Benzo[a]pyrene toxicokinetics in humans following dietary supplementation with 3,3'-diindolylmethane (DIM) or Brussels sprouts.

Utilizing the atto-zeptomole sensitivity of UPLC-accelerator mass spectrometry (UPLC-AMS), we previously demonstrated significant first-pass metabolism following escalating (25-250 ng) oral micro-dosing in humans of [14C]-benzo[a]pyrene ([14C]-BaP). The present study examines the potential for supplementation with Brussels sprouts (BS) or 3,3'-diindolylmethane (DIM) to alter plasma levels of [14C]-BaP and metabolites over a 48-h period following micro-dosing with 50 ng (5.4 nCi) [14C]-BaP. Volunteers were dosed with [14C]-BaP following fourteen days on a cruciferous vegetable restricted diet, or the same diet supplemented for seven days with 50 g of BS or 300 mg of BR-DIM® prior to dosing. BS or DIM reduced total [14C] recovered from plasma by 56-67% relative to non-intervention. Dietary supplementation with DIM markedly increased Tmax and reduced Cmax for [14C]-BaP indicative of slower absorption. Both dietary treatments significantly reduced Cmax values of four downstream BaP metabolites, consistent with delaying BaP absorption. Dietary treatments also appeared to reduce the T1/2 and the plasma AUC(0,∞) for Unknown Metabolite C, indicating some effect in accelerating clearance of this metabolite. Toxicokinetic constants for other metabolites followed the pattern for [14C]-BaP (metabolite profiles remained relatively consistent) and non-compartmental analysis did not indicate other significant alterations. Significant amounts of metabolites in plasma were at the bay region of [14C]-BaP irrespective of treatment. Although the number of subjects and large interindividual variation are limitations of this study, it represents the first human trial showing dietary intervention altering toxicokinetics of a defined dose of a known human carcinogen.

3,3'-Diindolylmethane Exhibits Significant Metabolism after Oral Dosing in Humans.

3,3'-Diindolylmethane (DIM), a major phytochemical derived from ingestion of cruciferous vegetables, is also a dietary supplement. In preclinical models, DIM is an effective cancer chemopreventive agent and has been studied in a number of clinical trials. Previous pharmacokinetic studies in preclinical and clinical models have not reported DIM metabolites in plasma or urine after oral dosing, and the pharmacological actions of DIM on target tissues is assumed to be solely via the parent compound. Seven subjects (6 males and 1 female) ranging from 26-65 years of age, on a cruciferous vegetable-restricted diet prior to and during the study, took 2 BioResponse DIM 150-mg capsules (45.3 mg DIM/capsule) every evening for one week with a final dose the morning of the first blood draw. A complete time course was performed with plasma and urine collected over 48 hours and analyzed by UPLC-MS/MS. In addition to parent DIM, two monohydroxylated metabolites and 1 dihydroxylated metabolite, along with their sulfate and glucuronide conjugates, were present in both plasma and urine. Results reported here are indicative of significant phase 1 and phase 2 metabolism and differ from previous pharmacokinetic studies in rodents and humans, which reported only parent DIM present after oral administration. 3-((1H-indole-3-yl)methyl)indolin-2-one, identified as one of the monohydroxylated products, exhibited greater potency and efficacy as an aryl hydrocarbon receptor agonist when tested in a xenobiotic response element-luciferase reporter assay using Hepa1 cells. In addition to competitive phytochemical-drug adverse reactions, additional metabolites may exhibit pharmacological activity highlighting the importance of further characterization of DIM metabolism in humans. SIGNIFICANCE STATEMENT: 3,3'-Diindolylmethane (DIM), derived from indole-3-carbinol in cruciferous vegetables, is an effective cancer chemopreventive agent in preclinical models and a popular dietary supplement currently in clinical trials. Pharmacokinetic studies to date have found little or no metabolites of DIM in plasma or urine. In marked contrast, we demonstrate rapid appearance of mono- and dihydroxylated metabolites in human plasma and urine as well as their sulfate and glucuronide conjugates. The 3-((1H-indole-3-yl)methyl)indolin-2-one metabolite exhibited significant aryl hydrocarbon receptor agonist activity, emphasizing the need for further characterization of the pharmacological properties of DIM metabolites.

