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. The sEH rapidly degrades EpFA; thus, inhibiting sEH increases EpFA in vivo and confers beneficial effects. This mechanism addresses disease states by shifting endoplasmic reticulum stress from promoting cellular senescence and inflammation toward cell survival and homeostasis. We describe the synthesis and optimization of EC5026 and its development through human Phase 1a trials with no drug-related adverse events. Additionally, we outline fundamental work leading to discovery of the analgesic and inflammation-resolving CYP450 branch of the arachidonate cascade.

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. Subsequent biotransformation results in urinary elimination of several conjugated metabolites. Glucuronide and sulfate conjugates of naphthols have been used as markers of naphthalene exposure but, as the current studies demonstrate, these assays provide a limited view of the range of metabolites generated from the parent hydrocarbon. Here, we present a liquid chromatography tandem mass spectrometry method for measurement of the glucuronide and sulfate conjugates of 1-naphthol as well as the mercapturic acids and N-acetyl glutathione conjugates from naphthalene epoxide. Standard curves were linear over 2 log orders. On column detection limits varied from 0.91 to 3.4 ng; limits of quantitation from 1.8 to 6.4 ng. The accuracy of measurement of spiked urine standards was -13.1 to + 5.2% of target and intra-day and inter-day variability averaged 7.2 (± 4.5) and 6.8 (± 5.0) %, respectively. Application of the method to urine collected from mice exposed to naphthalene at 15 ppm (4 hrs) showed that glutathione-derived metabolites accounted for 60-70% of the total measured metabolites and sulfate and glucuronide conjugates were eliminated in equal amounts. The method is robust and directly measures several major naphthalene metabolites including those derived from glutathione conjugation of naphthalene epoxide. The assays do not require enzymatic deconjugation, extraction or derivatization thus simplifying sample work up.

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. Airways and nasal olfactory epithelium from mice exposed to 15 ppm NA either for 4 h (acute) or for 4 h/day × 7 days (tolerant) were used for label-free protein quantitation by LC/MS/MS. Cytochrome P450 2F2 and secretoglobin 1A1 are decreased dramatically in airways of mice exposed for 4 h, a finding consistent with the fact that CYPs are localized primarily in Clara cells. A number of heat shock proteins and protein disulfide isomerases, which had previously been identified as adduct targets for reactive metabolites from several lung toxicants, were upregulated in airways but not olfactory epithelium of tolerant mice. Protein targets that are upregulated in tolerance may be key players in the pathophysiology associated with reactive metabolite protein adduction. All MS data have been deposited in the ProteomeXchange with identifier PXD000846 (http://proteomecentral.proteomexchange.org/dataset/PXD000846).

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. To obtain additional information on metabolic activation of TCC, this study characterized the reactive metabolites trapped as glutathione conjugates. Incubations were carried out with (14)C-labeled TCC, recombinant CYP1A1 or CYP1B1, coexpressed with cytochrome P450 reductase, glutathione-S-transferases (GSH), and an NADPH-generating system. Incubations containing CYP1A1, but not 1B1, led to formation of a single TCC-GSH adduct with a conversion rate of 1% of parent compound in 2 hours. Using high-resolution mass spectrometry and diagnostic fragmentation, the adduct was tentatively identified as 3,4-dichloro-3'-glutathionyl-4'-hydroxycarbanilide. These findings support the hypothesis that TCC is activated by oxidative dehalogenation and oxidation to a quinone imine. Incubations of TCDD-induced keratinocytes with (14)C-TCC yielded a minor radioactive peak coeluting with TCC-GSH. Thus, we conclude that covalent protein modification by TCC in TCDD-induced human keratinocyte incubations is mainly caused by activation of TCC by CYP1A1 via a dehalogenated TCC derivative as reactive species.

