this is the first report of the dose-response impact of a tomato food-product intervention in patients with prostate cancer on plasma phytoene and phytofluene. Although plasma phytoene-to-lycopene ratios mirrored that in the juice (1:10), plasma phytofluene:lycopene was 1:5, differing from the juice ratios of 1:20, which may suggest differing pharmacokinetics of phytofluene and phytoene. The dose-response effect of this product on plasma carotenoids provides a foundation for use in larger clinical trials. There are several limitations to our study. First, based on the prostate carotenoid data, the duration of feeding may have been too brief to change prostate carotenoid concentrations in these subjects with elevated baseline carotenoid status. Wait times at our clinic for curative cancer surgery limited the intervention period to ∼3 wk. Secondary analyses of prostate carotenoid outcomes provided insights. First, according to the Plateau Principle, it takes 3.3 half-lives to reach 90% of an expected steady-state concentration (51). The estimated half-life of lycopene in slow-turnover tissues, such as the prostate, is estimated to be 12 d (42); therefore, it may require ∼41 d to reach 90% of the steady-state prostate tissue lycopene concentration. The longer time to reach tissue than plasma carotenoid steady-state concentrations may also explain why baseline plasma lycopene and β-carotene concentrations were more strongly correlated with corresponding prostate carotenoid concentrations than were end-of-study plasma carotenoid concentrations. Second, the lack of a washout in the current study design may have affected some of our study results. We previously found that a similar amount (25–35 mg/d) of daily lycopene from tomato products provided for ∼23 d increased prostate lycopene by 2- to 3-fold compared with controls (48); that study engaged a 1-wk lycopene washout before a 3-wk intervention, and thus subjects had lower baseline lycopene status than in the current study. Indeed, prostate lycopene in the current control group (mean ± SD: 0.584 ± 0.096 nmol/g) was greater than that in the previous control group (mean ± SEM: 0.16 ± 0.06 nmol/g) (48). In the current study, in which subjects began with a higher lycopene status, we found that baseline plasma lycopene and β-carotene concentrations were more strongly correlated with end-of-study prostate carotenoid concentrations than were their end-of-study plasma lycopene and β-carotene concentrations. This suggests that, if at a high baseline carotenoid status, previous steady-state conditions may be more influential on prostate carotenoids than a short intervention. In addition, plasma carotenoid responses may have been repressed in this study due to an interaction with soy components of the drink. Zuniga and Erdman (52) previously found that soy germ consumed with tomato led to reduced serum and prostate lycopene compared with tomato feeding alone in rodents. Even so, Zuniga et al. (30) later found that a tomato and soy-germ combination was more effective than either component alone in reducing prostate cancer incidence in TRAMP mice. Furthermore, our studies (42, 48, 53) indicate that longer-term carotenoid interventions are required to change prostate carotenoid concentrations in those with a higher baseline carotenoid status. Finally, we were not able to procure malignant prostate tissue samples due to pathologic evaluation after surgery. Our previous study (54) showed that cancerous regions accumulate greater lycopene and all-trans β-carotene, but not α-carotene, β-cryptoxanthin, or lutein and zeaxanthin. Future studies to define the differential accumulation of phytoene, phytofluene, and isoflavones in cancerous compared with noncancerous regions would be of interest. To our knowledge, this is the