Environmentally persistent free radical-containing particulate matter competitively inhibits metabolism by cytochrome P450 1A2.

Combustion processes generate different types of particulate matter (PM) that can have deleterious effects on the pulmonary and cardiovascular systems. Environmentally persistent free radicals (EPFRs) represent a type of particulate matter that is generated after combustion of environmental wastes in the presence of redox-active metals and aromatic hydrocarbons. Cytochromes P450 (P450/CYP) are membrane-bound enzymes that are essential for the phase I metabolism of most lipophilic xenobiotics. The EPFR formed by chemisorption of 2-monochlorophenol to silica containing 5% copper oxide (MCP230) has been shown to generally inhibit the activities of different forms of P450s without affecting those of cytochrome P450 reductase and heme oxygenase-1. The mechanism of inhibition of rat liver microsomal CYP2D2 and purified rabbit CYP2B4 by MCP230 has been shown previously to be noncompetitive with respect to substrate. In this study, MCP230 was shown to competitively inhibit metabolism of 7-benzyl-4-trifluoromethylcoumarin and 7-ethoxyresorufin by the purified, reconstituted rabbit CYP1A2. MCP230 is at least 5- and 50-fold more potent as an inhibitor of CYP1A2 than silica containing 5% copper oxide and silica, respectively. Thus, even though PM generally inhibit multiple forms of P450, PM interacts differently with the forms of P450 resulting in different mechanisms of inhibition. P450s function as oligomeric complexes within the membrane. We also determined the mechanism by which PM inhibited metabolism by the mixed CYP1A2-CYP2B4 complex and found that the mechanism was purely competitive suggesting that the CYP2B4 is dramatically inhibited when bound to CYP1A2.

Effect of low temperature thermal treatment on soils contaminated with pentachlorophenol and environmentally persistent free radicals.

The effect of low temperature thermal treatment on soils from a former Superfund wood-treating site contaminated with pentachlorophenol (PCP) and the environmentally persistent free radical (EPFR), pentachlorophenoxyl, was determined. The pentachlorophenoxyl EPFRs' and the PCP molecules' chemical behavior were simultaneously monitored at temperatures ranging from 25 to 300 °C via electron paramagnetic resonance (EPR) spectroscopy and GC-MS analysis, respectively. Two types of thermal treatment were employed: a closed heating (oxygen-starved condition) where the soil was heated under vacuum and an open heating system (oxygen-rich conditions), where the soil was heated in ambient air. EPR analyses for closed heating indicated the EPFR concentration was 2-12 × 10(18) spins/g of soil, with a g-factor and line width (ΔHp-p) of 2.00311-2.00323 and 4.190-5.472 G, respectively. EPR analyses for the open heating soils revealed a slightly broader and weaker radical signal, with a concentration of 1-10 × 10(18) spins/g of soil, g-factor of 2.00327-2.00341, and ΔHp-p of 5.209-6.721 G. This suggested the open heating resulted in the formation of a more oxygen-centered structure of the pentachlorophenoxyl radical or additional, similar radicals. The EPFR concentration peaked at 10 × 10(18) spins/g of soil at 100 °C for open heating and 12 × 10(18) spins/g at 75 °C for closed heating. The half-lives of the EPFRs were 2-24 days at room temperature in ambient air. These results suggest low temperature treatment of soils contaminated with PCP can convert the PCP to potentially more toxic pentachlorophenoxyl EPFRs, which may persist in the environment long enough for human exposure.

Sorption selectivity in natural organic matter studied with nitroxyl paramagnetic relaxation probes.

