Impact of Pregnancy on the Pharmacokinetics of Dibenzo[def,p]chrysene in Mice

Abstract

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated during combustion. Dibenzo[def,p]chrysene (DBC) is a high molecular weight PAH classified as a 2B carcinogen by the International Agency for Research on Cancer. DBC crosses the placenta in exposed mice, causing carcinogenicity in offspring. We present pharmacokinetic data of DBC in pregnant and nonpregnant mice. Pregnant (gestational day 17) and nonpregnant female B6129SF1/J mice were exposed to 15mg/kg DBC by oral gavage. Subgroups of mice were sacrificed up to 48h postdosing, and blood, excreta, and tissues were analyzed for DBC and its major diol and tetrol metabolites. Elevated maximum concentrations and areas under the curve of DBC and its metabolites were observed in blood and tissues of pregnant animals compared with naïve mice. Using a physiologically based pharmacokinetic (PBPK) model, we found observed differences in pharmacokinetics could not be attributed solely to changes in tissue volumes and blood flows that occur during pregnancy. Measurement of enzyme activity in naïve and pregnant mice by activity-based protein profiling indicated a 2- to 10-fold reduction in activities of many of the enzymes relevant to PAH metabolism. Incorporating this reduction into the PBPK model improved model predictions. Concentrations of DBC in fetuses were one to two orders of magnitude below maternal blood concentrations, whereas metabolite concentrations closely resembled those observed in maternal blood.

Hepatic Cytochrome P450 Activity, Abundance, and Expression Throughout Human Development

“…ABPP utilizes chemical probes derived from aryl-or aliphatic-alkyne mechanism-based inhibitors of P450 enzymes, which directly report on the activity of P450 enzymes in subcellular fractions, whole cells, or organisms (Wright and Cravatt, 2007;Cravatt et al, 2008;Hollenberg et al, 2008;Wright et al, 2009;Crowell et al, 2013). Herein, we use a multiplexed labeling approach in which an aryl alkyne probe (P450-ABP1) (Wright and Cravatt, 2007) and an aliphatic alkyne probe (P450-ABP2) (Wright et al, 2009) are added simultaneously to human prenatal tissue microsome samples (Fig.…”

“…1). Multiplexed ABPP yields broad coverage of target enzyme families (Wiedner et al, 2012); this is particularly true in the P450 superfamily, in which individual P450 isoforms often display broad substrate specificity (Crowell et al, 2013). Aliphatic and aryl alkynes have been demonstrated to inhibit P450s from all major detoxifying P450 subfamilies (Wright et al, 2009).…”

“…Aliphatic and aryl alkynes have been demonstrated to inhibit P450s from all major detoxifying P450 subfamilies (Wright et al, 2009). In the presence of NADPH functionally active microsomal P450s directly oxidize the probes yielding reactive ketene moieties, which subsequently react irreversibly with the functional P450 (Wright and Cravatt, 2007;Wright et al, 2009;Crowell et al, 2013). Each probe also contains a second alkyne group that serves as a reactive handle for copper-catalyzed azide-alkyne cycloadditions ("click chemistry").…”

“…Control samples were prepared by heat-shock pretreatment of the microsome samples (98°C, 10 minutes) prior to addition of activity-based probes (ABPs) and NADPH. Following ABP incubation, the samples were reacted with a biotin-azide (30 mM) tag under click-chemistry conditions as described previously (Wright and Cravatt, 2007;Crowell et al, 2013). Prior to enrichment of probe-labeled proteins, sample protein amounts were normalized to 350 mg protein.…”

“…Activity-based protein profiling of P450s uses modified P450 mechanism-based inhibitors as probes to profile the functionally active cohort of enzymes directly in microsomes isolated from human liver tissues (Wright and Cravatt, 2007;Cravatt et al, 2008;Wright et al, 2009;Crowell et al, 2013;Ismail et al, 2013). The approach requires the catalytic activity of the P450s to form a covalent bond between P450 and probe, from which the targeted proteins can be identified by liquid chromatography-mass spectrometry (LC-MS)-based proteomics or fluorescent gel imaging.…”