Toxicokinetics of benzo[a]pyrene in humans: Extensive metabolism as determined by UPLC-accelerator mass spectrometry following oral micro-dosing.

Benzo[a]pyrene (BaP), is a known human carcinogen (International Agency for Research on Cancer (IARC) class 1). The remarkable sensitivity (zepto-attomole 14C in biological samples) of accelerator mass spectrometry (AMS) makes possible, with de minimus risk, pharmacokinetic (PK) analysis following [14C]-BaP micro-dosing of humans. A 46 ng (5 nCi) dose was given thrice to 5 volunteers with minimum 2 weeks between dosing and plasma collected over 72 h. [14C]-BaPeq PK analysis gave plasma Tmax and Cmax values of 1.25 h and 29-82 fg/mL, respectively. PK parameters were assessed by non- compartment and compartment models. Intervals between dosing ranged from 20 to 420 days and had little impact on intra-individual variation. DNA, extracted from peripheral blood mononuclear cells (PBMCs) of 4 volunteers, showed measurable levels (LOD ~ 0.5 adducts/1011 nucleotides) in two individuals 2-3 h post-dose, approximately three orders of magnitude lower than smokers or occupationally-exposed individuals. Little or no DNA binding was detectable at 48-72 h. In volunteers the allelic variants CYP1B1*1/*⁎1, *1/*3 or *3/*3 and GSTM1*0/0 or *1 had no impact on [14C]-BaPeq PK or DNA adduction with this very limited sample. Plasma metabolites over 72 h from two individuals (one CYP1B1*1/*1 and one CYP1B1*3/*3) were analyzed by UPLC-AMS. In both individuals, parent [14C]-BaP was a minor constituent even at the earliest time points and metabolite profiles markedly distinct. AMS, coupled with UPLC, could be used in humans to enhance the accuracy of pharmacokinetics, toxicokinetics and risk assessment of environmental carcinogens.

Daily Consumption of Oregon Hazelnuts Affects α-Tocopherol Status in Healthy Older Adults: A Pre-Post Intervention Study.

Background: Inadequate vitamin E and magnesium intakes are of concern for older adults owing to the associated incidence of age-related diseases. Objective: This study was designed to determine the extent to which a 16-wk intervention with hazelnuts alters vitamin E and magnesium status in a group of older men and women, and used a pre-post intervention design without a control group to adjust for temporal changes. Methods: Participants (n = 32 including 22 women; mean ± SD age: 63 ± 6 y) consumed hazelnuts (∼57 g/d) for 16 wk. Blood and urine samples and anthropomorphic measures were taken at the start and end of the intervention to determine plasma concentrations of α-tocopherol and serum concentrations of magnesium, lipids, glucose, insulin, and high-sensitivity C-reactive protein along with urinary vitamin E metabolites; several other micronutrients were measured by a lymphocyte proliferation assay. There were 3 primary endpoints, calculated as the mean changes in measurements between baseline and the end of the 16-wk intervention for 1) plasma α-tocopherol, 2) urinary α-carboxyethyl hydroxychromanol (α-CEHC; an α-tocopherol metabolite), and 3) serum magnesium. Results: Hazelnut consumption increased concentrations of the urinary α-tocopherol metabolite α-CEHC (mean ± SD: 0.84 ± 0.45 to 1.14 ± 0.50 µmol/g creatinine; P = 0.0006). In addition, hazelnut consumption increased serum concentrations of magnesium (+2.1%, P = 0.05), decreased concentrations of fasting glucose (-3.4%, P = 0.03) and LDL cholesterol (-6.0%, P = 0.02), and decreased total:HDL cholesterol ratios (-4.5%, P = 0.009). No significant changes were observed in blood pressure, lymphocyte proliferation assays, and serum concentrations of insulin, high-sensitivity C-reactive protein, triglyceride, α-tocopherol, or HDL cholesterol. Conclusions: Consuming hazelnuts improves a biomarker of vitamin E status in older adults. Vitamin E is a shortfall micronutrient, as identified by the Dietary Guidelines for Americans 2015-2020, which frequently is consumed at levels less than the Estimated Average Requirement of 12 mg/d; thus, hazelnuts should be considered as part of a healthy dietary pattern. This trial was registered at clinicaltrials.gov as NCT03485989.

