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Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure.

Naphthalene (NA) is a ubiquitous pollutant to which humans are widely exposed. 1,2-Dihydro-1,2-dihydroxynaphthalene (NA-dihydrodiol) is a major metabolite of NA generated by microsomal epoxide hydrolase (mEH). To investigate the role of the NA-dihydrodiol and subsequent metabolites (i.e. 1,2-naphthoquinone) in cytotoxicity, we exposed both male and female wild type (WT) and mEH null mice (KO) to NA by inhalation (5, 10, 20 ppm for 4h). NA-dihydrodiol was ablated in the KO mice. High-resolution histopathology was used to study site-specific cytotoxicity, and formation of naphthalene metabolites was measured by HPLC in microdissected airways. Swollen and vacuolated airway epithelial cells were observed in the intra- and extrapulmonary airways of all mice at and below the current OSHA standard (10 ppm). Female mice may be more susceptible to this acute cytotoxicity. In the extrapulmonary airways, WT mice were more susceptible to damage than KO mice, indicating that the metabolites associated with mEH-mediated metabolism could be partially responsible for cytotoxicity at this site. The level of cytotoxicity in the mEH KO mice at all airway levels suggests that non-mEH metabolites are contributing to NA cellular damage in the lung. Our results indicate that the apparent contribution of mEH-dependent metabolites to toxicity differs by location in the lung. These studies suggest that metabolites generated through the mEH pathway may be of minor importance in distal airway toxicity and subsequent carcinogenesis from NA exposure., (Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.)

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.

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., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

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.

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., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Background: The risks for infants and young children receiving inhaled corticosteroid (ICS) therapy are largely unknown. Recent clinical studies indicate that ICS therapy in pre-school children with symptoms of asthma result in decreased symptoms without influencing the clinical disease course, but potentially affect postnatal growth and development. The current study employs a primate experimental model to identify the risks posed by ICS therapy., Objective: To (1) establish whether ICS therapy in developing primate lungs reverses pulmonary pathobiology associated with allergic airway disease (AAD) and (2) define the impact of ICS on postnatal lung growth and development in primates., Methods: Infant rhesus monkeys were exposed, from 1 through 6 months, to filtered air (FA) with house dust mite allergen and ozone using a protocol that produces AAD (AAD monkeys), or to FA alone (Control monkeys). From three through 6 months, the monkeys were treated daily with ICS (budesonide) or saline., Results: Several AAD manifestations (airflow restrictions, lavage eosinophilia, basement membrane zone thickening, epithelial mucin composition) were reduced with ICS treatment, without adverse effects on body growth or adrenal function; however, airway branching abnormalities and intraepithelial innervation were not reduced. In addition, several indicators of postnatal lung growth and differentiation: vital capacity, inspiratory capacity, compliance, non-parenchymal lung volume and alveolarization, were increased in both AAD and Control monkeys that received ICS treatment., Conclusions and Clinical Relevance: Incomplete prevention of pathobiological changes in the airways and disruption of postnatal growth and differentiation of airways and lung parenchyma in response to ICS pose risks for developing primate lungs. These responses also represent two mechanisms that could compromise ICS therapy's ability to alter clinical disease course in young children., (© 2012 Blackwell Publishing Ltd.)

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.

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.

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.

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.

Substantial gaps exist in our knowledge of the metabolic clearance of therapeutic agents in horses. Accordingly, a cytochrome P450 monooxygenase in the 2C family was cloned from an equine liver, sequenced and expressed in a baculovirus expression system. Catalytic activities of the recombinant protein were measured with a number of substrates. The protein, assigned CYP2C92, displayed optimal catalytic activity with diclofenac using molar ratios of CYP2C92 to NADPH CYP450 reductase of 1:18. Addition of cytochrome b(5) to diclofenac incubations had no significant effect on metabolic turnover. CYP2C92 catalyzed diclofenac metabolism was 20-fold slower than the human counterpart, CYP2C9. CYP2C92 demonstrated comparable tolbutamide and (S)-warfarin hydroxylase activity compared to CYP2C9, upon addition of b(5) to the reactions. The results of this study demonstrate substantial interspecies differences in metabolism of substrates by CYP2C orthologues in the horse and human and support the need to fully characterize this enzyme system in equids.

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.

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.

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.

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.

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.

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. Quantitative RT-PCR studies confirmed the significant down-regulation of Isu1, CPOX and ferrochelatase at 10 weeks in mouse hearts. We observed that mutant cells were resistant to aminolevulinate-dependent toxicity, as expected if the heme pathway was inhibited. Consistent with this, we observed increased cellular protoporphyrin IX levels, reduced mitochondrial heme a and heme c levels and reduced activity of cytochrome oxidase, suggesting a defect between protoporphyrin IX and heme a. Fe-chelatase activities were similar in mutants and controls, whereas Zn-chelatase activities were slightly elevated in mutants, supporting the idea of an altered metal-specificity of ferrochelatase. These results suggest that frataxin deficiency causes defects late in the heme pathway. As ataxic symptoms occur in other diseases of heme deficiency, the heme defect we observe in frataxin-deficient cells could be primary to the pathophysiological process.

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. All available background subtraction and smoothing algorithms were tested using both data generated from one single 2-DE gel image, thus excluding experimental variance, and with authentic sets of replicate gels (n = 5). A slight shift of the image boundaries (the "cropping area") caused both programs to induce variance in protein spot quantification of otherwise identical gel images. The resulting variance for PDQuest (CV(mean) = 8%) was approximately twice that for Expression (CV(mean) = 4%). In authentic sets of replicate 2-DE gels (n = 5), the experimental variance confounded the software-induced variance to some extent. However, Expression still outperformed PDQuest, which exhibited software-induced variance as high as 25% of the total observed variance. Surprisingly, the complete omission of background subtraction algorithms resulted in the least amount of software-based variance. These data indicate that 2-DE gel analysis software constitutes a significant source of the variance observed in quantitative proteomics, and that the use of background subtraction algorithms can further increase the variance.