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. To examine sex differences in the acute nasal response to naphthalene, male and female F344 rats were nose-only exposed to 0, 1, 3, 10, or 30 ppm naphthalene vapor for 4 or 6 h. Following exposure, respiratory/transitional mucosa (RTM) and olfactory mucosa (OM) were isolated and analyzed for markers of oxidant/electrophilic stress and/or toxicity, including reduced/oxidized glutathione levels (GSH/GSSG), mRNA levels of electrophile-responsive genes, and epithelial cytoxicity (as measured by membrane permeability to ethidium homodimer-1). Naphthalene caused significant depletion of GSH in RTM and OM with no increase in GSSG. Cytotoxicity was apparent at concentrations of 15 and 30 ppm. No consistent sex differences were observed in these responses. Sex differences were observed in the induction of antielectrophilic genes in OM: glutamyl cysteine ligase (catalytic subunit) (Gclc), NADPH quinone oxidase 1 (Nqo1), and heme oxygenase 1 (Hmox1) were all induced to a greater extent in the male OM compared with the female. No consistent sex differences were observed in the RTM. Although the mechanism of the sex difference in the RTM adenoma response remains enigmatic, sex differences in the induction of antioxidant/electrophile-responsive genes may contribute to the heightened sensitivity of the female OM to the carcinogenic effects of naphthalene.

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. In some tissues, different enzymes with dissimilar Km and Vmax appeared to metabolize naphthalene. The rank order of Vmax (rat olfactory epithelium>mouse olfactory epithelium>murine airways>>rat airways) correlated well with tissue susceptibility to naphthalene. The Vmax in monkey alveolar subcompartment was 2% that in rat nasal olfactory epithelium. Rates of metabolism in nasal compartments of the monkey were low. The catalytic efficiencies of microsomes from known susceptible tissues/subcompartments are 10 and 250 fold higher than in rat airway and monkey alveolar subcompartments, respectively. Although the strong correlations between catalytic efficiencies and tissue susceptibility suggest that non-human primate tissues are unlikely to generate metabolites at a rate sufficient to produce cellular injury, other studies showing high levels of formation of protein adducts support the need for additional studies.

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. To better understand the chemical species produced when naphthalene metabolites react with proteins and peptides, we studied the formation and structure of the resulting adducts from the incubation of model peptides with naphthalene epoxide, naphthalene diol epoxide, 1,2-naphthoquinone, and 1,4-naphthoquinone using high resolution mass spectrometry. Identification of the binding sites, relative rates of depletion of the unadducted peptide, and selectivity of binding to amino acid residues were determined. Adduction occurred on the cysteine, lysine, and histidine residues, and on the N-terminus. Monoadduct formation occurred in 39 of the 48 reactions. In reactions with the naphthoquinones, diadducts were observed, and in one case, a triadduct was detected. The results from this model peptide study will assist in data interpretation from ongoing work to detect peptide adducts in vivo as markers of biologic effect.

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. All four reactive metabolites bound to Cys, Lys or His residues in actin and PDI. Cys17 of actin was the only residue adducted by all metabolites; there was substantial metabolite selectivity for the majority of adducted residues. Modifications of actin and PDI, following microsomal incubations containing ¹4C-naphthalene, were detected readily by 2D gel electrophoresis and phosphor imaging. However, target modifications on tryptic peptides from these isolated proteins could not be readily detected by MALDI/TOF/TOF and only three modified peptides were detected using high resolution-selective ion monitoring (HR-SIM). All the reactive metabolites investigated have the potential to modify several residues in a single protein, but even in tissues with very high rates of naphthalene activation, the extent of modification was too low to allow unambiguous identification of a significant number of modified residues in the isolated proteins.

