Human in Vivo Pharmacokinetics of [(14)C]Dibenzo[def,p]chrysene by Accelerator Mass Spectrometry Following Oral Microdosing

Abstract

Dibenzo(def,p)chrysene (DBC), (also known as dibenzo[a,l]pyrene), is a high molecular weight polycyclic aromatic hydrocarbon (PAH) found in the environment, including food, produced by the incomplete combustion of hydrocarbons. DBC, classified by IARC as a 2A probable human carcinogen, has a relative potency factor (RPF) in animal cancer models 30-fold higher than benzo[a]pyrene. No data are available describing the disposition of high molecular weight (>4 rings) PAHs in humans to compare to animal studies. Pharmacokinetics of DBC was determined in 3 female and 6 male human volunteers following oral microdosing (29 ng, 5 nCi) of [(14)C]-DBC. This study was made possible with highly sensitive accelerator mass spectrometry (AMS), capable of detecting [(14)C]-DBC equivalents in plasma and urine following a dose considered of de minimus risk to human health. Plasma and urine were collected over 72 h. The plasma Cmax was 68.8 ± 44.3 fg·mL(-1) with a Tmax of 2.25 ± 1.04 h. Elimination occurred in two distinct phases: a rapid (α)-phase, with a T1/2 of 5.8 ± 3.4 h and an apparent elimination rate constant (Kel) of 0.17 ± 0.12 fg·h(-1), followed by a slower (β)-phase, with a T1/2 of 41.3 ± 29.8 h and an apparent Kel of 0.03 ± 0.02 fg·h(-1). In spite of the high degree of hydrophobicity (log Kow of 7.4), DBC was eliminated rapidly in humans, as are most PAHs in animals, compared to other hydrophobic persistent organic pollutants such as, DDT, PCBs and TCDD. Preliminary examination utilizing a new UHPLC-AMS interface, suggests the presence of polar metabolites in plasma as early as 45 min following dosing. This is the first in vivo data set describing pharmacokinetics in humans of a high molecular weight PAH and should be a valuable addition to risk assessment paradigms.

Figure 1

Cumulative urinary elimination of [¹⁴C]-DBC_eq over time for six volunteers. All voided urine was collected over the 72 h of the study. A volumetric measurement was taken of urine pooled by time. A sample of 1 mL of urine was measured for [¹⁴C]-DBC_eq by AMS per pool, as described in Experimental Procedures. Volumetric corrections were made for total [¹⁴C]-DBC_eq elimination per pool. Results are recorded cumulatively.

Figure 2

[¹⁴C]-DBC_eq plasma elimination curve in 6 volunteers (A) and averaged (B). (A) Plasma levels of [¹⁴C]-DBC_eq over time are depicted for 6 individual volunteers as measured by AMS as described in Experimental Procedures. (B) This graph depicts the average and standard deviation of [¹⁴C]-DBC_eq in plasma for the same 6 volunteers as those in panel A.

Figure 3

UHPLC-AMS of [¹⁴C]-DBC and putative metabolites from plasma of volunteer 5 3 h following dosing. Plasma samples were extracted twice with 50 μL of ethyl acetate and the extract transferred to an HPLC vial with a 300 μL insert. The ethyl acetate was evaporated in a vacuum chamber and the residue reconstituted with 50 μL of acetonitrile prior to injection. Five microliters of plasma extract (volunteer 5, 3 h after dosing) was injected onto a Phenomenex Kinetex 2.6 μm C₁₈ 100 Å (Part no: 00f-4462-AN) column (150 × 2.1 mm) fitted with a C₁₈ guard column and eluted with 45% acetonitrile for 0–3 min followed by a linear gradient (3–10 min) to 100% acetonitrile, which was maintained from 10 to 13 min before a return to initial conditions (flow rate of 0.12 mL·min^–1 (25 °C)). Following passage through a dual channel UV/vis detector (280 and 315 nm), the eluent was deposited onto a moving nickel wire and the [¹⁴C]-DBC_eq converted to ¹⁴CO₂ prior to AMS analysis. Multiple channels allow for the simultaneous determination of A₂₈₀ and A₃₁₅ as well as counts of ¹²C and ¹⁴C. Only the ¹⁴C profile is shown here. The use of unlabeled parent DBC and standards (DBC-(±)-11,12-diol and DBC-(±)-11,12,13,14-tetraol) allow for tentative identification of ¹⁴C peaks (top panel). The polar putative metabolite eluting between 2 and 2.5 min is currently unknown but could be a conjugate formed by sulfotransferases or UDP-glucuronosyl transferase (sample not treated with sulfatase or β-glucuronidase prior to analysis). The plasma sample from volunteer 5 (lower panel) shows a detectable peak at 13.5 min, which is the retention time (top panel) of DBC-(±)-diol. The major ¹⁴C-containing peak in plasma at 3 h postdosing coelutes with parent DBC.

Figure 4

Pharmacokinetic profile of [¹⁴C]-DBC_eq in humans: comparison to a rat PBPK model. The ● symbols are plasma levels over time of [¹⁴C]-DBC_eq (with error bars depicting ± SD) following microdosing of humans with 5 nCi (29 ng) of [¹⁴C]-DBC. The solid curve is PBPK model prediction with metabolic rate scaled from the rat as calculated by Crowell et al.