Movement to the Clinic of Soluble Epoxide Hydrolase Inhibitor EC5026 as an Analgesic for Neuropathic Pain and for Use as a Nonaddictive Opioid Alternative.

This report describes the development of an orally active analgesic that resolves inflammation and neuropathic pain without the addictive potential of opioids. EC5026 acts on the cytochrome P450 branch of the arachidonate cascade to stabilize epoxides of polyunsaturated fatty acids (EpFA), which are natural mediators that reduce pain, resolve inflammation, and maintain normal blood pressure. EC5026 is a slow-tight binding transition-state mimic that inhibits the soluble epoxide hydrolase (sEH) at picomolar concentrations.

Simultaneous quantification of multiple urinary naphthalene metabolites by liquid chromatography tandem mass spectrometry.

Naphthalene is an environmental toxicant to which humans are exposed. Naphthalene causes dose-dependent cytotoxicity to murine airway epithelial cells but a link between exposure and human pulmonary disease has not been established. Naphthalene toxicity in rodents depends on P450 metabolism.

Alterations in the proteome of the respiratory tract in response to single and multiple exposures to naphthalene.

Protein adduction is considered to be critical to the loss of cellular homeostasis associated with environmental chemicals undergoing metabolic activation. Despite considerable effort, our understanding of the key proteins mediating the pathologic consequences from protein modification by electrophiles is incomplete. This work focused on naphthalene (NA) induced acute injury of respiratory epithelial cells and tolerance which arises after multiple toxicant doses to define the initial cellular proteomic response and later protective actions related to tolerance.

Metabolic activation of the antibacterial agent triclocarban by cytochrome P450 1A1 yielding glutathione adducts.

Triclocarban (3,4,4'-trichlorocarbanilide; TCC) is an antibacterial agent used in personal care products such as bar soaps. Small amounts of chemical are absorbed through the epidermis. Recent studies show that residues of reactive TCC metabolites are bound covalently to proteins in incubations with keratinocytes, raising concerns about the potential toxicity of this antimicrobial agent.

Sex differences in the acute nasal antioxidant/antielectrophilic response of the rat to inhaled naphthalene.

Naphthalene is a nasal carcinogen, inducing respiratory adenomas in male and olfactory neuroblastomas in female rats, respectively. The reasons for the site and sex-specific tumorigenic response are unknown. Naphthalene is bioactivated to electrophilic metabolites; cytotoxicity followed by regenerative cell proliferation is likely involved in the tumorigenic response.

Kinetics of naphthalene metabolism in target and non-target tissues of rodents and in nasal and airway microsomes from the Rhesus monkey.

Naphthalene produces species and cell selective injury to respiratory tract epithelial cells of rodents. In these studies we determined the apparent Km, Vmax, and catalytic efficiency (Vmax/Km) for naphthalene metabolism in microsomal preparations from subcompartments of the respiratory tract of rodents and non-human primates. In tissues with high substrate turnover, major metabolites were derived directly from naphthalene oxide with smaller amounts from conjugates of diol epoxide, diepoxide, and 1,2- and 1,4-naphthoquinones.

Characterization of model peptide adducts with reactive metabolites of naphthalene by mass spectrometry.

Naphthalene is a volatile polycyclic aromatic hydrocarbon generated during combustion and is a ubiquitous chemical in the environment. Short term exposures of rodents to air concentrations less than the current OSHA standard yielded necrotic lesions in the airways and nasal epithelium of the mouse, and in the nasal epithelium of the rat. The cytotoxic effects of naphthalene have been correlated with the formation of covalent protein adducts after the generation of reactive metabolites, but there is little information about the specific sites of adduction or on the amino acid targets of these metabolites.

Analysis of naphthalene adduct binding sites in model proteins by tandem mass spectrometry.

The electrophilic metabolites of the polyaromatic hydrocarbon naphthalene have been shown to bind covalently to proteins and covalent adduct formation correlates with the cytotoxic effects of the chemical in the respiratory system. Although 1,2-naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-napthoquinone have been identified as reactive metabolites of interest, the role of each metabolite in total covalent protein adduction and subsequent cytotoxicity remains to be established. To better understand the target residues associated with the reaction of these metabolites with proteins, mass spectrometry was used to identify adducted residues following (1) incubation of metabolites with actin and protein disulfide isomerase (PDI), and (2) activation of naphthalene in microsomal incubations containing supplemental actin or PDI.