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. In this study we investigated the formation of 1-NN protein adducts in the rat airway epithelium in vivo and examined how prior long-term ozone exposure affects adduct formation. Eight adducted proteins, several involved in cellular antioxidant defense, were identified. The extent of adduction of each protein was calculated, and two proteins, peroxiredoxin 6 and biliverdin reductase, were adducted at high specific activities (0.36-0.70 and 1.0 nmol adduct/nmol protein). Furthermore, the N-terminal region of calreticulin, known as vasostatin, was adducted only in ozone-exposed animals. Although vasostatin was adducted at relatively low specific activity (0.01 nmol adduct/nmol protein), the adduction only in ozone-exposed animals makes it a candidate protein for elucidating the synergistic toxicity between ozone and 1-NN. These studies identified in vivo protein targets for reactive 1-NN metabolites that are potentially associated with the mechanism of 1-NN toxicity and the synergistic effects of ozone.

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. Therefore, we developed a method that involves inflation of the lungs via the trachea with medium containing (14)C-naphthalene followed by incubation in situ. The viability of this preparation is supported by maintenance of glutathione levels, rates of naphthalene metabolism, and exclusion of ethidium homodimer-1 from airway epithelium. Following in situ incubation, the levels of adduct per milligram of protein were measured in proteins obtained from bronchoalveolar lavage, epithelial cells, and remaining lung. The levels of adducted proteins obtained in lavage and epithelial cells were similar and were 20-fold higher than those in residual lung tissue. (14)C-Labeled adducted proteins were identified by matrix-assisted laser desorption ionization-time-of-flight (MALDI-TOF) mass spectrometry (MS) and quadrupole-TOF MS/MS. Major adducted proteins include cytoskeletal proteins, proteins involved in folding and translocation, ATP synthase, extracellular proteins, redox proteins, and selenium binding proteins. We conclude that in situ incubation maintains structural integrity of the lung while allowing examination of reactive intermediate activation and interaction with target cell proteins of the lung. The proteins adducted and identified from in situ incubations were not the same proteins identified from microsomal incubations.

Naphthalene, a ubiquitous environmental contaminant, produces cytotoxicity in nonciliated bronchiolar epithelial (Clara) cells in mice; rats are refractory to lung cytotoxicity from naphthalene. In contrast, 1-nitronaphthalene is a potent toxicant in both species. Naphthalene is metabolized by CYP2F to a 1,2-epoxide, the first and obligate step in events leading to cytotoxicity. 1-Nitronaphthalene is metabolized to both the 5,6- and the 7,8-epoxides with the 7,8-epoxide predominating in lung. Previous studies have demonstrated recombinant CYP2F2 (mouse) to efficiently metabolize both naphthalene and 1-nitronaphthalene. To better understand the mechanism for the unique toxicity profiles for both compounds, a CYP2F ortholog (CYP2F4) was isolated from rat lung and expressed using a baculovirus system. Recombinant CYP2F4 efficiently generates 1R,2S-naphthalene oxide (K(m) = 3 microM, V(max) = 107 min(-1)) and the 5,6- and 7,8-epoxides of 1-nitronaphthalene (K(m) = 18 microM, V(max) = 25 min(-1) based on total generated glutathione conjugates). Kinetics and regio/stereoselectivity of rat CYP2F4 were indistinguishable from mouse CYP2F2. These results, combined with our recent immunomapping studies demonstrating minimal pulmonary CYP2F expression in rats, indicate that CYP2F expression is the factor most clearly associated with susceptibility to naphthalene-induced pneumotoxicity. CYP2F4 failed to display an enhanced ability to bioactivate 1-nitronaphthalene, an ability that could have potentially compensated for the lower CYP2F pulmonary expression levels in the rat, yet equal species susceptibilities. These results suggest the importance of other P450 enzymes in the epoxidation/bioactivation of 1-nitronaphthalene. Expression of recombinant CYP2F1 (human) yielded an immunoreactive protein with no detectable CO-difference spectrum suggesting inadequate heme incorporation.

Naphthalene is metabolized in the lung and liver to reactive intermediates by cytochrome P450 enzymes. These reactive species deplete glutathione, covalently bind to proteins, and cause necrosis in Clara cells of the lung. The importance of glutathione loss in naphthalene toxicity was investigated by using the glutathione prodrugs (glutathione monoethylester or cysteine-glutathione mixed disulfide) to maintain glutathione pools during naphthalene exposure. Mice given a single intraperitoneal injection of naphthalene (1.5 mmol/kg) were treated with either prodrug (2.5 mmol/kg) 30 min later. Both compounds effectively maintained glutathione levels and decreased naphthalene-protein adducts in the lung and liver. However, cysteine-glutathione mixed disulfide was more effective at preventing Clara cell injury. To study the prodrugs in Clara cells without the influence of hepatic naphthalene metabolism and circulating glutathione, dose-response and time-course studies were conducted with intrapulmonary airway explant cultures. Only the ester of glutathione raised GSH in vitro; however, both compounds limited protein adducts and cell necrosis. In vitro protection was not associated with decreased naphthalene metabolism. We conclude that (1) glutathione prodrugs can prevent naphthalene toxicity in Clara cells, (2) the prodrugs effectively prevent glutathione loss in vivo, and (3) cysteine-glutathione mixed disulfide prevents naphthalene injury in vitro without raising glutathione levels., (Copyright 2005 Wiley Periodicals, Inc.)

