**AWARDS ISSUED PRIOR TO JANUARY 20, 2025, WERE FUNDED UNDER PREVIOUS ADMINISTRATIONS AND MAY NOT REFLECT THE PRIORITIES AND POLICIES OF THE CURRENT ADMINISTRATION.** CANCER IS THE 2ND-LEADING CAUSE OF DEATH IN THE US, AND DIETARY FACTORS ARE IMPORTANT CANCER RISK FACTORS. CRUCIFEROUS VEGETABLES (CRUCIFERS) AND THEIR CONSTITUENT BIOLOGICALLY ACTIVE FOOD COMPONENTS, INCLUDING INDOLES AND ISOTHIOCYANATES (ITCS) SUCH AS SULFORAPHANE (SFN), APPEAR TO MODULATE CHRONIC DISEASE STATES INCLUDING CANCER. OVERALL THERE IS STRONG SUPPORT FOR A PREVENTIVE AND/OR MITIGATING IMPACT OF CRUCIFER-DERIVED ITCS AND INDOLES ON CANCER IN ANIMAL MODELS, BUT OBSERVATIONAL DATA TO DATE IN HUMANS ARE INCONSISTENT, AND THE IMPACTS OF CRUCIFEROUS VEGETABLE CONSUMPTION ON BREAST AND PROSTATE CANCER RISK REMAIN CONTROVERSIAL, AS RESULTS AND CONCLUSIONS FROM VARIOUS STUDIES DIFFER. SUCH DISCREPANCIES ARISE IN PART OWING TO METHODOLOGICAL LIMITATIONS OF ACCURATELY ASSESSING DIETARY EXPOSURES, WHICH THUS CONSTITUTE A MAJOR BARRIER IN THE FIELD. DESPITE WIDESPREAD USE, CLASSICAL DIETARY-INTAKE INSTRUMENTS INCLUDING FOOD FREQUENCY QUESTIONNAIRES (FFQ) AND OTHER DIETARY RECALL METHODS ARE SUBJECT TO WELL-KNOWN LIMITATIONS. THIS IS COUPLED WITH INADEQUATE UNDERSTANDING OF THE BIOAVAILABILITY AND ACTIVE METABOLITES OF DIETARY COMPOUNDS. TO CIRCUMVENT AND ADDRESS THESE ISSUES, BASED ON OUR PROMISING PRELIMINARY FINDINGS AND NOVEL TECHNOLOGIES, WE SEEK TO IDENTIFY UNIQUE AND QUANTIFIABLE BIOLOGICAL SIGNATURES OF CONSUMPTION OF SPECIFIC FOODS. FOOD TYPE-SPECIFIC CANDIDATE PHYTOCHEMICALS THAT CAN BE MEASURED IN PLASMA OR URINE AS BIOMARKERS OF INTAKE HAVE BEEN IDENTIFIED (E.G. ALKYLRESORCINOL LEVELS FOR WHOLE GRAINS), BUT KEY BARRIERS PERSIST TO IDENTIFYING FOOD-SPECIFIC CANDIDATE SINGLE BIOMARKERS, SUCH AS: 1) RARELY ARE SUCH SINGLE COMPOUNDS EXCLUSIVELY SPECIFIC FOR A SINGLE FOOD; AND 2) MANY SUCH PHYTOCHEMICALS ARE SHORT-LIVED AND RAPIDLY CLEARED FROM THE BODY. FOR EXAMPLE, THE BIOACTIVE ITC SFN, DERIVED FROM THE CRUCIFEROUS VEGETABLE BROCCOLI, IS CLEARED WITHIN 12-24 HR FROM THE CIRCULATION. THUS, DEPENDING ON THE TIMING OF SAMPLE COLLECTION, MEASURES OF SFN MAY OR MAY NOT REFLECT HABITUAL BROCCOLI INTAKE, MAKING IT A PROBLEMATIC BIOMARKER OF INTAKE. THIS EXAMPLE DEMONSTRATES