Debra A. Schmitz

Nrf2 Is a Key Transcription Factor That Regulates Antioxidant Defense in Macrophages and Epithelial Cells: Protecting against the Proinflammatory and Oxidizing Effects of Diesel Exhaust Chemicals

The proinflammatory effects of particulate pollutants, including diesel exhaust particles (DEP), are related to their content of redox cycling chemicals and their ability to generate oxidative stress in the respiratory tract. An antioxidant defense pathway, which involves phase II enzyme expression, protects against the pro-oxidative and proinflammatory effects of DEP. The expression of enzymes, including heme oxygenase-1 (HO-1) and GST, is dependent on the activity of a genetic antioxidant response element in their promoters. In this study we investigated the mechanism by which redox cycling organic chemicals, prepared from DEP, induce phase II enzyme expression as a protective response. We demonstrate that aromatic and polar DEP fractions, which are enriched in polycyclic aromatic hydrocarbons and quinones, respectively, induce the expression of HO-1, GST, and other phase II enzymes in macrophages and epithelial cells. We show that HO-1 expression is mediated through accumulation of the bZIP transcription factor, Nrf2, in the nucleus, and that Nrf2 gene targeting significantly weakens this response. Nrf2 accumulation and subsequent activation of the antioxidant response element is regulated by the proteasomal degradation of Nrf2. This pathway is sensitive to pro-oxidative and electrophilic DEP chemicals and is also activated by ambient ultrafine particles. We propose that Nrf2-mediated phase II enzyme expression protects against the proinflammatory effects of particulate pollutants in the setting of allergic inflammation and asthma.

Determination of Four Quinones in Diesel Exhaust Particles, SRM 1649a, and Atmospheric PM2.5 Special Issue of Aerosol Science and Technology on Findings from the Fine Particulate Matter Supersites Program

Quinones are reactive organic compounds and are known to initiate reactions associated with many toxicological events. Their presence in air pollution has been demonstrated, but routine quantitative measurements are lacking. A quantitative method for the determination of four quinones was developed using diesel exhaust particles (DEP) and National Institute of Standards and Technology (NIST) Standard Reference Material (SRM) 1649a. The method was then used to analyze ambient air samples from different sites in Southern California. After extraction in dichloromethane, the target compounds were converted to their stable diacetyl derivatives and determined by electron impact GC-MS using selected ion monitoring. Calibration plots were obtained with deuterium-labeled internal standards. The four quinones, 1,2-naphthoquinone (1,2-NQ), 1,4-naphthoquinone (1,4-NQ), 9,10-phenanthraquinone (9,10-PQ), and 9,10-anthraquinone (9,10-AQ), were quantified in DEP, in SRM 1649a, and in ambient air samples of PM2.5 collected in several rural and urban sampling locations upwind and downwind of major emission sources in Central Los Angeles. Mean concentration of individual target quinones ranged from 7.9–40.4 μg/g in the DEP, and from 5–730 pg/m3 in the PM2.5 samples. Precision (repeatability and reproducibility) varied from 2–22%. Further measurements of these species in future air samples should be considered in light of their potential health significance.

The adjuvant effect of ambient particulate matter is closely reflected by the particulate oxidant potential

Background It has been demonstrated that ambient particulate matter (PM) can act as an adjuvant for allergic sensitization. Redox-active organic chemicals on the particle surface play an important role in PM adverse health effects and may determine the adjuvant effect of different particle types according to their potential to perturb redox equilibrium in the immune system. Objectives We determined whether the adjuvant effect of ambient fine particles versus ultrafine particles (UFPs) is correlated to their prooxidant potential. Methods We have established an intranasal sensitization model that uses ambient PM as a potential adjuvant for sensitization to ovalbumin (OVA), which enhances the capacity for secondary OVA challenge to induce allergic airway inflammation. Results UFPs with a greater polycyclic aromatic hydrocarbon (PAH) content and higher oxidant potential enhanced OVA sensitization more readily than did fine particles. This manifests as enhanced allergic inflammation upon secondary OVA challenge, leading to eosinophilic inflammation and mucoid hyperplasia starting at the nasal turbinates all the way down to the small pulmonary airways. The thiol antioxidant N-acetyl cysteine was able to suppress some of these sensitization events. Conclusions The adjuvant effects of ambient UFP is determined by their oxidant potential, which likely plays a role in changing the redox equilibrium in the mucosal immune system.