Naphthalene (NA) is metabolized to highly reactive intermediates that are primarily detoxified by conjugation to glutathione (GSH). Intraperitoneal administration of naphthalene causes substantial loss of both hepatic and respiratory GSH, yet only respiratory tissues are injured in mice. The liver supplies GSH to other organs via the circulation, making it unclear whether respiratory GSH losses reflect in situ respiratory depletion or decreased hepatic supply. To address this concern, mice were exposed to naphthalene by inhalation (1.5-15 ppm; 2-4 h), thereby bypassing first-pass hepatic involvement. GSH levels and histopathology were monitored during the first 24 h after exposure. Half of the mice were given the GSH depletor diethylmaleate (DEM) 1 hour before naphthalene exposure. Lung and nasal GSH levels rapidly decreased (50-90%) in mice exposed to 15 ppm naphthalene, with cell necrosis throughout the respiratory tract becoming evident several hours later. Conversely, 1.5 ppm naphthalene caused moderate GSH loss and only injured the nasal olfactory epithelium. Neither naphthalene concentration depleted hepatic GSH. Animals pretreated with DEM showed significant GSH loss and injury in nasal and intrapulmonary airway epithelium at both naphthalene concentrations. DEM treatment, perhaps by causing significant GSH loss, decreased water-soluble naphthalene metabolite formation by 48% yet increased NA-protein adducts 193%. We conclude that (1) GSH depletion occurs in airways independent of hepatic function; (2) sufficient GSH is not supplied by the liver to maintain respiratory GSH pools, or to prevent injury from inhaled naphthalene; and (3) GSH loss precedes injury and increases protein adduct formation.

Naphthalene is a ubiquitous environmental contaminant that results in dose-dependent and tissue-, species-, and cell-selective necrosis of murine Clara cells upon exposure. Naphthalene is metabolized by CYP2F to a 1,2-epoxide, the first and obligate step in events leading to cytotoxicity. The studies reported here examine the relationship between levels of transcript (mRNA) and CYP2F protein in the respiratory tract of rodents with tissue susceptibility to injury. In both mice and rats, the lung contains more CYP2F transcript than liver; levels in kidney were undetectable. Mice expressed 4- and 8-fold greater CYP2F transcript in lung and liver tissue, respectively, than rats. Quantitative immunoblot blot analysis of CYP2F in airway subcompartments revealed mice to have 30- (minor daughters/terminal bronchioles), 20- (major daughter), 40- (trachea), and 6- (parenchyma) fold higher levels of CYP2F protein than rats. Within the lungs of both rodent species, the highest CYP2F expression was found in the distal airways. The kidney contained undetectable amounts of CYP2F; multiple immunoreactive bands in liver precluded quantification. The olfactory epithelium contains the greatest amount of cytochrome P450 protein of all tissues studied in the rat, consistent with the observed pattern of in vivo injury. Overall, these studies in rodents demonstrate a strong association between CYP2F expression levels and susceptibility to naphthalene-induced cytotoxicity. Of all primate tissues studied, only the nasal ethmoturbinates contain quantifiable amounts of CYP2F, roughly 10- and 20-fold less than the corresponding tissues in rats and mice, respectively. These results suggest that rhesus macaques may be refractory to naphthalene-induced pulmonary injury.

A key challenge in measuring gene expression changes in the lung in response to site-selective toxicants is differentiating between target and nontarget areas. The toxicity for the cytotoxicant 1-nitronaphthalene is highly localized in the airway epithelium. Target cells comprise but a fraction of the total lung cell mass; measurements from whole lung homogenates are not likely to reflect what occurs at the target site. Additionally, the use of generic microarrays to measure expression in airway epithelium may not provide a good representation of transcripts present at the site of toxic action. cDNA libraries from airway and alveolar subcompartments of rat lung were sequenced for the development of a custom microarray representative of these lung regions. We identified 7,460 nonredundant rat lung sequences. Nearly 30% of the sequences on this array are not present on the Affymetrix Rat GeneChip 230. A 20,000-element microarray was developed that delineates differences in gene expression between subcompartments. This is the first in a series of articles employing this microarray for detecting gene expression changes during acute injury produced by 1-nitronaphthalene and subsequent repair.

Recent developments in genomics, proteomics, and metabolomics hold substantial promise for understanding cellular responses to toxicants. Gene expression profiling is now considered standard procedure, but numerous publications reporting a lack of correlation between mRNA and protein expression emphasize the importance of conducting parallel proteomics studies. The cellular complexity of the lung presents great challenges for in vivo proteomics, and improved isolation methods for proteins from specific lung cell phenotypes are required. To address this issue, we have developed a novel method for isolation of rodent airway epithelial cell proteins that facilitates in vivo proteomics studies of two target-cell pheno-types of the lung, Clara cells and ciliated cells. The airway epithelial cell proteins are reproducibly solubilized, leaving the underlying basement membrane and smooth muscle intact as shown by histopathological analyses. The method yields epithelial cell-specific proteins in fivefold higher concentrations and reduces the yield of nonepithelial cell proteins 13-fold compared with homogenates from microdissected airways. In addition, 36% more protein spots were detectable by two-dimensional gel electrophoresis.