Free flow electrophoresis separation and AMS quantitation of C-naphthalene-protein adducts.

Naphthalene is a volatile aromatic hydrocarbon to which humans are exposed from a variety of sources including mobile air sources and cigarette smoke. Naphthalene produces dose- (concentration) dependent injury to airway epithelial cells of murine lung which is observed at concentrations well below the current occupational exposure standard. Toxicity is dependent upon the cytochrome P450 mediated metabolic activation of the parent substrate to unstable metabolites which become bound covalently to tissue proteins.

Formation of covalently bound protein adducts from the cytotoxicant naphthalene in nasal epithelium: species comparisons.

Background: Naphthalene is a volatile hydrocarbon that causes dose-, species-, and cell type-dependent cytotoxicity after acute exposure and hyperplasia/neoplasia after lifetime exposures in rodents. Toxicity depends on metabolic activation, and reactive metabolite binding correlates with tissue and site susceptibility.

Objectives: We compared proteins adducted in nasal epithelium from rats and rhesus macaques in vitro.

Protein thiol oxidation in murine airway epithelial cells in response to naphthalene or diethyl maleate.

Naphthalene (NA) is a semivolatile aromatic hydrocarbon to which humans are exposed from a variety of sources. NA results in acute cytotoxicity to respiratory epithelium in rodents. Cytochrome P450-dependent metabolic activation to form reactive intermediates and loss of soluble cellular thiols (glutathione) are critical steps in NA toxicity, but the precise mechanisms by which this chemical results in cellular injury remain unclear.

Upper respiratory tract uptake of naphthalene.

Naphthalene is a nasal toxicant and carcinogen in the rat. Upper respiratory tract (URT) uptake of naphthalene was measured in the male and female F344 rat at exposure concentrations of 1, 4, 10, or 30 ppm at inspiratory flow rates of 150 or 300 ml/min. To assess the potential importance of nasal cytochrome P450 (CYP) metabolism, groups of rats were pretreated with the CYP inhibitor 5-phenyl-1-pentyne (PP) (100 mg/kg, ip).

Toxicity and metabolism of methylnaphthalenes: comparison with naphthalene and 1-nitronaphthalene.

Naphthalene and close structural analogues have been shown to cause necrosis of bronchiolar epithelial cells in mice by both inhalation exposure and by systemic administration. Cancer bioassays of naphthalene in mice have demonstrated a slight increase in bronchiolar/alveolar adenomas in female mice, and in inflammation and metaplasia of the olfactory epithelium in the nasal cavity. Similar work in rats demonstrated a significant, and concentration-dependent increase in the incidence of respiratory epithelial adenomas and neuroblastomas in the nasal epithelium of both male and female rats.

Measurement of protein sulfhydryls in response to cellular oxidative stress using gel electrophoresis and multiplexed fluorescent imaging analysis.

The significance of free radicals in biology has been established by numerous investigations spanning a period of over 40 years. Whereas there are many intracellular targets for these radical species, the importance of cysteine thiol posttranslational modification has received considerable attention. The current studies present a highly sensitive method for measurement of the posttranslational modification of protein thiols.

Development of metabolically stable inhibitors of Mammalian microsomal epoxide hydrolase.

The microsomal epoxide hydrolase (mEH) plays a significant role in the metabolism of xenobiotics such as polyaromatic toxicants. Additionally, polymorphism studies have underlined a potential role of this enzyme in relation to a number of diseases, such as emphysema, spontaneous abortion, eclampsia, and several forms of cancer. We recently demonstrated that fatty amides, such as elaidamide, represent a new class of potent inhibitors of mEH.

TRX-ASK1-JNK signaling regulation of cell density-dependent cytotoxicity in cigarette smoke-exposed human bronchial epithelial cells.

Cigarette smoke is a major environmental air pollutant that injures airway epithelium and incites subsequent diseases including chronic obstructive pulmonary disease. The lesion that smoke induces in airway epithelium is still incompletely understood. Using a LIVE/DEAD cytotoxicity assay, we observed that subconfluent cultures of bronchial epithelial cells derived from both human and monkey airway tissues and an immortalized normal human bronchial epithelial cell line (HBE1) were more susceptible to injury by cigarette smoke extract (CSE) and by direct cigarette smoke exposure than cells in confluent cultures.

Hemin rescues adrenodoxin, heme a and cytochrome oxidase activity in frataxin-deficient oligodendroglioma cells.