THAT NOVEL APPROACHES ARE CRITICALLY NEEDED TO CHARACTERIZE INDIVIDUALS' INTAKES OF SPECIFIC FOODS TO BETTER UNDERSTAND THE ASSOCIATION OF FOODS WITH DISEASE RISK AND IDENTIFY AT-RISK POPULATIONS. TO ADDRESS THIS NEED, WE WILL USE A METABOLOMICS-BASED APPROACH, WHICH CAN IDENTIFY COMBINATIONS OF FOOD-DERIVED METABOLITES CAPABLE OF PROVIDING MORE ACCURATE, FOOD-SPECIFIC MEASURES OF CONSUMPTION, AND COMBINE IT WITH DATA-DRIVEN PREDICTION MODELS AND NOVEL MACHINE-LEARNING ALGORITHMS. ADVANTAGES OF THIS DATA-DRIVEN APPROACH IN BIOLOGICAL FLUIDS INCLUDE A) REMOVING RECALL BIAS; B) ACCOUNTING FOR INDIVIDUAL AND FOOD COMPOSITION VARIABLES THAT IMPACT BIOAVAILABILITY, C) ACCOUNTING FOR POSSIBLE FOOD INTERACTIONS, AND D) ACCOUNTING FOR METABOLISM DIFFERENCES, INCLUDING MICROBIAL BREAKDOWN PRODUCTS; AND E) PROVIDING A MORE PRECISE AND ACCURATE ASSESSMENT OF FOOD INTAKE. THE OVERAL,L GOAL OF THE PROPOSED STUDY IS TO IDENTIFY RELIABLE AND ACCURATE MOLECULAR BIOLOGICAL SIGNATURES OF CRUCIFEROUS VEGETABLE INTAKE. OUR CENTRAL HYPOTHESIS IS THAT BY COMBINING METABOLOMICS WITH DATA- AND MACHINE LEARNING-BASED PREDICTION MODELS, WE CAN IDENTIFY METABOLIC SIGNATURES THAT ESTIMATE INTAKE OF BROCCOLI AND RELATE THEIR UNBIASED MEASURE OF DIETARY INTAKE MORE MEANINGFULLY THAN DO FOOD INTAKE QUESTIONNAIRES. WE WILL UTILIZE DEUTERIUM-LABELED BROCCOLI SPROUTS IN COMBINATION WITH MASS SPECTROMETRY (MS)-BASED METABOLOMICS APPROACHES TO QUANTIFY AND DIFFERENTIATE BETWEEN BROCCOLI-SPECIFIC METABOLITES AND THEIR INTERACTIONS WITH THEIR MOLECULAR TARGETS OF ACTION. TO DEMONSTRATE THE FEASIBILITY OF OUR APPROACH, WE WILL PRESENT DATA SHOWING THAT 1) OUR LABELLED BROCCOLI SPROUTS CONTAIN DEUTERIUM-LABELED GLUCOSINOLATES (PRECURSORS OF ITCS) AND OTHER LABELED PHYTOCHEMICALS REPRESENTING MAJOR BIOSYNTHETIC PATHWAYS THAT ARE STABLE, 2) WE HAVE IDENTIFIED MICROBIAL-DERIVED METABOLITES IN HUMAN STOOL SAMPLES FOLLOWING INCUBATION OF BROCCOLI DIGESTS AND IDENTIFIED MICROBIAL DERIVED ITCS (SFN-NITRILE AND ERUCIN-NITRILE) IN PLASMA FROM HUMANS WHO CONSUMED BROCCOLI SPROUTS, 3) THAT THESE MICROBIAL METABOLITES ARE INDICATIVE OF BROCCOLI INTAKE AS LATE AS 12-48 H POST-CONSUMPTION AND 4) NOVEL MACHINE LEARNING APPROACHES OFFER ADVANTAGES OVER TRADITIONAL MULTIVARIATE METHODS.