The mechanisms of toxicant-mediated lung injury and repair are influenced by the considerable spatial heterogeneity that exists within the conducting airways of the lungs. As a result of this heterogeneity, significant differences and similarities in gene expression are observed throughout lung subcompartments. RNA-based technologies such as real-time reverse transcription polymerase chain reaction (real-time RT-PCR) and cDNA microarray analysis of gene expression provide valuable clues to understanding the mechanisms of toxicant-induced injury. Isolating RNA from lung subcompartments has previously involved considerable time and labor-intensive processes that limit the number of animals that could be processed in a day. The aim of this study was to determine if intact, high-quality RNA could be preserved in situ over a period of time to delay the need to immediately perform site-specific lung subcompartment microdissections and RNA isolations. Two hours after 1-nitronaphthalene treatment, rat lungs were inflated with and stored in RNA preservation solution and stored at 4 degrees C for 7 days. RNA was isolated from the lung subcompartments isolated by microdissection. After 7 days of storage, the RNA was intact, of high quality, and could be used for real-time RT-PCR to examine heterogeneous gene expression in the lung subcompartments. In summary, this simplified technique of in situ RNA preservation and site-specific lung subcompartment microdissection allows the isolation of intact, high-quality RNA that may be used with molecular RNA-based technologies that will significantly accelerate our understanding of pulmonary injury and repair mechanisms.

The involvement of reactive metabolites in cancer and cellular necrosis has been well established. The nucleophile, glutathione, provides a major mechanism of intracellular protection from electrophilic metabolites. Conjugation with glutathione to generate stable, water-soluble metabolites has been utilized to determine the nature and rates of formation of precursor reactive metabolites. In addition, because activities of the glutathione transferases may play a key role in tissue/cellular susceptibilities to electrophilic compounds, measurement of catalytic activities of these proteins can play an important role in discerning the underlying mechanisms of cell-selective toxicities. This unit outlines HPLC methods found to provide good separation of glutathione conjugates and includes two additional procedures that can be utilized in experiments where high throughput assays are needed for measuring transferase activities.

The present study describes the pharmacokinetics and metabolism of pirfenidone (PD), a compound which has been shown to have significant antifibrotic effects in rodent models of pulmonary and cardiac fibrosis. Despite the fact that this compound is currently in phase II clinical trials, little data are available on the metabolism and disposition of this agent in rodents or humans. Radioactive PD [benzene ring (14)C(U)] was administered i.v. to mice at 40 mg PD/kg body weight, and animals were killed at varying times for determination of parent compound and metabolites in various tissues. The disappearance of parent compound from the plasma followed apparent 2-compartment elimination kinetics with a terminal elimination half-life of 8.6 min. Cl (0.10 ml/min/g) and V(d(ss)) (0.67 ml/g) indicated that PD was rapidly distributed in body water. This is consistent with the finding that peak tissue radioactivity occurred within 5 min following the i.v. administration of [(14)C]-PD and that well-perfused tissues, kidney>liver>lung have much higher levels of parent compound and metabolites than did fat. Two peaks isolated from plasma samples by HPLC yielded mass spectra that were consistent with initial oxidation to the alcohol followed by further metabolism to the carboxylic acid. The radioactivity recovered in the 24 h urine samples averaged 97% of the administered dose and none of that was associated with the parent compound. The short plasma half-life of parent compound in mice supports the need for additional studies in humans where the compound has been shown to have clinical benefits., (Copyright 2002 John Wiley & Sons, Ltd.)

Repeated exposures to the Clara cell cytotoxicant naphthalene (NA) result in target cell populations that become refractory to further injury. To determine whether tolerance occurs from specific adaptations favoring glutathione (GSH) resynthesis without broad shifts in cellular phenotype, mice were administered NA for 21 days. We found that gamma-glutamylcysteine synthetase (gamma-GCS) was induced in tolerant Clara cells by repeated exposures to NA. Treating tolerant mice with buthionine sulfoximine, a gamma-GCS inhibitor, eliminates resistance acquired by repeated exposures to NA. Broad phenotypic shifts were not present. Marker proteins of differentiation declined over the first 3 days in the development of tolerance, but returned to control levels at 14 and 21 days. Epithelial organizational structure and internal organelle composition in Clara cells from tolerant mice were similar compared to corn oil-treated controls, while subtle shifts in organelle distribution were present. We conclude that induction of gamma-GCS expression is coordinated with the development of NA tolerance, but induction of NA tolerance does not markedly alter Clara cell differentiation, epithelial organization, or organelle composition in bronchiolar epithelium.

Current OSHA standards for naphthalene exposure are set at 10 ppm (time-weighted average) with a standard threshold exposure concentration of 15 ppm. While several studies have thoroughly delineated the time course and dose response of injury by naphthalene administered ip, the pattern and severity of injury by inhalation exposure are unknown. These studies compare the regiospecific and dose-dependent cytotoxicity of naphthalene after inhalation exposure. Mice and rats were exposed for 4 h to naphthalene vapor at concentrations of 0-110 ppm. In rats, no injury was observed in the lung epithelium at exposure concentrations up to 100 ppm. Exposures as low as 2 ppm produced proximal airway injury in mice, with increased severity in a concentration-dependent fashion up to 75 ppm. Terminal airways of exposed mice exhibited little or no injury at low concentrations (1-3 ppm). Exposures of 8.5 ppm or higher were required to produce injury to Clara cells in the terminal airways. In contrast, administration of naphthalene (300 mg/kg) extended the injury pattern toward the lobar bronchus. We conclude (1) the pattern of injury to naphthalene is highly dependent on the route of exposure, (2) lung injury to inhaled naphthalene is species dependent, and (3) Clara cells of mouse airways are exquisitely sensitive to inhaled naphthalene at concentrations well below the current OSHA standard for human exposure., (Copyright 2001 Academic Press.)