Mutations in the frataxin gene cause neurodegeneration and demyelination in Friedreich's ataxia. We showed earlier that frataxin deficiency causes primary iron-sulfur cluster defects, and later causes defects in heme and cytochrome c hemoprotein levels. Iron-sulfur (Fe/S) clusters are required in two enzymes of heme biosynthesis in humans i.

Asthma/allergic airways disease: does postnatal exposure to environmental toxicants promote airway pathobiology?

Unlabelled: The recent, dramatic increase in the incidence of childhood asthma suggests a role for environmental contaminants in the promotion of interactions between allergens and the respiratory system of young children. To establish whether exposure to an environmental stressor, ozone (O3), and an allergen, house dust mite (HDMA), during early childhood promotes remodeling of the epithelial-mesenchymal trophic unit (EMTU) of the tracheobronchial airway wall by altering postnatal development, infant rhesus monkeys were exposed to cyclic episodes of filtered air (FA), HDMA, O3, or HDMA plus O3. The following alterations in the EMTU were found after exposure to HDMA, O3, or HDMA plus O3: (1) reduced airway number; (2) hyperplasia of bronchial epithelium; (3) increased mucous cells; (4) shifts in distal airway smooth muscle bundle orientation and abundance to favor hyperreactivity; (5) interrupted postnatal basement membrane zone differentiation; (6) modified epithelial nerve fiber distribution; and (7) reorganization of the airway vascular and immune system.

Identification of proteins adducted by reactive metabolites of naphthalene and 1-nitronaphthalene in dissected airways of rhesus macaques.

Naphthalene and 1-nitronaphthalene are ambient air pollutants, which undergo P450-dependent bioactivation in the lung. Reactive metabolites of naphthalene and 1-nitronaphthalene covalently bind to proteins, and the formation of covalent adducts correlates with airway epithelial cell injury in rodent models. These studies were designed to identify protein adducts generated from these reactive metabolites within distal respiratory airways.

Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis.

2-DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2-DE analysis.

Software-induced variance in two-dimensional gel electrophoresis image analysis.

Experimental variability in 2-DE is well documented, but little attention has been paid to variability arising from postexperimental quantitative analyses using various 2-DE software packages. The performance of two 2-DE analysis software programs, Phoretix 2D Expression v2004 (Expression) and PDQuest 7.2 (PDQuest), was evaluated in this study.

Frataxin deficiency alters heme pathway transcripts and decreases mitochondrial heme metabolites in mammalian cells.

Deficiency of the frataxin mRNA alters the transcriptome, triggering neuro- and cardiodegeneration in Friedreich's ataxia. We microarrayed murine frataxin-deficient heart tissue, liver tissue and cardiocytes and observed a transcript down-regulation to up-regulation ratio of nearly 2:1 with a mitochondrial localization of transcriptional changes. Combining all mouse and human microarray data for frataxin-deficient cells and tissues, the most consistently decreased transcripts were mitochondrial coproporphyrinogen oxidase (CPOX) of the heme pathway and mature T-cell proliferation 1, a homolog of yeast COX23, which is thought to function as a mitochondrial metallochaperone.

Identification of proteins adducted by reactive naphthalene metabolites in vitro.

Metabolic activation of inert chemicals to electrophilic intermediates has been correlated with the incidence and severity of cytotoxicity. The current studies have identified several proteins adducted by reactive metabolites of the lung toxicant, naphthalene. Proteins isolated from microsomal incubations of (14)C-naphthalene were separated by 2-DE, proteins were blotted to PVDF membranes and radioactive proteins were localized by storage phosphor analysis.

Characterization of a structurally intact in situ lung model and comparison of naphthalene protein adducts generated in this model vs lung microsomes.

Airway epithelial cells are a susceptible site for injury by ambient air toxicants such as naphthalene that undergo P450-dependent metabolic activation. The metabolism of naphthalene in Clara cells to reactive intermediates that bind covalently to proteins correlates with cell toxicity. Although several proteins adducted by reactive naphthalene metabolites were identified in microsomal incubations, new methods that maintain the structural integrity of the lung are needed to examine protein targets.

In vivo effects of ozone exposure on protein adduct formation by 1-nitronaphthalene in rat lung.

The incidence of serious photochemical smog events is steadily growing in urban environments around the world. The electrophilic metabolites of 1-nitronaphthalene (1-NN), a common air pollutant in urban areas, have been shown to bind covalently to proteins. 1-NN specifically targets the airway epithelium, and the toxicity is synergized by prior long-term ozone exposure in rat.