first report of prostate phytoene and phytofluene concentrations (0.2–0.3 nmol/g) in response to a tomato product intervention. Although these are minor carotenoids in the juice, they accumulate to nearly half the level of lycopene in the prostate, consistent with previous kinetic studies in rodent models (53, 55, 56) and in humans, suggesting that phytoene is more bioavailable and more rapidly taken up by slow-turnover tissue pools than lycopene (42, 57). Urinary isoflavones are a marker of soy food intake (58), but, to our knowledge, the relation between urinary isoflavones and tissue concentrations has not been explored. Urinary isoflavone metabolite (dhDaidzein, dhGenistein, ODMA) concentrations correlated with their respective tissue concentrations, whereas the parent isoflavones (genistein and daidzein) did not. In particular, urinary ODMA may be a useful biomarker of prostatic ODMA, because it was excreted by most subjects. Finally, the patterns of parent isoflavone and metabolite urinary excretion clustered into groups similar to our previous report (24). We postulate that these metabolic phenotypes represent the combination of host genetics and microbiome determinants of isoflavone metabolism. Critically, the application of statistical and bioinformatic techniques to isoflavone metabolites may provide insight into how these various phenotypes relate to health outcomes. This study is the first, to our knowledge, to report prostatic isoflavone concentrations after a food-based soy intervention study. We observed a nonsignificant (P > 0.1) dose-response effect for genistein and daidzein, which is unsurprising given the person-to-person heterogeneity observed. Tissue isoflavone concentrations in the current study are lower than in previous reports with botanical dietary supplements resulting in genistein and daidzein concentrations of ∼1–2.4 nmol/g (59–61). However, these studies were of shorter duration (3–14 d), which could also affect isoflavone metabolic rate by the host or colonic microbiota. In sum, isoflavones do accumulate in prostatic tissue, although with significant individual variability, as is characteristic of phytochemicals. Although not a primary outcome, we measured PSA kinetics as a marker of prostate cancer status and found a trend supporting a dietary impact for the 2-cans/d dose in reducing PSA (Figure 4). We observed a prolongation of PSA doubling time in a previous short-term study (24), and others have reported favorable changes in PSA after a tomato-based intervention (50, 62). To conclude, an optimized tomato-soy juice reliably increases plasma carotenoids and urinary markers of soy isoflavone intake with minimal grade I toxicity events. Complementary genetic and microbiome studies can define the key predictors of plasma and tissue exposure responses. Future studies may utilize this characterized product for longer-term interventions to determine efficacy for slowing disease development or progression in high-risk individuals or progression in patients with low-risk prostate cancer on active surveillance. Acknowledgments We thank Caryn Fasko, Anna Maria Bittoni, Ashley Schmitz, and Christina Simpson for assistance in editing and entering patient compliance, 3-d diet records, and anthropometric data, and Jennifer Ahn-Jarvis for analyzing the juice isoflavone content. The authors’ responsibilities were as follows—SKC, SJS, and EMG: designed the study; SKC, SJS, KMR, REK, EMG, and NEM: formulated the research questions; SKC, RRB and RA: recruited the subjects; DMF: developed, grew, and harvested the FG99-218 tomatoes; SKC, EMG, and RA: carried out the clinical study; EMG, REK, LW, and KMR: analyzed the samples; GSY, EMG, KMR, NEM, JT-A, SJS, and SKC: analyzed the data; EMG, NEM, and SKC: drafted the manuscript; and all