Human Microdosing with Carcinogenic Polycyclic Aromatic Hydrocarbons: In Vivo Pharmacokinetics of Dibenzo[def,p]chrysene and Metabolites by UPLC Accelerator Mass Spectrometry

Abstract

Metabolism is a key health risk factor following exposures to pro-carcinogenic polycyclic aromatic hydrocarbons (PAHs) such as dibenzo[def,p]chrysene (DBC), an IARC classified 2A probable human carcinogen. Human exposure to PAHs occurs primarily from the diet in nonsmokers. However, little data is available on the metabolism and pharmacokinetics in humans of high molecular weight PAHs (≥4 aromatic rings), including DBC. We previously determined the pharmacokinetics of DBC in human volunteers orally administered a microdose (29 ng; 5 nCi) of [¹⁴C]-DBC by accelerator mass spectrometry (AMS) analysis of total [¹⁴C] in plasma and urine. In the current study, we utilized a novel "moving wire" interface between ultraperformance liquid chromatography (UPLC) and AMS to detect and quantify parent DBC and its major metabolites. The major [¹⁴C] product identified in plasma was unmetabolized [¹⁴C]-DBC itself (C_max = 18.5 ±15.9 fg/mL, T_max= 2.1 ± 1.0 h), whereas the major metabolite was identified as [¹⁴C]-(+/-)-DBC-11,12-diol (C_max= 2.5 ±1.3 fg/mL, T_max= 1.8 h). Several minor species of [¹⁴C]-DBC metabolites were also detected for which no reference standards were available. Free and conjugated metabolites were detected in urine with [¹⁴C]-(+/-)-DBC-11,12,13,14-tetraol isomers identified as the major metabolites, 56.3% of which were conjugated (C_max= 35.8 ± 23.0 pg/pool, T_max = 6-12 h pool). [¹⁴C]-DBC-11,12-diol, of which 97.5% was conjugated, was also identified in urine (C_max = 29.4 ± 11.6 pg/pool, T_max = 6-12 h pool). Parent [¹⁴C]-DBC was not detected in urine. This is the first data set to assess metabolite profiles and associated pharmacokinetics of a carcinogenic PAH in human volunteers at an environmentally relevant dose, providing the data necessary for translation of high dose animal models to humans for translation of environmental health risk assessment.

Figure 1. Metabolic Activation of DBC

Pro-carcinogenic DBC is activated to reactive intermediate metabolites by several enzymatic processes. Peroxidase activation can form a radical DBC species that adduct DNA. The CYP1B1 or CYP1A1 pathways can convert DBC to several metabolites that form DNA adducts, be conjugated for elimination or further metabolized to more reactive intermediates. The primary DBC carcinogenic metabolite is the (−)-anti-trans-11,12-diol-13,14-epoxide. The 11,12-trans-dihydrodiol is additionally a substrate for to aldo-keto reductase (AKR) formation of a catechol which can then undergo redox cycling. As information on the fate of DBC quinones was not available we relied on the prototypical PAH, benzo[a]pyrene (BaP), for which evidence has been published with respect to DNA adduction and further metabolism by UDP-glucuronosyl transferase (UGT), NADPH quinone oxidoreductase (NQO) and sulfatase (SULT)^–. It is acknowledge that the reactivity and metabolism of DBC quinones may differ from BaP.

Figure 2. [¹⁴C]-DBC Metabolite Profile in Plasma from a Representative Volunteer 0.75 hours after Dosing

A. [¹⁴C] particles were detected from plasma extracts by AMS (left axis). The [¹⁴C] particles detected per peak were converted to fg DBC · mL⁻¹ using the specific activity of [¹⁴C]-DBC and sample processing correction factors. [¹²C] measurements were used to determine and remove the endogenous biological [¹⁴C] (right axis). B. Non-labeled DBC and DBC metabolite standards provided retention time data utilizing PDA detection.

Figure 3. Profiles of Plasma [¹⁴C]-DBC and [¹⁴C]-DBC Metabolites over 24 Hours in a Representative Volunteer

The plasma metabolite profile over the first 24 hours following dosing was compared to [¹⁴C]-DBC in plasma. The most abundant species in plasma was parent [¹⁴C]-DBC, with peaks eluting with the solvent front (more polar than tetrols), putatively identified as conjugates of [¹⁴C]-DBC metabolites.

Figure 4. β-Glucuronidase Cleavage of Urine DBC Metabolites

A. UPLC-AMS analysis of the 6–12 hour urine pool, not treated with β-glucuronidase, from a representative volunteer. Non-β-glucuronidase treated urine contains few retained metabolites and a low [¹⁴C] signal. B. UPLC-AMS analysis of urine from the same pool, treated with β-glucuronidase, contained several metabolites that were free to be extracted, separated, and detected.

Figure 5. Urine [¹⁴C]-DBC Metabolite Profile from a Representative Volunteer over 72 Hours

A. The urine [¹⁴C]-DBC metabolite profile from extracts of urine not treated with β-glucuronidase, or B. from β-glucuronidase (with sulfatase activity)-treated urine extracts. The [¹⁴C]-DBC-DHD is only present following enzyme treatment, indicating that it is in a conjugated form in urine. The putative [¹⁴C]-DBC-dione is increased following enzyme treatment only slightly, while other metabolite fractions were mostly present following enzymatic cleavage, indicating that they exist in the conjugated form in urine.