One of the presumed roles of intracellular glutathione (GSH) is the protection of cells from injury by reactive intermediates produced by the metabolism of xenobiotics. To establish whether GSH depletion is a critical step in the initiation of events that lead to cytotoxicity by P450-activated cytotoxicants, naphthalene, a well-defined Clara cell cytotoxicant, was administered to mice (200 mg/kg) by intraperitoneal injection. Shortly after injection (1, 2, and 3 h), intracellular GSH content was assessed by high performance liquid chromatography or quantitative epifluorescent imaging microscopy and compared with the degree of cytotoxicity as assessed by high resolution histopathology. In highly susceptible airways (distal bronchioles), GSH decreased by 50% in 1 h. Cytoplasmic vacuolization was not visible until 2 h, when GSH had decreased by an additional 50%. By 3 h, cytoplasmic blebbing was extensive. In minimally susceptible airways (lobar and proximal bronchi), GSH depletion varied widely within the population; a small proportion of the cells lost greater than 50% of their GSH by 2 h and a significant percentage of the cells retained most of their GSH throughout the entire 3 h. Cytoplasmic vacuolization was apparent in some of the cells at 2 h but not visible in any cells at 3 h. We conclude that (1) loss of intracellular GSH is an early event that precedes initial signs of cellular damage in Clara cell cytotoxicity; (2) this pattern of loss in relation to early injury is found both in highly susceptible and minimally susceptible airway sites; (3) there is wide cell-to-cell heterogeneity in the response; (4) the heterogeneity in the response profile varies between populations in highly susceptible and minimally susceptible sites; and (5) once the intracellular GSH concentration within the entire cell population drops below a certain threshold, the initial phase of injury becomes irreversible.

To establish whether allergic asthma could be induced experimentally in a nonhuman primate using a common human allergen, three female rhesus monkeys (Macaca mulatta) were sensitized with house dust mite (Dermatophagoides farinae) allergen (HDMA) by subcutaneous injection, followed by four intranasal sensitizations, and exposure to allergen aerosol 3 hours per day, 3 days per week for up to 13 weeks. Before aerosol challenge, all three monkeys skin-tested positive for HDMA. During aerosol challenge with HDMA, sensitized monkeys exhibited cough and rapid shallow breathing and increased airway resistance, which was reversed by albuterol aerosol treatment. Compared to nonsensitized monkeys, there was a fourfold reduction in the dose of histamine aerosol necessary to produce a 150% increase in airway resistance in sensitized monkeys. After aerosol challenge, serum levels of histamine were elevated in sensitized monkeys. Sensitized monkeys exhibited increased levels of HDMA-specific IgE in serum, numbers of eosinophils and exfoliated cells within lavage, and elevated CD25 expression on circulating CD4(+) lymphocytes. Intrapulmonary bronchi of sensitized monkeys had focal mucus cell hyperplasia, interstitial infiltrates of eosinophils, and thickening of the basement membrane zone. We conclude that a model of allergic asthma can be induced in rhesus monkeys using a protocol consisting of subcutaneous injection, intranasal instillation, and aerosol challenge with HDMA.

To test whether exposure to ozone alters pulmonary cytochrome P450 monooxygenase-mediated metabolism of xenobiotics, rates of 1-nitronaphthalene (1-NN) metabolism were measured in microsomes prepared from trachea, intrapulmonary airways, and distal lung of rats exposed to filtered air (FA) or ozone (O(3)) (0.8 ppm 8 h/day for 90 days). Regioisomeric glutathione conjugates derived from intermediate epoxides were measured by HPLC. Compared with FA, rates of glutathione conjugate formation in distal lung (including the central acinus) were elevated 2-fold in O(3)-exposed rats. Activity for cytochrome P450 2B, the isozyme thought to be responsible for the metabolic activation of 1-NN, was increased 3-fold in the distal lung of O(3)- compared with FA-exposed rats. There was a 2 +/- 0. 5-fold increase in immunodetectable CYP 2B protein in microsomes from the same lung subcompartment (P <.05). Immunodetectable protein was expressed in nonciliated epithelial (or "Clara") cells and not associated with ciliated epithelial cells. No differences between O(3)- and FA-exposed rats were noted in 1-NN metabolism or CYP 2B activity in trachea or intrapulmonary airways. This study emphasizes that cellular and biochemical alterations associated with long-term O(3) exposure vary considerably by location within the lung. Long-term exposure to O(3) elevates both CYP 2B activity and 1-NN metabolism in an airway-specific manner.

Glutathione S-transferases (GSTs) and epoxide hydrolases (EHs) protect cells from exogenous insult by detoxifying electrophilic compounds. Little is known about these enzyme systems during postnatal lung development. This study was designed to help establish whether the heightened neonatal susceptibility of the lung to bioactivated cytotoxicants is the result of inadequate ability to detoxify reactive intermediates. We compared the distribution of immunoreactive protein and enzymatic activity of GSTs and EHs in isolated distal airways during pre- and postnatal development in lungs of mice from 16 days gestation to 9 weeks postnatal age (adult). GST alpha, mu, and pi class protein expression in fetal and postnatal lung varied by isozyme and age. Isozymes alpha and mu are expressed at low levels before birth, high levels on postnatal day 7, low levels between postnatal days 14 and 21, high levels at postnatal day 28, and slightly lower levels in adults. Immunoreactive protein of isozyme pi has a peak expression on gestational day 18 and again on postnatal day 4, is undetectable at postnatal day 21, and is at peak levels in the adult mouse lung. GST activity in distal airways increased with age. Microsomal EH protein expression increased in intensity with age, while activity was similar in airways from all ages. We conclude that in the mouse lung (1) cellular expression of glutathione S-transferase varies by age and isozyme and does not increase with increasing age, (2) airway glutathione S-transferase activity increases with increasing age and does not correlate with immunoreactive protein expression, and (3) airway microsomal epoxide hydrolase activity does not increase, even though immunoreactive protein expression does increase with age., (Copyright 2000 Academic Press.)