authors: read and approved the final manuscript. Notes Supported by the National Cancer Institute of the NIH by R01CA112632 (principal investigator: SKC). Additional resources have been provided through the National Cancer Institute P30CA01605-supported Molecular Carcinogenesis and Chemoprevention Program and the Nutrient and Phytochemical Analytic Shared Resource and the National Center for Advancing Translational Sciences by the award UL1TR001070-supported Clinical Research Center. NEM was funded by the National Center for Complementary and Integrative Health and Office of Dietary Supplements under award K99/R00 AT008576 and by the USDA–Agricultural Research Service under CRIS 3092-51000-056-03S. Additional support was provided by The Ohio State University's Food Innovation Center, The Center for Advancement of Functional Foods Research and Entrepreneurship, and The Ohio State University James Development funds [Prostate Cancer Prevention and Treatment Fund (302024), Bionutrition and Cancer Prevention Fund (310684), and Hammond Cancer Research Fund (262914)]. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH or the USDA. Author disclosures: EMG, NEM, DMF, SJS, LW, JT-A, REK, KMR, GSY, RA, RRB, and SKC, no conflicts of interest. Supplemental Data 1 and 2 are available from the “Supplementary data” link in the online posting of the article and from the same link in the online table of contents at https://academic.oup.com/jn/. Present address for REK: Department of Human Sciences, 1787 Neil Avenue, 067 Campbell Hall, Columbus, OH 43210. Present address for RA: OhioHealth Robotic Urologic Surgeons, Dublin Methodist Hospital, 7450 Hospital Drive, Suite 300, Dublin, OH 43016. Abbreviations used: dhDaidzein, dihydrodaidzein; dhGenistein, dihydrogenistein; ODMA, O-desmethylangolensin; PSA, prostate-specific antigen; 6-OH-ODMA, 6-hydroxy-O-desmethylangolensin. References 1. Grainger E , Clinton SK , Giovannucci E . Diet and nutrition in the etiology and prevention of cancer . In: Bast RC , Croce CM , Hait WN , Hong WK , Kufe DW , Piccart-Gebhart M , Pollock R , Weichselbaum RR , Want H , Holland JF , editors. Cancer medicine . Hoboken (NJ) : John Wiley and Sons, Inc. ; 2017 : 415 – 31 . 2. Giovannucci E , Ascherio A , Rimm EB , Stampfer MJ , Colditz GA , Willett WC . Intake of carotenoids and retinol in relation to risk of prostate cancer . J Natl Cancer Inst 1995 ; 87 ( 23 ): 1767 – 76 . Google Scholar Crossref Search ADS PubMed 3. Graff RE , Pettersson A , Lis RT , Ahearn TU , Markt SC , Wilson KM , Rider JR , Fiorentino M , Finn S , Kenfield SA et al. Dietary lycopene intake and risk of prostate cancer defined by ERG protein expression . Am J Clin Nutr 2016 ; 103 ( 3 ): 851 – 60 . Google Scholar Crossref Search ADS PubMed 4. Zu K , Mucci L , Rosner BA , Clinton SK , Loda M , Stampfer MJ , Giovannucci E . Dietary lycopene, angiogenesis, and prostate cancer: a prospective study in the prostate-specific antigen era . J Natl Cancer Inst 2014 ; 106 ( 2 ): djt430 . Google Scholar Crossref Search ADS PubMed 5. Key TJ , Appleby PN , Travis RC , Albanes D , Alberg AJ , Barricarte A , Black A , Boeing H , Bueno-de-Mesquita HB , Chan JM et al. Carotenoids, retinol, tocopherols, and prostate cancer risk: pooled analysis of 15 studies . Am J Clin Nutr 2015 ; 102 ( 5 ): 1142 – 57 . Google Scholar Crossref Search ADS PubMed 6. World Cancer Research Fund/American Institute for Cancer Research . Food, nutrition, physical activity and the prevention of cancer: a global perspective . Washington (DC) : American Institute for Cancer Research; 2007 . 7. World Cancer Research Fund International/American Institute for Cancer Research . Diet, nutrition, physical activity, and prostate cancer. Continuous Update Project Expert Report . 20 14 . 