Multivitamin/Multimineral Supplementation Prevents or Reverses Decline in Vitamin Biomarkers and Cellular Energy Metabolism in Healthy Older Men: A Randomized, Double-Blind, Placebo-Controlled Study.

Despite the reported prevalence of micronutrient deficiencies in older adults, it is not yet established whether multivitamin/multimineral (MV/MM) supplements improve blood micronutrient status in individuals over the age of 65. Therefore, a cohort of 35 healthy men (>67 years) was recruited for an MV/MM supplementation trial. The primary endpoint was, as an indicator of micronutrient status, changes in blood micronutrient biomarkers from baseline to at least six months of supplementation with MV/MM or placebo. The secondary endpoint was basal O2 consumption in monocytes as an indicator of cellular metabolism. MV/MM supplementation improved blood concentrations of pyridoxal phosphate, calcifediol, α-tocopherol, and ß-carotene concentrations throughout the cohort. By contrast, those in the placebo group generally showed declines in blood vitamin concentrations and an increased prevalence of suboptimal vitamin status during the study period. On the other hand, MV/MM supplementation did not significantly affect blood mineral concentrations, i.e., calcium, copper, iron, magnesium, and zinc. Interestingly, MV/MM supplementation prevented the decline in monocyte O2 consumption rate. Overall, MV/MM use improves or prevents declines in vitamin, but not mineral, status and limits declines in cellular O2 consumption, which may have important implications for metabolism and immune health in healthy older men.

Impact of phenanthrene co-administration on the toxicokinetics of benzo[a]pyrene in humans. UPLC-accelerator mass spectrometry following oral microdosing.

Current risk assessments for environmental carcinogens rely on animal studies utilizing doses orders of magnitude higher than actual human exposures. Epidemiological studies of people with high exposures (e.g., occupational) are of value, but rely on uncertain exposure data. In addition, exposures are typically not to a single chemical but to mixtures, such as polycyclic aromatic hydrocarbons (PAHs). The extremely high sensitivity of accelerator mass spectrometry (AMS) allows for dosing humans with known carcinogens with de minimus risk. In this study UPLC-AMS was used to assess the toxicokinetics of [14C]-benzo[a]pyrene ([14C]-BaP) when dosed alone or in a binary mixture with phenanthrene (Phe). Plasma was collected for 48 h following a dose of [14C]-BaP (50 ng, 5.4 nCi) or the same dose of [14C]-BaP plus Phe (1250 ng). Following the binary mixture, Cmax of [14C]-BaP significantly decreased (4.4-fold) whereas the volume of distribution (Vd) increased (2-fold). Further, the toxicokinetics of twelve [14C]-BaP metabolites provided evidence of little change in the metabolite profile of [14C]-BaP and the pattern was overall reduction consistent with reduced absorption (decrease in Cmax). Although Phe was shown to be a competitive inhibitor of the major hepatic cytochrome P-450 (CYP) responsible for metabolism of [14C]-BaP, CYP1A2, the high inhibition constant (Ki) and lack of any increase in unmetabolized [14C]-BaP in plasma makes this mechanism unlikely to be responsible. Rather, co-administration of Phe reduces the absorption of [14C]-BaP through a mechanism yet to be determined. This is the first study to provide evidence that, at actual environmental levels of exposure, the toxicokinetics of [14C]-BaP in humans is markedly altered by the presence of a second PAH, Phe, a common component of environmental PAH mixtures.