Repeated exposures to Clara cell cytotoxicants, such as naphthalene (NA), render target cell populations resistant to further acute injury. Previous studies suggest that alterations in bioactivation enzymes in target sites (bronchioles) of tolerant mice are insufficient to account for the marked reduction in susceptibility. Mice were made tolerant by seven daily injections of NA. GSH in the terminal airways was 2.7-fold greater in tolerant mice than in vehicle controls and a NA (300 mg/kg) challenge dose did not produce injury. Tolerant mice, allowed to recuperate for 96 h after the seventh NA injection, were again susceptible to NA injury, and terminal airway GSH levels had declined to control levels. To determine whether alterations in GSH resynthesis account for tolerance, the activity of gamma-glutamylcysteine synthetase (gamma-GCS) was measured or mice were treated with a combination of buthionine sulfoximine (BSO), a gamma-GCS inhibitor, and NA. gamma-GCS activity was elevated in resistant airways of tolerant mice. Tolerant mice treated with both BSO and NA appeared as susceptible to injury as NA-challenged controls. We conclude that GSH is critical for Clara cell resistance to NA injury in tolerant mice because: 1) GSH levels in target airways from NA-tolerant animals are elevated; 2) after a 96-h recuperation period, tolerant mice had lower GSH levels and are again susceptible to NA injury; 3) alterations in the activity of gamma-GCS correspond with changes in susceptibility to NA injury; and 4) inhibition of gamma-GCS with BSO increases susceptibility to NA injury in tolerant mice.

Clara-cell populations show a high degree of variation in susceptibility to injury by bioactivated cytotoxicants. Because glutathione (GSH) is critical for detoxification of electrophilic metabolites, heterogeneity in Clara cell GSH levels may lead to a wide range of cytotoxic responses. This study was designed to define the distinct GSH pools within Clara cells, characterize heterogeneity within the population, and examine whether heterogeneity contributes to susceptibility. Using fluorescent imaging combined with high-performance liquid chromatography analysis, semiquantitative measurements were obtained by evaluation of GSH using monochlorobimane and monobromobimane. In steady-state conditions, the GSH measured in isolated cells was in the femtomole range, but varied 4-fold between individual cells. Clara cells analyzed in situ and in vitro confirmed this heterogeneity. The response of these cells to compounds that modulate GSH was also variable. Diethylmaleate depleted GSH, whereas GSH monoethylester augmented it. However, both acted nonuniformly in isolated Clara cells. The depletion of intracellular GSH caused a striking decrease in cell viability upon incubation with naphthalene (NA). The sulfhydryl-binding fluorochrome BODIPY, which colocalized with tetramethylrosamine, a mitochondrial dye, demonstrated by confocal microscopy that cellular sulfhydryls are highest in the mitochondria, next-highest in cytoplasm, and lowest in the nucleus. These pools responded differently to modulators of GSH. We concluded that the steady-state intracellular GSH of Clara cells exists in distinct pools and is highly heterogeneous within the population, and that the heterogeneity of GSH levels corresponds closely to the response of Clara cells to injury by NA.

1-Nitronaphthalene (1-NN) is a mutagenic nitroaromatic that has been detected in emissions from both heavy- and light-duty diesel engines, as well as in urban airborne particles. 1-NN is a cytochrome P450-bioactivated, nonciliated bronchiolar epithelial (Clara) cell cytotoxicant. Our recent studies demonstrated that 1-NN was metabolized by rat lung and liver microsomal enzymes to six 1-NN GSH conjugates via intermediate C(5),C(6)- and C(7),C(8)-epoxides. These studies examined the metabolism of 1-NN in mouse, and compared the differences in rates of 1-NN GSH conjugate formation between the two species. HPLC radioactivity profiles demonstrated that seven different conjugates were generated in mouse lung and liver microsomal incubations. Six of the seven conjugates corresponded with those observed in incubations with rat microsomes. Mass spectrometry of the new conjugate yielded a m/z 497 (M+H) and identical daughter ions as in the other six conjugates when analyzed by mass spectrometry in electrospray positive ion mode. The major conjugate generated in mouse and rat lung microsomal incubations was conjugate 4 (1-nitro-7-glutathionyl-8-hydroxy-7, 8-dihydronaphthalene). In comparison, the formation of conjugate 6 (1-nitro-5-hydroxy-6-glutathionyl-5,6-dihydronaphthalene) predominated in mouse liver, whereas in rat liver, conjugate 5, a diastereomer of conjugate 6, was generated at the highest rate. We concluded that the rates of formation of regio- and stereoisomeric epoxides from 1-NN differed substantially in target and nontarget tissues, but there was no clear pattern of correlation of tissue susceptibility to the rate or metabolite produced.