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. Nearly 70 proteins have been identified as forming adducts with reactive naphthalene metabolites using in vitro systems but very little work has been conducted in vivo because reasonably large amounts (100 µCi) of (14)C labeled parent compound must be administered to generate detectable adduct levels on storage phosphor screens following separation of labeled proteins by 2 D gel electrophoresis. The work described here was done to provide proof of concept that protein separation by free flow electrophoresis followed by AMS detection of protein fractions containing protein bound reactive metabolites would provide adducted protein profiles in animals dosed with trace quantities of labeled naphthalene. Mice were administered 200 mg/kg naphthalene intraperitoneally at a calculated specific activity of 2 DPM/nmol (1 pCi/nmol) and respiratory epithelial tissue was obtained by lysis lavage 4 hr post injection. Free flow electrophoresis (FFE) separates proteins in the liquid phase over a large pH range (2.5-11.5) using low molecular weight acids and bases to modify the pH. The apparatus separates fractions into standard 96-well plates that can be used in other protein analysis techniques. The buffers of the fractions have very high carbon content, however, and need to be dialyzed to yield buffers compatible with (14)C-AMS. We describe the processing techniques required to couple FFE to AMS for quantitation of protein adducts.

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. METHODS: Adducted proteins recovered from incubations of nasal epithelium and 14C-naphthalene were separated by two-dimensional (2D) gel electrophoresis and imaged to register radioactive proteins. We identified proteins visualized by silver staining on complementary non-radioactive gels by peptide mass mapping. RESULTS: The levels of reactive metabolite binding in incubations of rhesus ethmo-turbinates and maxillo-turbinates are similar to those in incubations of target tissues, including rat septal/-olfactory regions and murine dissected airway incubations. We identified 40 adducted spots from 2D gel separations of rat olfactory epithelial proteins; 22 of these were non-redundant. In monkeys, we identified 19 spots by mass spectrometry, yielding three non-redundant identifications. Structural proteins (actin/tubulin) were prominent targets in both species. CONCLUSIONS: In this study we identified potential target proteins that may serve as markers closely associated with toxicity. The large differences in previously reported rates of naphthalene metabolism to water-soluble metabolites in dissected airways from mice and monkeys are not reflected in similar differences in covalent adduct formation in the nose. This raises concerns that downstream metabolic/biochemical events are very similar between the rat, a known target for naphthalene toxicity and tumorigenicity, and the rhesus macaque, a species similar to the human.

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. Protein thiols are likely targets of reactive NA metabolites. Loss of these, through adduction or thiol oxidation mechanisms, may be important underlying mechanisms for NA toxicity. To address the hypothesis that loss of thiols on specific cellular proteins is critical to NA-induced cytotoxicity, we compared reduced to oxidized thiol ratios in airway epithelial cell proteins isolated from lungs of mice treated with NA or the nontoxic glutathione depletor, diethyl maleate (DEM). At 300 mg/kg doses, NA administration resulted in a greater than 85% loss of glutathione levels in the airway epithelium, which is similar to the loss observed after DEM treatment. Using differential fluorescent maleimide labeling followed by 2DE separation of proteins, we identified more than 35 unique proteins that have treatment-specific differential sulfhydryl oxidation. At doses of NA and DEM that produce similar levels of glutathione depletion, Cy3/Cy5 labeling ratios were statistically different for 16 nonredundant proteins in airway epithelium. Proteins identified include a zinc finger protein, several aldehyde dehydrogenase variants, beta-actin, and several other structural proteins. These studies show distinct patterns of protein thiol alterations with the noncytotoxic DEM and the cytotoxic NA.

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). In vitro metabolism of naphthalene was similar in nasal tissues from both genders and was reduced by 80% by the inhibitor. URT uptake in female rats was concentration dependent with uptake efficiencies (flow 150 ml/min) of 56, 40, 34, and 28% being observed at inspired concentrations of 1, 4, 10, and 30 ppm, respectively. A similar effect was observed in male rats (flow 150 ml/min) with uptake efficiencies of 57, 49, 37, and 36% being observed. Uptake was more efficient in the male than female rat, likely due to their larger size (226 vs. 144 g). Uptake of naphthalene was significantly reduced by inhibitor pretreatment with the effect being greater at the lower inspired concentrations. Specifically, in pretreated female rats (150 ml/min), URT uptake averaged 25, 29, and 26% at inspired concentrations of 4, 10, and 30 ppm, respectively. Thus, the concentration dependence of uptake was virtually abolished by PP pretreatment. These results provide evidence that nasal CYP metabolism of naphthalene contributes to URT scrubbing of this vapor and is also involved in the concentration dependence of uptake that is observed.