8. Cohen JH , Kristal AR , Stanford JL . Fruit and vegetable intakes and prostate cancer risk . J Natl Cancer Inst 2000 ; 92 ( 1 ): 61 – 8 . Google Scholar Crossref Search ADS PubMed 9. Giovannucci E . Does prostate-specific antigen screening influence the results of studies of tomatoes, lycopene, and prostate cancer risk? J Natl Cancer Inst 2007 ; 99 ( 14 ): 1060 – 2 . Google Scholar Crossref Search ADS PubMed 10. International Agency for Research on Cancer; WHO; International Association of Cancer Registries . Cancer incidence in five continents . Lyon (France) : IARC Press ; 2005 . 11. Miyanaga N , Akaza H , Hinotsu S , Fujioka T , Naito S , Namiki M , Takahashi S , Hirao Y , Horie S , Tsukamoto T et al. Prostate cancer chemoprevention study: an investigative randomized control study using purified isoflavones in men with rising prostate-specific antigen . Cancer Sci 2012 ; 103 ( 1 ): 125 – 30 . Google Scholar Crossref Search ADS PubMed 12. Boileau TW , Liao Z , Kim S , Lemeshow S , Erdman JW Jr. , Clinton SK . Prostate carcinogenesis in N-methyl-N-nitrosourea (NMU)-testosterone-treated rats fed tomato powder, lycopene, or energy-restricted diets . J Natl Cancer Inst 2003 ; 95 ( 21 ): 1578 – 86 . Google Scholar Crossref Search ADS PubMed 13. Liu AG , Juvik JA , Jeffery EH , Berman-Booty LD , Clinton SK , Erdman JW Jr . Enhancement of broccoli indole glucosinolates by methyl jasmonate treatment and effects on prostate carcinogenesis . J Med Food 2014 ; 17 ( 11 ): 1177 – 82 . Google Scholar Crossref Search ADS PubMed 14. Tan HL , Thomas-Ahner JM , Moran NE , Cooperstone JL , Erdman JW Jr. , Young GS , Clinton SK . beta-Carotene 9′,10′ oxygenase modulates the anticancer activity of dietary tomato or lycopene on prostate carcinogenesis in the TRAMP model . Cancer Prev Res 2017 ; 10 ( 2 ): 161 – 9 . Google Scholar Crossref Search ADS 15. Zhou JR , Gugger ET , Tanaka T , Guo Y , Blackburn GL , Clinton SK . Soybean phytochemicals inhibit the growth of transplantable human prostate carcinoma and tumor angiogenesis in mice . J Nutr 1999 ; 129 ( 9 ): 1628 – 35 . Google Scholar Crossref Search ADS PubMed 16. Mein JR , Lian F , Wang XD . Biological activity of lycopene metabolites: implications for cancer prevention . Nutr Rev 2008 ; 66 ( 12 ): 667 – 83 . Google Scholar Crossref Search ADS PubMed 17. Sies H , Stahl W . Lycopene: antioxidant and biological effects and its bioavailability in the human . Proc Soc Exp Biol Med 1998 ; 218 ( 2 ): 121 – 4 . Google Scholar Crossref Search ADS PubMed 18. Wan L , Tan HL , Thomas-Ahner JM , Pearl DK , Erdman JW Jr. , Moran NE , Clinton SK . Dietary tomato and lycopene impact androgen signaling- and carcinogenesis-related gene expression during early TRAMP prostate carcinogenesis . Cancer Prev Res 2014 ; 7 ( 12 ): 1228 – 39 . Google Scholar Crossref Search ADS 19. Lesinski GB , Reville PK , Mace TA , Young GS , Ahn-Jarvis J , Thomas-Ahner J , Vodovotz Y , Ameen Z , Grainger E , Riedl K et al. Consumption of soy isoflavone enriched bread in men with prostate cancer is associated with reduced proinflammatory cytokines and immunosuppressive cells . Cancer Prev Res 2015 ; 8 ( 11 ): 1036 – 44 . Google Scholar Crossref Search ADS 20. Singh AV , Franke AA , Blackburn GL , Zhou JR . Soy phytochemicals prevent orthotopic growth and metastasis of bladder cancer in mice by alterations of cancer cell proliferation and apoptosis and tumor angiogenesis . Cancer Res 2006 ; 66 ( 3 ): 1851 – 8 . Google Scholar Crossref Search ADS PubMed 21. Wang S , DeGroff VL , Clinton SK . Tomato and soy polyphenols reduce insulin-like growth factor-I-stimulated rat prostate cancer cell proliferation and apoptotic resistance in vitro via inhibition of intracellular signaling pathways involving tyrosine kinase . J Nutr 2003 ; 133 ( 7 ): 2367 – 76 . Google Scholar Crossref Search ADS PubMed 