Sorption site selectivity and mechanism in natural organic matter (NOM) were addressed spectroscopically by the sorption of paramagnetic nitroxyl compounds (spin probes) of different polarity, TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) and HTEMPO (4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl). The sorbents were Pahokee peat, Beulah-Zap lignite, and a polystyrene-poly(vinyl methyl ether) (PS-PVME) polymer blend representing the mixed aliphatic-aromatic, polar-nonpolar character of NOM. Nuclear-electron spin interaction serves as an efficient relaxation pathway, resulting in attenuation of the (13)C-CP/TOSS NMR signal for (13)C nuclei in proximity to the N-O· group (r(-6) dependence). In the natural solids the spin probes sorbed more specifically (greater isotherm nonlinearity) and had lower rotational mobility (broader electron paramagnetic resonance signals) than in PS-PVME. Titration with spin probe indicated almost no selectivity for the different carbon functional groups of PS-PVME, and little to no selectivity for the different carbon moieties of Pahokee and Beulah, including aromatic, alkyl, O-alkyl, di-O-alkyl, and O-methyl. In any case, sorption site selectivity of spin probes to NOM was always weaker than partition selectivity found in model solvent-water (toluene, hexadecane, anisole, octanol) and cellulose-water systems. The results indicate little or no preferential sorption in NOM based on functional group chemistry or putative microdomain character, but rather are consistent with the filling of pores whose walls have an average chemical environment reflecting the bulk chemical composition of the solid. This work demonstrates for the first time the use of paramagnetic probes to study sorption specificity.

Detection of environmentally persistent free radicals at a superfund wood treating site.

Environmentally persistent free radicals (EPFRs) have previously been observed in association with combustion-generated particles and airborne PM(2.5) (particulate matter, d < 2.5um). The purpose of this study was to determine if similar radicals were present in soils and sediments at Superfund sites. The site was a former wood treating facility containing pentachlorophenol (PCP) as a major contaminant. Both contaminated and noncontaminated (just outside the contaminated area) soil samples were collected. The samples were subjected to the conventional humic substances (HS) extraction procedure. Electron paramagnetic resonance (EPR) spectroscopy was used to measure the EPFR concentrations and determine their structure for each sample fraction. Analyses revealed a ∼30× higher EPFR concentration in the PCP contaminated soils (20.2 × 10(17) spins/g) than in the noncontaminated soil (0.7 × 10(17) spins/g). Almost 90% of the EPFR signal originated from the minerals/clays/humins fraction. GC-MS analyses revealed ∼6500 ppm of PCP in the contaminated soil samples and none detected in the background samples. Inductively coupled plasma-atomic emission spectrophotometry (ICP-AES) analyses revealed ∼7× higher concentrations of redox-active transition metals, in the contaminated soils than the noncontaminated soil. Vapor phase and liquid phase dosing of the clays/minerals/humins fraction of the soil with PCP resulted in an EPR signal identical to that observed in the contaminated soil, strongly suggesting the observed EPFR is pentachlorophenoxyl radical. Chemisorption and electron transfer from PCP to transition metals and other electron sinks in the soil are proposed to be responsible for EPFR formation.

Phytosampling-a supplementary tool for particulate matter (PM) speciation characterization.

Ambient air particulate matter (PM) and PM-associated environmentally persistent free radicals (EPFRs) have been documented to contribute to pollution-related health effects. Studies of ambient air PM potentially bear artifacts stemming from the collection methods. We have investigated the applicability of PM phytosampling (PHS) as a supplementary tool to a classic PM sampler in respect of achieving better PM chemical composition assessment (primarily organic fraction). Phytosampling is a static PM collection method relying on the particle entrapment by the plant's leaf through electrostatic forces and surface trichomes. We have investigated the differences in the EPFR and polycyclic aromatic hydrocarbon (PAH) speciation and concentration on ambient air PM for PHS and high-volume PM sampler (HVS). The advantages of PHS are easy particle recovery from the matrix, collection under natural environmental conditions, and the ability to apply a dense collection network to accurately represent spatial pollutant distribution. The experimental results show that the PHS can provide valuable speciation information, sometimes different from that observed for HVS. For PM collected by PHS, we detected the larger contribution of oxygen-centered EPFRs, different decay behavior, and more consistent PAH distribution between different PM sizes compared to the PM from HVS. These results indicate that the isolation of samples from the ambient during HVS sampling and exposure to high-volume airflow may alter the chemical composition of the samples, while the PHS method could provide details on the original speciation and concentration and be more representative of the PM surface. However, PHS cannot evaluate an absolute air concentration of PM, so it serves as an excellent supplementary tool to work in conjunction with the standard PM collection method.

Polybrominated Diphenylethers (PBDEs) in ambient air samples at the electronic waste (e-waste) reclamation site.