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. Although the studies on the acute toxicity of the methylnaphthalene derivatives are more limited, it appears that the species selective toxicity associated with naphthalene administration also is observed with methylnaphthalenes. Chronic administration of the methylnaphthalenes, however, failed to demonstrate the same oncogenic potential as that observed with naphthalene. The information available on the isopropylnaphthalene derivatives suggests that they are not cytotoxic. Like the methylnaphthalenes, 1-nitronaphthalene causes lesions in both Clara and ciliated cells. However, the species selective lung toxicity observed in the mouse with both naphthalene and the methylnaphthalenes is not seen with 1-nitronaphthalene. With 1-nitronaphthalene, the rat is far more susceptible to parenteral administration of the compound than mice. The wide-spread distribution of these compounds in the environment and the high potential for low level exposure to humans supports a need for further work on the mechanisms of toxicity in animal models with attention to whether these processes are applicable to humans. Although it is tempting to suppose that the toxicity and mechanisms of toxicity of the alkylnaphthalenes and nitronaphthalenes are similar to naphthalene, there is sufficient published literature to suggest that this may not be the case. Certainly the enzymes involved in the metabolic activation of each of these substrates are likely to differ. The available data showing extensive oxidation of the aromatic nucleus of naphthalene, nitronaphthalene and the methylnaphthalenes (with some oxidation of the methyl group) contrast with the isopropylnaphthalene derivatives, where the major metabolites involve side chain oxidation. Overall, these data support the view that ring epoxidation is a key step in the process involved in cytotoxicity. Whether the epoxide itself or a downstream metabolite mediates the toxic effects is still not clear even with naphthalene, the best studied of this group of compounds. Additional work is needed in several areas to further assess the potential human health consequences of exposure to these agents. These studies should involve the definition of the extent and severity of methylnaphthalene toxicity after single dose exposures with attention to both the nasal and respiratory epithelia. The cytochromes P450 responsible for the initial activation of these agents in rodents with subsequent complimentary studies in primate models should help determine whether key metabolic processes responsible for toxicity occur also in primates. Finally, the precise involvement of reactive metabolite formation and adduction of cellular proteins in toxicity will be important in not only assessing the potential for human toxicity, but also in developing an understanding of the genetic and environmental factors which could alter the toxicity of these agents.

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. This method is based on labeling of proteins with monofunctional maleimide dyes followed by 2D gel electrophoresis to separate proteins and multiplexed fluorescent imaging analysis. The method correctly interrogates the thiol/disulfide ratio present in commercially available proteins. Exposure of pulmonary airway epithelial cells to high concentrations of menadione or t-butyl hydroperoxide resulted in the modification of cysteines in more than 141 proteins of which 60 were subsequently identified by MALDI-TOF/TOF MS. Although some proteins were modified similarly by these two oxidants, several showed detectably different maleimide ratios in response to these two agents. Proteins that were modified by one or both oxidants include those involved in transcription, protein synthesis and folding, and cell death/growth. In conclusion, these studies provide a novel procedure for measuring the redox status of cysteine thiols on individual proteins with a clearly demonstrated applicability to interactions of chemicals with pulmonary epithelial cells.