22. Sargeant AM , Klein RD , Rengel RC , Clinton SK , Kulp SK , Kashida Y , Yamaguchi M , Wang X , Chen CS . Chemopreventive and bioenergetic signaling effects of PDK1/Akt pathway inhibition in a transgenic mouse model of prostate cancer . Toxicol Pathol 2007 ; 35 ( 4 ): 549 – 61 . Google Scholar Crossref Search ADS PubMed 23. Zhou JR , Yu L , Zhong Y , Blackburn GL . Soy phytochemicals and tea bioactive components synergistically inhibit androgen-sensitive human prostate tumors in mice . J Nutr 2003 ; 133 ( 2 ): 516 – 21 . Google Scholar Crossref Search ADS PubMed 24. Ahn-Jarvis JH , Clinton SK , Grainger EM , Riedl KM , Schwartz SJ , Lee ML , Cruz-Cano R , Young GS , Lesinski GB , Vodovotz Y . Isoflavone pharmacokinetics and metabolism after consumption of a standardized soy and soy-almond bread in men with asymptomatic prostate cancer . Cancer Prev Res 2015 ; 8 ( 11 ): 1045 – 54 . Google Scholar Crossref Search ADS 25. Varinska L , Gal P , Mojzisova G , Mirossay L , Mojzis J . Soy and breast cancer: focus on angiogenesis . Int J Mol Sci 2015 ; 16 ( 5 ): 11728 – 49 . Google Scholar Crossref Search ADS PubMed 26. Karsli-Ceppioglu S , Ngollo M , Adjakly M , Dagdemir A , Judes G , Lebert A , Boiteux JP , Penault LF , Bignon YJ , Guy L et al. Genome-wide DNA methylation modified by soy phytoestrogens: role for epigenetic therapeutics in prostate cancer? Omics 2015 ; 19 ( 4 ): 209 – 19 . Google Scholar Crossref Search ADS PubMed 27. DeVita VT , Hellman S , Rosenberg SA . Cancer, principles & practice of oncology . Philadelphia : Lippincott, Williams, & Wilkins ; 2005 . 28. Hong WK ; American Association for Cancer Research . Holland Frei cancer medicine 8 . Shelton (CT) : People's Medical Pub. House ; 2010 . 29. Canene-Adams K , Lindshield BL , Wang S , Jeffery EH , Clinton SK , Erdman JW Jr . Combinations of tomato and broccoli enhance antitumor activity in Dunning r3327-h prostate adenocarcinomas . Cancer Res 2007 ; 67 ( 2 ): 836 – 43 . Google Scholar Crossref Search ADS PubMed 30. Zuniga KE , Clinton SK , Erdman JW Jr . The interactions of dietary tomato powder and soy germ on prostate carcinogenesis in the TRAMP model . Cancer Prev Res 2013 ; 6 ( 6 ): 548 – 57 . Google Scholar Crossref Search ADS 31. Grainger EM , Schwartz SJ , Wang S , Unlu NZ , Boileau TW , Ferketich AK , Monk JP , Gong MC , Bahnson RR , DeGroff VL et al. A combination of tomato and soy products for men with recurring prostate cancer and rising prostate specific antigen . Nutr Cancer 2008 ; 60 ( 2 ): 145 – 54 . Google Scholar Crossref Search ADS PubMed 32. Bohn T , Blackwood M , Francis D , Tian Q , Schwartz SJ , Clinton SK . Bioavailability of phytochemical constituents from a novel soy fortified lycopene rich tomato juice developed for targeted cancer prevention trials . Nutr Cancer 2013 ; 65 ( 6 ): 919 – 29 . Google Scholar Crossref Search ADS PubMed 33. Giovannucci E , Rimm EB , Liu Y , Stampfer MJ , Willett WC . A prospective study of tomato products, lycopene, and prostate cancer risk . J Natl Cancer Inst 2002 ; 94 ( 5 ): 391 – 8 . Google Scholar Crossref Search ADS PubMed 34. Shu XO , Zheng Y , Cai H , Gu K , Chen Z , Zheng W , Lu W . Soy food intake and breast cancer survival . JAMA 2009 ; 302 ( 22 ): 2437 – 43 . Google Scholar Crossref Search ADS PubMed 35. Kopec RE , Riedl KM , Harrison EH , Curley RW Jr. , Hruszkewycz DP , Clinton SK , Schwartz SJ . Identification and quantification of apo-lycopenals in fruits, vegetables, and human plasma . J Agric Food Chem 2010 ; 58 ( 6 ): 3290 – 6 . Google Scholar Crossref Search ADS PubMed 36. Xu SY , Shoemaker CF , Luh BS . Effect of break temperature on rheological properties and microstructure of tomato juices and pastes . J Food Sci 1986 ; 51 ( 2 ): 399 . Google Scholar Crossref Search ADS 37. Barona J , Jones JJ , Kopec RE , Comperatore M , Andersen C , Schwartz SJ , Lerman RH , Fernandez ML . A Mediterranean-style low-glycemic-load diet increases plasma carotenoids and decreases LDL oxidation in women with metabolic syndrome . J Nutr Biochem 2012 ; 23 ( 6 ): 609 – 15 . Google Scholar Crossref Search ADS PubMed 38. Cooperstone JL , Ralston RA , Riedl KM , Haufe TC , Schweiggert RM , King SA , Timmers CD , Francis DM , Lesinski GB , Clinton SK et al. Enhanced bioavailability of lycopene when consumed as cis-isomers from tangerine compared to red tomato juice, a randomized, cross-over clinical trial . Mol Nutr Food Res 2015 ; 59 ( 4 ): 658 – 69 . Google Scholar Crossref Search ADS PubMed 39. Zhang D , Fan C , Zhang J , Zhang CH . Nonparametric methods for measurements below detection limit . Stat Med 2009 ; 28 ( 4 ): 700 – 15 . Google Scholar Crossref Search ADS PubMed 40. Mickey RM , Greenland S . The impact of confounder selection criteria on effect estimation . Am J Epidemiol 1989 ; 129 ( 1 ): 125 – 37 . Google Scholar Crossref Search ADS PubMed 41. Anupongsanugool E , Teekachunhatean S , Rojanasthien N , Pongsatha S , Sangdee C . Pharmacokinetics of isoflavones, daidzein and genistein, after ingestion of soy beverage compared with soy extract capsules in postmenopausal Thai women . BMC Clin Pharmacol 2005 ; 5 : 2 . Google Scholar Crossref Search ADS PubMed 42. Moran NE , Cichon MJ , Riedl KM , Grainger EM , Schwartz SJ , Novotny JA , Erdman JW Jr. , Clinton SK . Compartmental and noncompartmental modeling of 13C-lycopene absorption, isomerization, and distribution kinetics in healthy adults . Am J Clin Nutr 2015 ; 102 ( 6 ): 1436 – 49 . Google Scholar Crossref Search ADS PubMed 43. Fisher B , Redmond C , Brown A , Wolmark N , Wittliff J , Fisher ER , Plotkin D , Bowman D , Sachs S , Wolter J et al. Treatment of primary breast cancer with chemotherapy and tamoxifen . N Engl J Med 1981 ; 305 ( 1 ): 1 – 6 . Google Scholar Crossref Search ADS PubMed 44. Albanes D , Heinonen OP , Taylor PR , Virtamo J , Edwards BK , Rautalahti M , Hartman AM , Palmgren J , Freedman LS , Haapakoski J et al. Alpha-Tocopherol and beta-carotene supplements and lung cancer incidence in the alpha-tocopherol, beta-carotene cancer prevention study: effects of base-line characteristics and study compliance . J Natl Cancer Inst 1996 ; 88 ( 21 ): 1560 – 70 . Google Scholar Crossref Search ADS PubMed 45. Klein EA , Thompson IM Jr. , Tangen CM , Crowley JJ , Lucia MS , Goodman PJ , Minasian LM , Ford LG , Parnes HL , Gaziano JM et al. Vitamin E and the risk of prostate cancer: the Selenium and Vitamin E Cancer Prevention Trial (SELECT) . JAMA 2011 ; 306 ( 14 ): 1549 – 56 . Google Scholar Crossref Search ADS PubMed 46. Lazarevic B , Hammarstrom C , Yang J , Ramberg H , Diep LM , Karlsen SJ , Kucuk O , Saatcioglu F , Tasken KA , Svindland A . The effects of short-term genistein intervention on prostate biomarker expression in patients with localised prostate cancer before radical prostatectomy . Br J Nutr 2012 ; 108 ( 12 ): 2138 – 47 . Google Scholar Crossref Search ADS PubMed 47. Pierce JP , Faerber S , Wright FA , Rock CL , Newman V , Flatt SW , Kealey S , Jones VE , Caan BJ , Gold EB et al. A randomized trial of the effect of a plant-based dietary pattern on additional breast cancer events and survival: the Women's Healthy Eating and Living (WHEL) Study . Control Clin Trials 2002 ; 23 ( 6 ): 728 – 56 . Google Scholar Crossref Search ADS PubMed 48. Grainger EM , Hadley CW , Moran NE , Riedl KM , Gong MC , Pohar K , Schwartz SJ , Clinton SK . A comparison of plasma and prostate lycopene in response to typical servings of tomato soup, sauce or juice in men before prostatectomy . Br J Nutr 2015 ; 114 ( 4 ): 1 – 12 . Google Scholar Crossref Search ADS PubMed 49. Williams AW , Boileau TW , Erdman JW Jr . Factors influencing the uptake and absorption of carotenoids . Proc Soc Exp Biol Med 1998 ; 218 ( 2 ): 106 – 8 . Google Scholar Crossref Search ADS PubMed 50. Paur I , Lilleby W , Bohn SK , Hulander E , Klein W , Vlatkovic L , Axcrona K , Bolstad N , Bjoro T , Laake P et al. Tomato-based randomized controlled trial in prostate cancer patients: effect on PSA . Clin Nutr 2017 ; 36 ( 3 ): 672 – 9 . Google Scholar Crossref Search ADS PubMed 51. Atkinson AJ Jr. , Huang S-M , Lertora JJL , Markey SP . Principles of clinical pharmacology . 