Objective: To determine hepatic and pulmonary phase-I and phase-II enzyme activities in horses., Sample Population: Pulmonary and hepatic tissues from 22 horses that were 4 months to 32 years old., Procedure: Pulmonary and hepatic tissues from horses were used to prepare cytosolic (glutathione S-transferase and soluble epoxide hydrolase) and microsomal (cytochrome P450 monooxygenases) enzymes. Rates of microsomal metabolism of ethoxyresorufin, pentoxyresorufin, and naphthalene were determined by high-performance liquid chromatography. Activities of glutathione S-transferase and soluble epoxide hydrolase were determined spectrophotometrically. Cytochrome P450 content was determined by carbon monoxide bound-difference spectrum of dithionite-reduced microsomes. Activity was expressed relative to total protein concentration., Results: Microsomal protein and cytochromeP450 contents were detectable in all horses and did not vary with age. Hepatic ethoxyresorufin metabolism was detected in all horses; by comparison, pulmonary metabolism of ethoxyresorufin and hepatic and pulmonary metabolism of pentoxyresorufin were detected at lower rates. Rate of hepatic naphthalene metabolism remained constant with increasing age, whereas rate of pulmonary naphthalene metabolism was significantly lower in weanlings (ie, horses 4 to 6 months old), compared with adult horses. Hepatic glutathione S-transferase activity (cytosol) increased with age; however, these changes were not significant. Pulmonary glutathione S-transferase activity (cytosol) was significantly lower in weanlings than adult horses. Hepatic and pulmonary soluble epoxide hydrolase did not vary with age of horses., Conclusions and Clinical Relevance: Activity of cytochrome P450 isoforms that metabolize naphthalene and glutathione S-transferases in lungs are significantly lower in weanlings than adult horses, which suggests reduced ability of young horses to metabolize xenobiotics by this organ.

1-Nitronaphthalene (1-NN) is a mutagenic nitroaromatic which has been detected in emissions from both heavy- and light-duty diesel engines, as well as in urban airborne particles. 1-NN is a cytochrome P450-bioactivated, nonciliated bronchiolar epithelial (Clara) cell cytotoxicant. These studies examined the metabolism of 1-NN to electrophilic metabolites which were trapped as glutathione conjugates in highly susceptible (lung) and less susceptible (liver) tissues of the rat. Significant depletion of reduced glutathione was observed at all levels of tracheobronchial airways of rats treated with 200 mg/kg 1-NN, ip. This observation of depleted glutathione was consistent with the HPLC radioactivity profiles demonstrating six glutathione conjugates isolated from liver and lung microsomal incubations with 1-NN, [(3)H]glutathione, and glutathione S-transferase. Mass spectrometry of all six metabolites in electrospray positive ion mode yielded an ion of m/z 497 (M + H), and daughter ions of m/z 479 (loss of water), m/z 306 (glutathione), and m/z 177 (loss of the nitro group and formation of hydroxy naphthalene thiolate ion), demonstrating the formation of hydroxy-dihydroglutathionyl derivatives presumably via intermediate epoxide(s). Proton nuclear magnetic resonance spectroscopy identified four different regioisomeric conjugates from lung and liver microsomal incubations: 1-nitro-7-glutathionyl-8-hydroxy-7, 8-dihydronaphthalene, 1-nitro-7-hydroxy-8-glutathionyl-7, 8-dihydronaphthalene, 1-nitro-5-hydroxy-6-glutathionyl-5, 6-dihydronaphthalene, and 1-nitro-5-glutathionyl-6-hydroxy-5, 6-dihydronaphthalene. HPLC radioactivity profiles demonstrated that major conjugates generated in the lung were derived from the C(7), C(8)-epoxide, whereas the most prominent metabolites in the liver were derived from the C(5),C(6)-epoxide.

Despite their substantially lower levels relative to hepatic tissue, pulmonary cytochrome P-450 (CYP) monooxygenases play an important role in the metabolic activation of substrates that cause lung injury. The target- and species-selective toxicity of a number of pulmonary toxicants has been attributed to the presence and distribution of activating enzymes with high kcat in target airways of susceptible species. However, experimental demonstration of these concepts and quantitative assessment of the contribution of individual CYP isoforms is lacking. This study was undertaken to characterize the catalytic activities of CYP2F2 with naphthalene, a murine Clara cell toxicant, as well as with other xenobiotics that either undergo metabolic activation to cytotoxic intermediates or that function as "isoform-selective" substrates. Recombinant CYP2F2 was produced using the baculovirus expression vector system in Spodoptera frugiperda and Trichoplusia ni cells, accounting up to approximately 20% of the total cellular protein. Incubations containing naphthalene, recombinant CYP2F2, NADPH-cytochrome P-450 oxidoreductase, and NADPH-regenerating system metabolized naphthalene with a high degree of stereoselectivity to 1R, 2S-naphthalene oxide (66:1 enantiomeric ratio). The Km and kcat values, along with the specificity constant, for naphthalene metabolism by recombinant CYP2F2 were 3 microM, 104 min-1, and 5.8 x 10(5) M-1 s-1, respectively. Recombinant CYP2F2 also metabolized ethoxyresorufin, pentoxyresorufin, p-nitrophenol, and 1-nitronaphthalene at easily detectable levels. The results from this work suggest that CYP2F2 1) plays a key role in the species- and cell-selective toxicity of naphthalene and 2) efficiently metabolizes a number of other substrates, including the lung toxicant 1-nitronaphthalene.