Electrophilic and redox properties of diesel exhaust particles

The adverse health effects of air pollutants have been associated with their redox and electrophilic properties. Although the specific chemical species involved in these effects are not known, the characterization of their general physical and chemical properties is important to our understanding of the mechanisms by which they cause health problems. This manuscript describes results of a study examining the partition properties of these activities in aqueous and organic media. The water and dichloromethane (DCM) solubility of redox active and electrophilic constituents of seven diesel exhaust particle (DEP) samples were determined with assays developed earlier in this laboratory. The constituents exhibiting redox activity, which included both metals and nonmetal species, were associated with the particles in the aqueous suspensions. Portions of the redox active compounds were also DCM-soluble. In contrast, the electrophilic constituents included both water-soluble and DCM-soluble species. The role of quinones or quinone-like compounds in redox and electrophilic activities of the DCM-soluble constituents was assessed by reductive acetylation, a procedure that inactivates quinones. The results from this experiment indicated that most of the activities in the organic extract were associated with quinone-like substances. The partition properties of the reactive species are important in exposure assessment since the toxicokinetics of particles and solutes are quite distinct.

REDOX AND ELECTROPHILIC PROPERTIES OF VAPOR- AND PARTICLE-PHASE COMPONENTS OF AMBIENT AEROSOLS

Particulate matter (PM) has been the primary focus of studies aiming to understand the relationship between the chemical properties of ambient aerosols and adverse health effects. Size and chemical composition of PM have been linked to their oxidative capacity which has been postulated to promote or exacerbate pulmonary and cardiovascular diseases. But in the last few years, new studies have suggested that volatile and semi-volatile components may also contribute to many adverse health effects. The objectives of this study were: (i) assess for the first time the redox and electrophilic potential of vapor-phase components of ambient aerosols and (ii) evaluate the relative contributions of particle- and vapor-fractions to the hazard of a given aerosol. To achieve these objectives vapor- and particle-phase samples collected in Riverside (CA) were subjected to three chemical assays to determine their redox and electrophilic capacities. The results indicate that redox active components are mainly associated with the particle-phase, while electrophilic compounds are found primarily in the vapor-phase. Vapor-phase organic extracts were also capable of inducing the stress responding protein, heme-oxygenase-1 (HO-1), in RAW264.7 murine macrophages. These results demonstrate the importance of volatile components in the overall oxidative and electrophilic capacity of aerosols, and point out the need for inclusion of vapors in future health and risk assessment studies.

Atmospheric Distribution of Gas- and Particle-Phase Quinones in Southern California

Quinones are reactive organic compounds known to initiate reactions associated with a host of toxicological events. Their presence in different atmospheres has been demonstrated although their sources remain uncertain. As a result of their reactivity and instability during chemical analysis, only a limited number of studies have reported on atmospheric concentrations of quinones in ambient air. Furthermore, besides the limited information on quinones associated with particulate matter, no previous studies have quantified vapor-phase quinones. We report vapor- and particle-phase concentrations of 1,2- and 1,4-naphthoquinones (1,2-NQ, 1,4-NQ), 9,10-phenanthraquinone (9,10-PQ), and 9,10-anthraquinone (9,10-AQ), measured over a 5-year period in Southern California. The results showed that vapor-phase concentrations of the target quinones were in general higher than those in the particle-phase. Vapor-phase concentrations ranged from 80 pg/m 3 for the AQ to 1747 pg/m 3 for the 1,4-NQ, and the particle-phase concentrations between 13 pg/m 3 for the 1,2-NQ and 250 pg/m 3 for 9,10-AQ. The target quinones were found to be distributed between vapor- and particle-phase, with the exception of 9,10-PQ found only in the particle-phase. The differences observed in the concentrations among sites and seasons suggest different source contributions; source sites were dominated by primary sources, while downwind locations showed a high contribution from photochemical activity.

Caudate-putamen dopamine and stereotypy response profiles after intravenous and subcutaneous amphetamine.

