Superfund Research Program

Elucidating Metabolic and Physicochemical Mechanisms of PAH Susceptibility in Toxicity Test Systems and Humans

Project Leader: Jordan N. Smith (Pacific Northwest National Laboratory)
Co-Investigator: Justin Teeguarden (Pacific Northwest National Laboratory)
Grant Number: P42ES016465
Funding Period: 2009-2025

Project-Specific Links

Final Progress Reports

Year:   2019  2012 

Studies and Results Year 4 (final year of initial funding cycle)

Since previous studies completed in year 3 demonstrated that pregnancy significantly altered the pharmacokinetics of DBC in mice that could not be explained by changes in physiology alone, the research team recruited Aaron Wright, Ph.D., an organic chemist at PNNL, to the project to assess the relative activities of key enzymes important to PAH metabolism using a novel, mass spectrometry based functional proteomics approach. This approach--activity based protein profiling (ABPP)--provides an unprecedented, simultaneous assessment of >75 microsomal enzyme activities, including many of the P450s, epoxide hydrolases, glutathione S-transferases, and glucuronosyl transferases that are involved in PAH activation and detoxification. Initial results demonstrated that higher levels of DBC and its diol and tetraol metabolites found in blood and tissues of pregnant mice vs. their non-pregnant counterparts were primarily due to significant pregnancy-induced decreases in the activities of P450 enzymes involved in PAH metabolism. Manuscripts are currently being developed describing this approach and how it impacts the understanding of PAH metabolism. Since this technique is sensitive (as little as 50 mg of microsomal protein are needed to characterize enzyme activities in mouse or human tissues using mass spectrometry), the research team initiated ontogeny studies of active enzymes in fetal tissues from humans and fetal, neonatal, juvenile, and adult tissues from mice to expand the PBPK models to include all life stages.

In addition the research team previously found that concentrations of unmetabolized DBC in fetuses from mice administered DBC at a single dose (15 mg/kg) orally on gestational day 17 were an order of magnitude less than maternal blood concentrations while concentrations of the diol metabolite, the immediate precursor to the most carcinogenic diol-epoxide metabolite, eventually exceeded maternal blood concentrations over time. No differences in concentrations of DBC or metabolites were observed between fetuses with responsive versus non-responsive AhR genotypes. Therefore, an additional in vivo study was conducted with DBC in pregnant mice to measure DBC and its diol and tetraol metabolites in target tissues (liver, lung, and thymus) within the developing fetus along with ABPP analyses of tissue enzyme activities to determine whether genotype is a factor in target site dosimetry. While analyses of these tissues are in progress, preliminary results indicate that genotype is indeed a factor in metabolite levels in target tissues.

In vitro metabolism studies with DBC and BaP were completed in liver tissues from male rats, female mice (pregnant and naïve), and female humans. The comparative rates of DBC and BaP metabolism were greatest in mice followed by rats, then humans. Furthermore, the metabolic rates for DBC were lower in liver microsomes from pregnant mice, confirming results from the ABPP assays in Aim 1. A manuscript is being developed to describe these results.

In collaboration with Environmental PAH Mixtures as Skin and Transplacental Carcinogens, the first ultra low-dose human pharmacokinetic study with DBC (or any high-molecular weight PAH) was completed with nine human volunteers. DBC was quickly absorbed after oral consumption of a gelatin capsule containing ~5 ng of [⁴C]-DBC achieving peak plasma concentrations of 33.9 ± 22.1 fg DBC Eq/ml by 2.3 ± 0.9 hr. No significant differences were detected in plasma or urine ¹⁴C concentrations between males (n=6) and females (n=3). Clearance of ¹⁴C from plasma was bi-phasic with a rapid alpha phase where most of the absorbed dose was cleared from plasma within 12-24 hr with a half-life of 3.5 ± 2.3 hr. This was followed by a prolonged beta elimination phase with residual, low level ¹⁴C concentrations that may represent protein bound material, with an apparent half-life of 160.1 ± 322.0 hr. Only 1.24 ± 0.49% of the administered dose was accounted for in urine collected for up to 72 hr post-dosing. Renal clearance of ¹⁴C (12.9 ± 8.1 ml/min) was ~12% of published measures of glomerular filtration indicating the potential for extensive resorption of filtered metabolites by proximal tubules. A manuscript is currently being developed to describe these results.

The research team revised the first PBPK models for any high molecular weight PAH for BaP and DBC to include recent results from Aims 1-3. A manuscript is currently being developed to describe these refinements. Importantly, by using the metabolic rate constants determined in human liver microsomes in Aim 2, researchers were able to successfully simulate the kinetics of DBC in human volunteers from Aim 3 assuming that the majority of ¹⁴C in plasma was unmetabolized DBC as the research group has shown in the mouse.

Significance

The establishment of exposure and cleanup guidelines for PAHs found at Superfund sites have historically relied upon toxicity and carcinogenicity studies conducted in laboratory animals. Unfortunately, little is known about potentially important differences between laboratory animals and humans in how PAHs are absorbed by the body, distributed to various organs and tissues, metabolized through either activation or detoxification pathways, and eventually eliminated: the so-called "ADME" processes that can drive adverse responses under real-world exposure conditions. Regulatory agencies now recommend physiologically based pharmacokinetic (PBPK) models for bridging this gap because they integrate chemical-specific ADME processes with species-specific anatomy and physiology. Since the research team developed the first PBPK models for any high molecular weight PAH encountered at Superfund sites in year three, two important technological advances--accelerator mass spectrometry (AMS) and activity based protein profiling (ABPP)--enabled researchers to expand their models to address potentially susceptible populations (e.g. the developing fetus and neonate) and evaluate them under relevant human exposure conditions. Using AMS, the research group completed the first controlled exposure study with human volunteers exposed to ultra low, background doses of [¹⁴C]-DBC. The results from this study confirmed the team's ability to predict DBC pharmacokinetics in humans using knowledge of human anatomy and physiology along with project-specific in vitro DBC metabolism studies in mouse and human tissues and in vivo pharmacokinetic studies in mice. Using ABPP, the investigators also discovered that the activities of many of enzymes involved in PAH metabolism are reduced in mice during pregnancy and that these changes have a dramatic impact on PAH pharmacokinetics. The research team is now working to compare the ontogeny of many of these metabolic enzymes in target tissues from mice and humans from early fetal development through adulthood and how these activities are affected by PAH exposures to create full life-stage specific PBPK models for animals and humans. The results from these ontogeny studies have broad impact beyond PAH risk-related research as >75 enzymes involved in a broad range of drug and chemical metabolism will be included in the analyses.

With a focus on developing the first PBPK models for high molecular weight PAHs that are of greatest concern at superfund sites, this project provides the SRP Center and its stakeholders with state-of-the-art, quantitative and integrative computational tools that are needed to assess potential adverse responses and susceptibility factors in humans across all stages of life that are associated with environmental levels of PAHs. The proposed research project is therefore consistent with two of the four mandated SRP research areas involving the development of advanced techniques for health effects and risk assessment research. SRP stakeholders have also identified the need for research leading to improved understanding of the impact on potential susceptible populations and identified children as one such group. The development of PBPK models that includes transplacental dosimetry, neonatal growth and development, as well as adult PBPK models directly addresses this need.