Sulforaphane Bioavailability in Healthy Subjects Fed a Single Serving of Fresh Broccoli Microgreens.

Cruciferous vegetable consumption is associated with numerous health benefits attributed to the phytochemical sulforaphane (SFN) that exerts antioxidant and chemopreventive properties, among other bioactive compounds. Broccoli sprouts, rich in SFN precursor glucoraphanin (GRN), have been investigated in numerous clinical trials. Broccoli microgreens are similarly rich in GRN but have remained largely unexplored. The goal of this study was to examine SFN bioavailability and the microbiome profile in subjects fed a single serving of fresh broccoli microgreens. Eleven subjects participated in a broccoli microgreens feeding study. Broccoli microgreens GRN and SFN contents and stability were measured. Urine and stool SFN metabolite profiles and microbiome composition were examined. Broccoli microgreens had similar GRN content to values previously reported for broccoli sprouts, which was stable over time. Urine SFN metabolite profiles in broccoli microgreens-fed subjects were similar to those reported previously in broccoli sprouts-fed subjects, including the detection of SFN-nitriles. We also reported the detection of SFN metabolites in stool samples for the first time. A single serving of broccoli microgreens did not significantly alter microbiome composition. We showed in this study that broccoli microgreens are a significant source of SFN. Our work provides the foundation for future studies to establish the health benefits of broccoli microgreens consumption.

Benzo[a]pyrene (BaP) metabolites predominant in human plasma following escalating oral micro-dosing with [14C]-BaP.

Benzo[a]pyrene (BaP) is formed by incomplete combustion of organic materials (petroleum, coal, tobacco, etc.). BaP is designated by the International Agency for Research on Cancer as a group 1 known human carcinogen; a classification supported by numerous studies in preclinical models and epidemiology studies of exposed populations. Risk assessment relies on toxicokinetic and cancer studies in rodents at doses 5-6 orders of magnitude greater than average human uptake. Using a dose-response design at environmentally relevant concentrations, this study follows uptake, metabolism, and elimination of [14C]-BaP in human plasma by employing UPLC - accelerator mass spectrometry (UPLC-AMS). Volunteers were administered 25, 50, 100, and 250 ng (2.7-27 nCi) of [14C]-BaP (with interceding minimum 3-week washout periods) with quantification of parent [14C]-BaP and metabolites in plasma measured over 48 h. [14C]-BaP median Tmax was 30 min with Cmax and area under the curve (AUC) approximating dose-dependency. Marked inter-individual variability in plasma pharmacokinetics following a 250 ng dose was seen with 7 volunteers as measured by the Cmax (8.99 ± 7.08 ng × mL-1) and AUC0-48hr (68.6 ± 64.0 fg × hr-1 × mL-1). Approximately 3-6% of the [14C] recovered (AUC0-48 hr) was parent compound, demonstrating extensive metabolism following oral dosing. Metabolite profiles showed that, even at the earliest time-point (30 min), a substantial percentage of [14C] in plasma was polar BaP metabolites. The best fit modeling approach identified non-compartmental apparent volume of distribution of BaP as significantly increasing as a function of dose (p = 0.004). Bay region tetrols and dihydrodiols predominated, suggesting not only was there extensive first pass metabolism but also potentially bioactivation. AMS enables the study of environmental carcinogens in humans with de minimus risk, allowing for important testing and validation of physiologically based pharmacokinetic models derived from animal data, risk assessment, and the interpretation of data from high-risk occupationally exposed populations.

The Effect of a Multivitamin and Mineral Supplement on Immune Function in Healthy Older Adults: A Double-Blind, Randomized, Controlled Trial.