We compared the behavioral and caudate–putamen extracellular dopamine responses following intravenous (3.6 mg/kg) and subcutaneous (8 mg/kg) amphetamine administration using 2‐min microdialysate sampling intervals, and doses of the drug selected to achieve comparable maximal brain concentrations. Following intravenous amphetamine, dopamine peaked within the first 2 min, then declined with a first‐order decay rate of 0.018 ± 0.007 min−1. Following subcutaneous amphetamine, dopamine achieved maximum concentrations at 9 min and remained near peak levels for about 30 min before declining with a first‐order decay rate of 0.019 ± 0.008 min−1. Maximal brain amphetamine levels and peak dopamine concentrations were equivalent following either route of drug administration. In contrast to the short latency to maximal extracellular dopamine, the onset of oral stereotypies was delayed until about 30 min following both routes of drug administration. Furthermore, in contrast to the behavioral response to amphetamine, apomorphine administration resulted in the rapid appearance of oral stereotypies within 5–10 min after drug administration. These results suggest that although caudate–putamen dopamine receptor activation may be a critical factor in the expression of focused oral stereotypies, other effects of amphetamine may interfere with the ability of animals to exhibit these behaviors. Synapse 31:125–133, 1999.

Metabolism of methylenedioxyphenyl compounds by Rabbit liver preparations: Participation of different cytochrome P450 isozymes in the demethylenation reaction

The cytochrome P450-mediated oxidative demethylenation of the benzo-1,3-dioxoles (methylenedioxyphenyl compounds, MDPs), methylenedioxybenzene (MDB), methylenedioxyamphetamine (MDA), and methylenedioxymethamphetamine (MDMA), by rabbit liver microsomes and cytochrome P450IIB4 (CYP2B4) was examined. Material balance studies indicated that demethylenation to catechol derivatives is a major metabolic pathway for MDB, MDA and MDMA. The reactions required NADPH and were inhibited by CO/O2 (4:1, v/v). Biphasic double-reciprocal plots of MDMA, MDA and MDB oxidation suggested participation of more than one isozyme of cytochrome P450 in the reaction. Phenobarbital (PB) induction was selective in that the Vmax values for MDB were increased but not those for MDA and MDMA. Exposure of liver microsomes from PB-pretreated animals to phencyclidine (PCP) markedly suppressed MDB oxidation but had little effect on MDA and MDMA demethylenation. Reconstitution experiments with CYP2B4 demonstrated that MDB is a good substrate for the isozyme; but the relative demethylenation activities for MDA and MDMA were 1 and 2% of that for MDB. These results indicate that the PB-inducible isozymes such as CYP2B4 appear to play an important role in MDB demethylenation, whereas MDA and MDMA oxidation is mediated mainly by constitutive isozymes.

Pharmacokinetic and pharmacodynamic properties of some phencyclidine analogs in rats

The pharmacodynamics and pharmacokinetics of three phencyclidine analogs, differing from phencyclidine (PCP) only in the nature of the amine structure, were determined after intravenous doses of equimolar amounts to rats. The purpose of the study was to assess the role of pharmacokinetics in the in vivo potency of the compounds. The compounds examined were phenylcyclohexyl-pyrrolidine (PCPY), diethylamine (PCDE), ethylamine (PCE), and phencyclohexylamine (PCA). The behavior responses monitored included ataxia and others previously shown to be characteristic of PCP. In contrast to their relative affinities for the MK 801 binding site, the behavioral potencies of PCE, PCDE and PCPY were comparable to PCP. The major discrepancy occurred with PCDE, whose affinity for the NMDA receptor was 1/20th of PCP. The pharmacokinetic studies showed that the discrepancy between in vivo and in vitro activity of PCDE could be partially accounted for by its conversion to PCE, a relatively potent PCP-like agent.

Induction of phencyclidine metabolism by phencyclidine, ketamine, ethanol, phenobarbital and isosafrole.

The in vitro metabolism of phencyclidine (PCP) was investigated in 9000 g supernatant fractions of both control and PCP-, ketamine-, ethanol-, phenobarbital- or isosafrole-pretreated rats. Levels of PCP, trans-4-phenyl-4-piperidinocyclohexanol (I), 1-(1-phenylcyclohexyl)-4-hydroxypiperidine (II), N-(5-hydroxypentyl)-1-phenylcyclohexylamine (IX), and 5-(1-phenylcyclohexylamino)-valeric acid (X) were monitored by gas chromatographic analysis in all cases. The inhibition of metabolism by N2, CO, SKF-525A or 2,4-dichloro-6-phenylphenoxyethylamine (DPEA), or deletion of NADPH or protein, implied the involvement of cytochrome P-450 in the reactions. The various inducing agents affected the metabolism of PCP in different ways, implying that at least several isozymes of cytochrome P-450 were involved in the total metabolism. The majority of the consumed PCP was not accounted for by the measured metabolites so that some other metabolic pathways of major quantitative importance must be operative.