Exposure to an Environmental Mixture of Polycyclic Aromatic Hydrocarbons Induces Hepatic Cytochrome P450 Enzymes in Mice.

Cytochrome P450 enzymes (CYPs) play an important role in bioactivating or detoxifying polycyclic aromatic hydrocarbons (PAHs), common environmental contaminants. While it is widely accepted that exposure to PAHs induces CYPs, effectively increasing rates of xenobiotic metabolism, dose- and time-response patterns of CYP induction are not well-known. In order to better understand dose- and time-response relationships of individual CYPs following induction, we exposed B6129SF1/J mice to single or repeated doses (2-180 µmol/kg/d) of benzo[a]pyrene (BaP) or Supermix-10, a mixture of the top 10 most abundant PAHs found at the Portland Harbor Superfund Site. In hepatic microsomes from exposed mice, we measured amounts of active CYPs using activity-based protein profiling and total CYP expression using global proteomics. We observed rapid Cyp1a1 induction after 6 h at the lowest PAH exposures and broad induction of many CYPs after 3 daily PAH doses at 72 h following the first dose. Using samples displaying Cyp1a1 induction, we observed significantly higher metabolic affinity for BaP metabolism (Km reduced 3-fold), 3-fold higher intrinsic clearance, but no changes to the Vmax. Mice dosed with the highest PAH exposures exhibited 1.7-5-fold higher intrinsic clearance rates for BaP compared to controls and higher Vmax values indicating greater amounts of enzymes capable of metabolizing BaP. This study demonstrates exposure to PAHs found at superfund sites induces enzymes in dose- and time-dependent patterns in mice. Accounting for specific changes in enzyme profiles, relative rates of PAH bioactivation and detoxification, and resulting risk will help translate internal dosimetry of animal models to humans and improve risk assessments of PAHs at superfund sites.

Benzo[ a]pyrene Induction of Glutathione S-Transferases: An Activity-Based Protein Profiling Investigation.

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous environmental contaminants generated from combustion of carbon-based matter. Upon ingestion, these molecules can be bioactivated by cytochrome P450 monooxygenases to oxidized toxic metabolites. Some of these metabolites are potent carcinogens that can form irreversible adducts with DNA and other biological macromolecules. Conjugative enzymes, such as glutathione S-transferases or UDP-glucuronosyltransferases, are responsible for the detoxification and/or facilitate the elimination of these carcinogens. While responses to PAH exposures have been extensively studied for the bioactivating cytochrome P450 enzymes, much less is known regarding the response of glutathione S-transferases in mammalian systems. In this study, we investigated the expression and activity responses of murine hepatic glutathione S-transferases to benzo[ a]pyrene exposure using global proteomics and activity-based protein profiling for chemoproteomics, respectively. Using this approach, we identified several enzymes exhibiting increased activity including GSTA2, M1, M2, M4, M6, and P1. The activity of one GST enzyme, GSTA4, was found to be downregulated with increasing B[ a]P dose. Activity responses of several of these enzymes were identified as being expression-independent when comparing global and activity-based data sets, possibly alluding to as of yet unknown regulatory post-translational mechanisms.

Multifunctional Activity-Based Protein Profiling of the Developing Lung.

Lung diseases and disorders are a leading cause of death among infants. Many of these diseases and disorders are caused by premature birth and underdeveloped lungs. In addition to developmentally related disorders, the lungs are exposed to a variety of environmental contaminants and xenobiotics upon birth that can cause breathing issues and are progenitors of cancer. In order to gain a deeper understanding of the developing lung, we applied an activity-based chemoproteomics approach for the functional characterization of the xenometabolizing cytochrome P450 enzymes, active ATP and nucleotide binding enzymes, and serine hydrolases using a suite of activity-based probes (ABPs). We detected P450 activity primarily in the postnatal lung; using our ATP-ABP, we characterized a wide range of ATPases and other active nucleotide- and nucleic acid-binding enzymes involved in multiple facets of cellular metabolism throughout development. ATP-ABP targets include kinases, phosphatases, NAD- and FAD-dependent enzymes, RNA/DNA helicases, and others. The serine hydrolase-targeting probe detected changes in the activities of several proteases during the course of lung development, yielding insights into protein turnover at different stages of development. Select activity-based probe targets were then correlated with RNA in situ hybridization analyses of lung tissue sections.

Activity-Based Probes for Isoenzyme- and Site-Specific Functional Characterization of Glutathione S-Transferases.

Glutathione S-transferases (GSTs) comprise a diverse family of phase II drug metabolizing enzymes whose shared function is the conjugation of reduced glutathione (GSH) to endo- and xenobiotics. Although the conglomerate activity of these enzymes can be measured, the isoform-specific contribution to the metabolism of xenobiotics in complex biological samples has not been possible. We have developed two activity-based probes (ABPs) that characterize active GSTs in mammalian tissues. The GST active site is composed of a GSH binding "G site" and a substrate binding "H site". Therefore, we developed (1) a GSH-based photoaffinity probe (GSTABP-G) to target the "G site", and (2) an ABP designed to mimic a substrate molecule and have "H site" activity (GSTABP-H). The GSTABP-G features a photoreactive moiety for UV-induced covalent binding to GSTs and GSH-binding enzymes. The GSTABP-H is a derivative of a known mechanism-based GST inhibitor that binds within the active site and inhibits GST activity. Validation of probe targets and "G" and "H" site specificity was carried out using a series of competition experiments in the liver. Herein, we present robust tools for the characterization of enzyme- and active site-specific GST activity in mammalian model systems.