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. While these compounds are very active on recombinant mEH in vitro, they are quickly inactivated in liver extracts reducing their value in vivo. We investigated the effect of structural changes on mEH inhibition potency and microsomal stability. Results obtained indicate that the presence of a small alkyl group alpha to the terminal amide function and a thio-ether beta to this function increased mEH inhibition by an order of magnitude while significantly reducing microsomal inactivation. The addition of a hydroxyl group 9-10 carbons from the terminal amide function resulted in better inhibition potency without improving microsomal stability. The best compound obtained, 2-nonylsulfanyl-propionamide, is a competitive inhibitor of mEH with a K I of 72 nM. Furthermore, this new inhibitor significantly reduces mEH diol production in ex vivo lungs exposed to naphthalene, underlying the usefulness of the inhibitors described herein. These novel inhibitors could be valuable tools to investigate the physiological and biological roles 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. Scraping confluent cultures also caused an enhanced cell injury predominately in the leading edge of the scraped confluent cultures by CSE. Cellular ATP levels in both subconfluent and confluent cultures were drastically reduced after CSE exposure. In contrast, GSH levels were significantly reduced only in subconfluent cultures exposed to smoke and not in confluent cultures. Western blot analysis demonstrated ERK activation in both confluent and subconfluent cultures after CSE. However, activation of apoptosis signal-regulating kinase 1 (ASK1), JNK, and p38 were demonstrated only in subconfluent cultures and not in confluent cultures after CSE. Using short interfering RNA (siRNA) to JNK1 and JNK2 and a JNK inhibitor, we attenuated CSE-mediated cell death in subconfluent cultures but not with an inhibitor of the p38 pathway. Using the tetracycline (Tet)-on inducible approach, overexpression of thioredoxin (TRX) attenuated CSE-mediated cell death and JNK activation in subconfluent cultures. These results suggest that the TRX-ASK1-JNK pathway may play a critical role in mediating cell density-dependent CSE cytotoxicity.

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.e. in ferrochelatase and adrenodoxin. However, decreases in ferrochelatase activity have not been observed in frataxin-deficient HeLa cells or patient lymphoblasts. We knocked down frataxin in oligodendroglioma cells using siRNA, which produced significant defects in the activity of the Fe/S cluster enzymes adrenodoxin and aconitase, the adrenodoxin product heme a, and cytochrome oxidase, for which heme a serves as a prosthetic group. Exogenous hemin produced a significant rescue of adrenodoxin, aconitase, heme a levels and cytochrome oxidase activity. Thus hemin rescues iron-sulfur cluster defects that are the result of frataxin-deficiency, perhaps as a consequence of increasing the pool of bioavailable iron, and thus should be more fully tested for beneficial effects in Friedreich's ataxia models.

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. CONCLUSIONS: cyclic challenge of infants to toxic stress during postnatal lung development modifies the EMTU. This exacerbates the allergen response to favor development of intermittent airway obstruction associated with wheeze. And, exposure of infants during early postnatal lung development initiates compromises in airway growth and development that persist or worsen as growth continues, even with cessation of exposure.

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. Distal bronchioles and parenchyma from rhesus monkeys were incubated with [(14)C]naphthalene or [(14)C]1-nitronaphthalene. Proteins were separated by 2-DE, blotted to PVDF membranes, and adducted proteins imaged by storage phosphor analysis. MS of in-gel tryptic digests identified numerous adducted proteins including: eight cytoskeletal proteins, two chaperone proteins, seven metabolic enzymes, one redox protein, two proteins involved in ion balance and cell signaling, and two extracellular proteins. While many proteins are adducted by both naphthalene and 1-nitronaphthalene, some are unique to the individual toxicant and airway subcompartment. Although the role which adduction of these proteins plays in cytotoxicity was not evaluated, these studies provide candidate proteins for future work designed to determine the importance of protein adducts in the mechanisms of toxicity and for developing biomarkers useful in determining the relevance of findings in animal models to exposed human populations.

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. Protein samples are spiked with an Alexa-labeled internal standard (ALIS) prior to separation with 2-DE. Due to the high extinction coefficient of the Alexa-fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2-DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II-tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten-fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1-4 ng) and dynamic ranges (10(3)). ALIS is a powerful normalization method for quantitative 2-DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance-variance compared to normalization through division by the total spot volume.