Polybrominated Diphenylethers (PBDEs) were used as flame-retardants in various building materials, plastic and other polymers, airplanes, electronics etc. All or some of their congeners have been already banned in many countries, due to their persistency and adverse health effects. In this study, we are focusing on the e-wastes as a source of emission of PBDEs in ambient air during reclamation processes. The ambient air particulate matter (PM) samples were collected at and near e-waste reclamation site in Bangkok, Thailand. Results showed the presence of various homologues viz: tri, tetra, penta, hexa, and hepta-PBDEs on both PM2.5 and Total Suspended Particle (TSP) samples. The comparison of samples as a function of distance from reclamation site indicated elevated levels of PBDEs in the close proximity to e-waste site. Interestingly, a shift in the congener pattern was observed with lower brominated PBDEs being more prevalent on nearby off-site samples as compared to the PM collected at the e-waste site. The total penta-PBDEs concentration is about double on e-waste site PM2.5 compared to control site samples. For TSP, tetra, penta, and hepta-PBDEs congeners are at higher concentrations at e-waste sites and its vicinity compared to reference sites. Overall, a clear trend can be observed indicating a debromination of PBDEs to more toxic tri and tetra congeners during reclamation process and PBDEs are being translocated from treated materials to ambient air PM. BDE 30 congener is identified as a specific marker of thermal reclamation processes of e-wastes as a most stable degradation product. This work indicates potential hazards related to the reclamation of e-wastes and remediation of sites containing PBDEs. In particular, thermal treatment methods can lead to congener transformation and increased emissions of more toxic lower-brominated congeners.

Formation of environmentally persistent free radical (EPFR) in iron(III) cation-exchanged smectite clay.

Environmentally persistent free radicals (EPFRs) have been found at a number of Superfund sites, with EPFRs being formed via a proposed redox process at ambient environmental conditions. The possibility of such a redox process taking place at ambient environmental conditions is studied utilizing a surrogate soil system of phenol and iron(III)-exchanged calcium montmorillonite clay, Fe(III)CaM. Sorption of phenol by the Fe(III)CaM is demonstrated by Fourier-transformed infra-red (FT-IR) spectroscopy, as evidenced by the peaks between 1345 cm(-1) and 1595 cm(-1), and at lower frequencies between 694 cm(-1) and 806 cm(-1), as well as X-ray diffraction (XRD) spectroscopy, as shown by an increase in interlayer spacing within Fe(III)CaM. The formation and characterization of the EPFRs is determined by electron paramagnetic resonance (EPR) spectroscopy, showing phenoxyl-type radical with a g-factor of 2.0034 and ΔHP-P of 6.1 G at an average concentration of 7.5 × 10(17) spins per g. EPFRs lifetime data are indicative of oxygen and water molecules being responsible for EPFR decay. The change in the oxidation state of the iron redox center is studied by X-ray absorption near-edge structure (XANES) spectroscopy, showing that 23% of the Fe(III) is reduced to Fe(II). X-ray photoemission spectroscopy (XPS) results confirm the XANES results. These findings, when combined with the EPFR concentration data, demonstrate that the stoichiometry of the EPFR formation under the conditions of this study is 1.5 × 10(-2) spins per Fe(II) atom.

Model System Study of Environmentally Persistent Free Radicals Formation in a Semiconducting Polymer Modified Copper Clay System at Ambient Temperature.