Older adults are at increased risk for vitamin and mineral deficiencies that contribute to age-related immune system decline. Several lines of evidence suggest that taking a multi-vitamin and mineral supplement (MVM) could improve immune function in individuals 55 and older. To test this hypothesis, we provided healthy older adults with either an MVM supplement formulated to improve immune function (Redoxon® VI, Singapore) or an identical, inactive placebo control to take daily for 12 weeks. Prior to and after treatment, we measured (1) their blood mineral and vitamin status (i.e., vitamin C, zinc and vitamin D); (2) immune function (i.e., whole blood bacterial killing activity, neutrophil phagocytic activity, and reactive oxygen species production); (3) immune status (salivary IgA and plasma cytokine/chemokine levels); and (4) self-reported health status. MVM supplementation improved vitamin C and zinc status in blood and self-reported health-status without altering measures of immune function or status or vitamin D levels, suggesting that healthy older adults may benefit from MVM supplementation. Further development of functional assays and larger study populations should improve detection of specific changes in immune function after supplementation in healthy older adults. Clinical Trials Registration: ClinicalTrials.gov #NCT02876315.

Pharmacokinetics of [14C]-Benzo[a]pyrene (BaP) in humans: Impact of Co-Administration of smoked salmon and BaP dietary restriction.

Benzo[a]pyrene (BaP), a polycyclic aromatic hydrocarbon (PAH), is a known human carcinogen. In non-smoking adults greater than 95% of BaP exposure is through diet. The carcinogenicity of BaP is utilized by the U.S. EPA to assess relative potency of complex PAH mixtures. PAH relative potency factors (RPFs, BaP = 1) are determined from high dose animal data. We employed accelerator mass spectrometry (AMS) to determine pharmacokinetics of [14C]-BaP in humans following dosing with 46 ng (an order of magnitude lower than human dietary daily exposure and million-fold lower than animal cancer models). To assess the impact of co-administration of food with a complex PAH mixture, humans were dosed with 46 ng of [14C]-BaP with or without smoked salmon. Subjects were asked to avoid high BaP-containing diets and a 3-day dietary questionnaire given to assess dietary exposure prior to dosing and three days post-dosing with [14C]-BaP. Co-administration of smoked salmon, containing a complex mixture of PAHs with an RPF of 460 ng BaPeq, reduced and delayed absorption. Administration of canned commercial salmon, containing very low amounts of PAHs, showed the impacts on pharmacokinetics were not due to high amounts of PAHs but rather a food matrix effect.

Perceptions of the Environment and Health Among Members of the Confederated Tribes of the Umatilla Indian Reservation.

Indigenous cultures perceive the natural environment as an essential link between traditional cultural practices, social connectedness, identity, and health. Many tribal communities face substantial health disparities related to exposure to environmental hazards. Our study used qualitative methods to better understand the Confederated Tribes of the Umatilla Indian Reservation (CTUIR) members' perspectives about their environment and its connections with their health including views on environmental health hazards. Three 90-minute focus group sessions with a total of 27 participants were held to elicit opinions on meanings of health and how the environment interacts with health. A systematic text analysis was used to derive themes across focus groups. Participants expressed a holistic view of health that included environmental, physical, mental, spiritual, and social components. A healthy natural environment was identified as an essential component of a healthy individual and a healthy community. Participants also described many environmental health concerns including second-hand smoke, outdoor smoke, diesel exhaust, mold, pesticides, contaminated natural foods, and toxic wastes from the Hanford nuclear site and methamphetamine labs. Many believe the identified environmental hazards contribute to diseases in their community. The natural environment is an important resource to CTUIR members and plays an integral role in achieving and maintaining health. Knowledge about the values and concerns of the community are useful to the tribal and federal governments, health professionals, environmental health practitioners, and community members who seek to achieve sustainable and healthy rural Native communities.