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.

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.

Azithromycin sorption and biodegradation in a simulated California river system.

Azithromycin (AZ) is a widely-used macrolide antibiotic that is continually deposited into natural waterways by sewage effluent. Though recognized as an emerging contaminant of concern, little is known about its fate and transport in aquatic systems. American River soils and water were used to determine degradation of AZ in microcosms simulating flooded (anaerobic) and non-flooded (aerobic) California watershed conditions. Under aerobic conditions the degradation rate constant (k=0.0084 ± 0.0039 day-1) and DT50 (82.52 ± 56.54 days) were calculated, as AZ disappearance indicated potential degradation. However, based on concurrent product appearance, less than one percent of the parent degraded over 150 days. Throughout the experiment microbial growth was observed by culturing in tryptic soy broth despite antibiotic addition and soil being autoclaved. Sorption likely contributes to AZ recalcitrance, thus the soil-water partition coefficient (log Kd = 2.18 Lkg-1), Freundlich sorption and desorption coefficients (log Kf = 1.90 ± 0.14 and log Kfd = 2.51 ± 0.30, respectively), and organic-carbon-normalized distribution coefficient (log Koc = 4.25 Lkg-1) were also calculated. Based on these results, AZ degradation in aquatic systems will likely be very limited and transport will fluctuate based on the extent of soil-water saturation or bulk movement of sediment.