This paper systematically investigates how environmentally persistent free radicals (EPFRs) are formed in a phenol contaminated model soil. Poly-p-phenylene (PPP) modified and copper-loaded montmorillonite (MMT) clays were developed and used as models of soil organic matter and the clay mineral component, respectively, with phenol being employed as a precursor pollutant. The polymer modification of the clays was carried out via surface-confined Kumada catalyst-transfer chain-growth polymerization. The presence and location of the polymer were confirmed by a combination of thermogravimetric analysis (TGA), Raman spectroscopy, and X-ray diffraction data. EPFRs were formed by the Cu(II)-clay (Cu(II)CaMMT) and poly-p-phenylene-Cu(II)clay (PPP-Cu(II)CaMMT) composite systems under environmentally relevant conditions. The g-factor and concentration of EPFRs formed by the Cu(II)CaMMT and PPP-Cu(II)CaMMT systems were found to be 2.0034 and 1.22 × 1017 spins/g and 2.0033 and 1.58 × 1017spins/g, respectively. These g-factors are consistent with the formation of phenoxyl radicals. Extended X-Ray absorption fine structure (EXAFS) analysis shows that there are distinct differences in the local stuctures of the phenoxyl radicals associated with only the Cu(II) redox centers and those formed in the presences of the PPP polymer. X-ray absorption near edge spectroscopy (XANES) results provided evidence for the reduction of Cu(II) to Cu(I) in the EPFR forming process. The 1/e lifetimes of the formed EPFRs revealed a decay time of ~20 h for the Cu(II)CaMMT system and a two-step decay pattern for the PPP-Cu(II)CaMMT system with decay times of ~13.5 h and ~55.6 h. Finally, the generation of reactive oxygen species (hydroxyl radical; •OH) by these clay systems was also investigated, with higher concentrations of •OH detected for the phenol-dosed Cu(II)CaMMT and PPP-Cu(II)CaMMT systems, compared to the non-EPFR containing undosed PPP-Cu(II)CaMMT system.

Inhibition of cytochrome P450 2B4 by environmentally persistent free radical-containing particulate matter.

Combustion processes generate particulate matter (PM) that can affect human health. The presence of redox-active metals and aromatic hydrocarbons in the post-combustion regions results in the formation of air-stable, environmentally persistent free radicals (EPFRs) on entrained particles. Exposure to EPFRs has been shown to negatively influence pulmonary and cardiovascular functions. Cytochromes P450 (P450/CYP) are endoplasmic reticulum resident proteins that are responsible for the metabolism of foreign compounds. Previously, it was shown that model EPFRs, generated by exposure of silica containing 5% copper oxide (CuO-Si) to either dicholorobenzene (DCB230) or 2-monochlorophenol (MCP230) at ≥ 230 °C, inhibited six forms of P450 in rat liver microsomes (Toxicol. Appl. Pharmacol. (2014) 277:200-209). In this study, the inhibition of P450 by MCP230 was examined in more detail by measuring its effect on the rate of metabolism of 7-ethoxy-4-trifluoromethylcoumarin (7EFC) and 7-benzyloxyresorufin (7BRF) by the purified, reconstituted CYP2B4 system. MCP230 inhibited the CYP2B4-mediated metabolism of 7EFC at least 10-fold more potently than non-EPFR controls (CuO-Si, silica, and silica generated from heating silica and MCP at 50 °C, so that EPFRs were not formed (MCP50)). The inhibition by EPFRs was specific for the P450 and did not affect the ability of the redox partner, P450 reductase (CPR) from reducing cytochrome c. All of the PM inhibited CYP2B4-mediated metabolism noncompetitively with respect to substrate. When CYP2B4-mediated metabolism of 7EFC was measured as a function of the CPR concentration, the mechanism of inhibition was competitive. EPFRs likely inhibit CYP2B4-mediated substrate metabolism by physically disrupting the CPR·P450 complex.

Assessment of environmentally persistent free radicals in soils and sediments from three Superfund sites.

We previously reported the presence of environmentally persistent free radicals (EPFRs) in pentachlorophenol (PCP) contaminated soils at a closed wood treatment facility site in Georgia. The reported EPFRs were pentachlorophenoxyl radicals formed on soils under ambient conditions via electron transfer from PCP to electron acceptors in the soil. In this study, we present results for soil and sediment samples from additional Superfund sites in Montana and Washington. Paramagnetic centers associated with different chemical environments were characterized by distinct g-factors and line widths (ΔHp-p). EPFR concentrations in contaminated samples were ~30×, ~12×, and ~2× higher than background samples at the Georgia, Montana, and Washington sites, respectively. EPR signals in the Montana contaminated soils were very similar to those previously observed for pentachlorophenol contaminated soils at the Georgia site, i.e., g = 2.00300 and ΔHp-p = 6.0 G, whereas signals in the Washington sediment samples were similar to those previously observed for other PAH contaminated soils, i.e., g = 2.00270 and ΔHp-p = 9.0 G. Total carbon content measurements exhibited direct correlation with EPFR concentration. The presence of radicals in sites contaminated a decade to a century ago suggests continuous formation of EPFRs from molecular contaminants in the soil and sediment.