3 rd ed . San Diego (CA) : Elsevier ; 2012 . 52. Zuniga KE , Erdman JW Jr . Combined consumption of soy germ and tomato powders results in altered isoflavone and carotenoid bioavailability in rats . J Agric Food Chem 2011 ; 59 ( 10 ): 5335 – 41 . Google Scholar Crossref Search ADS PubMed 53. Moran NE , Clinton SK , Erdman JW Jr . Differential bioavailability, clearance, and tissue distribution of the acyclic tomato carotenoids lycopene and phytoene in Mongolian gerbils . J Nutr 2013 ; 143 ( 12 ): 1920 – 6 . Google Scholar Crossref Search ADS PubMed 54. Clinton SK , Emenhiser C , Schwartz SJ , Bostwick DG , Williams AW , Moore BJ , Erdman JWJ . cis-trans Lycopene isomers, carotenoids, and retinol in the human prostate . Cancer Epidemiol Biomarkers Prev 1996 ; 5 ( 10 ): 823 – 33 . Google Scholar PubMed 55. Conlon LE , King RD , Moran NE , Erdman JW Jr . Coconut oil enhances tomato carotenoid tissue accumulation compared to safflower oil in the Mongolian gerbil (Meriones unguiculatus) . J Agric Food Chem 2012 ; 60 ( 34 ): 8386 – 94 . Google Scholar Crossref Search ADS PubMed 56. Campbell JK , Engelmann NJ , Lila MA , Erdman JW Jr . Phytoene, phytofluene, and lycopene from tomato powder differentially accumulate in tissues of male Fisher 344 rats . Nutr Res 2007 ; 27 ( 12 ): 794 – 801 . Google Scholar Crossref Search ADS PubMed 57. Moran NE , Novotny JA , Cichon MJ , Riedl KM , Rogers RB , Grainger EM , Schwartz SJ , Erdman JW Jr. , Clinton SK . Absorption and distribution kinetics of the 13C-labeled tomato carotenoid phytoene in healthy adults . J Nutr 2015 ; 146 ( 2 ): 368 – 76 . Google Scholar Crossref Search ADS PubMed 58. Morimoto Y , Beckford F , Franke AA , Maskarinec G . Urinary isoflavonoid excretion as a biomarker of dietary soy intake during two randomized soy trials . Asia Pac J Clin Nutr 2014 ; 23 ( 2 ): 205 – 9 . Google Scholar PubMed 59. Gardner CD , Oelrich B , Liu JP , Feldman D , Franke AA , Brooks JD . Prostatic soy isoflavone concentrations exceed serum levels after dietary supplementation . Prostate 2009 ; 69 ( 7 ): 719 – 26 . Google Scholar Crossref Search ADS PubMed 60. Rannikko A , Petas A , Raivio T , Janne OA , Rannikko S , Adlercreutz H . The effects of short-term oral phytoestrogen supplementation on the hypothalamic-pituitary-testicular axis in prostate cancer patients . Prostate 2006 ; 66 ( 10 ): 1086 – 91 . Google Scholar Crossref Search ADS PubMed 61. Guy L , Vedrine N , Urpi-Sarda M , Gil-Izquierdo A , Al-Maharik N , Boiteux JP , Scalbert A , Remesy C , Botting NP , Manach C . Orally administered isoflavones are present as glucuronides in the human prostate . Nutr Cancer 2008 ; 60 ( 4 ): 461 – 8 . Google Scholar Crossref Search ADS PubMed 62. Bowen P , Chen L , Stacewicz-Sapuntzakis M , Duncan C , Sharifi R , Ghosh L , Kim HS , Christov-Tzelkov K , van Breemen R . Tomato sauce supplementation and prostate cancer: lycopene accumulation and modulation of biomarkers of carcinogenesis . Exp Biol Med 2002 ; 227 ( 10 ): 886 – 93 . Google Scholar Crossref Search ADS © 2018 American Society for Nutrition. This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Journal

Journal of NutritionOxford University Press

Published: Jan 1, 2019

References

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