Acute pulmonary epithelial injury produced by short-term exposure to ozone varies by site within the tracheobronchial tree. To test whether this variability is related to the local dose of ozone at the tissue site or to local concentrations of glutathione, we exposed adult male rhesus monkeys for 2 h to filtered air or to 0.4 or 1.0 ppm ozone generated from 18O2. Following exposure, lungs were split into lobes and specimens were selected by microdissection so that measurements could be made on airway tissue of similar branching history, including trachea, proximal (generation one or two) and distal (generation six or seven) intrapulmonary bronchi, and proximal respiratory bronchioles. One half of the lung was lavaged for analysis of extracellular components. In monkeys exposed to filtered air, the concentration of reduced glutathione (GSH) varied throughout the airway tree, with the proximal intrapulmonary bronchus having the lowest concentration and the parenchyma having the highest concentration. Exposure to 1.0 ppm ozone significantly reduced GSH only in the respiratory bronchiole, whereas exposure to 0.4 ppm increased GSH only in the proximal intrapulmonary bronchus. Local ozone dose (measured as excess 18O) varied by as much as a factor of three in different airways of monkeys exposed to 1.0 ppm, with respiratory bronchioles having the highest concentration and the parenchyma the lowest concentration. In monkeys exposed to 0.4 ppm, the ozone dose was 60% to 70% less than in the same site in monkeys exposed to 1.0 ppm. Epithelial disruption was present to some degree in all airway sites, but not in the parenchyma, in animals exposed to 1.0 ppm ozone. The mass of mucous and ciliated cells decreased in all airways, and necrotic and inflammatory cells increased. At 0.4 ppm, epithelial injury was minimal, except in the respiratory bronchiole, where cell loss and necrosis occurred, and was 50% that found in monkeys exposed to 1.0 ppm ozone. We conclude that there is a close association between site-specific O3 dose, the degree of epithelial injury, and glutathione depletion at local sites in the tracheobronchial tree.

The distribution of pulmonary cytochrome P450 (P450 or CYP) isoforms has been investigated primarily in immunohistochemical studies, which are neither quantitative nor reflective of the functions of these enzymes. Studies of enzyme activities have been performed using whole-lung homogenates or isolated cells, but there is little information on the regioselective expression of P450 monooxygenases. The aims of this study were to compare the activities of P450 monooxygenases in different lung subcompartments in two commonly studied animal models, i.e. rats and monkeys, and to explore the possibility that inducing agents would result in activity up-regulation that is highly site-selective, using rats as a model. Microdissection techniques were used to separate the airways from blood vessels and lung parenchyma. In rats, CYP1A1 (ethoxyresorufin) and CYP2B (pentoxyresorufin) dealkylase activities were highest in the parenchyma, whereas CYP2E1 (p-nitrophenol) hydroxylase activity was highest in the airways. P450 reductase activities were similar in airways and parenchyma and were lower in trachea. In monkeys, no significant site-selective differences in CYP1A1 and CYP2B1 activities were found. In contrast, CYP2E1 activity was higher in the distal bronchioles and parenchyma than in the proximal airways. P450 reductase activities were similar in microsomes prepared from all subcompartments of monkey lung. Induction of rat CYP1A1 activity by beta-naphthoflavone (administered ip) was much greater in the airways and lung parenchyma ( approximately 30-fold) than in the liver ( approximately 10-fold) or trachea ( approximately 2.5-fold). Oral administration of phenobarbital or acetone increased CYP2B and CYP2E1 activities in rat liver but had no significant effect on P450 activities in subcompartments of rat lung. These findings support the conclusion that there are regiospecific and species-specific differences in the activities of P450 isoforms and that the inducibility of rat pulmonary P450s is dependent on the isoform and lung region.

The distal trachea and centriacinus of the lung are primary sites of acute injury during short-term ozone exposure; long-term exposure yields cells in these areas that are resistant to high doses of oxidant gases. Epithelial cells located in primary sites for ozone injury are also targets for chemicals that undergo cytochrome P450 (CYP)-dependent activation. These studies were designed to compare the effects of ozone exposure on pulmonary CYP2E1 in susceptible and nonsusceptible sites within the airway tree of lung. CYP2E1 activity was measured in well-defined regions of airways using p-nitrophenol, a CYP2E1-selective substrate, with HPLC/ electrochemical detection of the p-nitrocatechol. Alterations in distribution of CYP2E1 were evaluated by immunohistochemistry. CYP2E1 activities were highest in the distal bronchioles and minor daughter airways but were much lower in the lobar bronchi/ major daughter airways and trachea. Immediately after short-term ozone exposures (8 h, 1 ppm), CYP2E1 activities were elevated only in the lobar bronchi/major daughter airways. These activities remained above the filtered air control at 1 day but returned to control levels by 2 days. Immunohistochemical assessment of CYP2E1 protein in ozone and filtered air-exposed animals was consistent with the activity measurements. After long-term ozone exposures (90 days, 1 ppm), CYP2E1 activities were decreased in the major and minor daughter airways. These studies indicate that CYP2E1 activities vary substantially by airway level. However, ozone exposure only results in minimal alterations in activity with varying concentration of ozone, length of exposure, and time after exposure in any of the lung subcompartments examined.

Various studies indicate that cytodifferentiation of Clara cells and development of pulmonary cytochrome P450 (CYP) monooxygenases occur postnatally. The timing of these events is species-specific. Neonatal mice are more susceptible than adult mice are to Clara cell injury by naphthalene, but little is known about the postnatal development of Clara cells and CYP in mice. This study was designed to determine the developmental pattern of Clara cell differentiation and CYP expression in mice. Lungs from mice aged 16 days gestation to 63 days postnatal (DPN) were studied. Clara cell secretory protein (CC10) expression in nonciliated cells was detected earlier in proximal airways than in distal airways, but reached adult levels at 14 DPN in all airway levels. Cilia-associated tubulin expression closely followed the onset of CC10 expression, as did expression of CYP reductase. CYP2B protein expression appeared and differentiated earlier in bronchi than in bronchioles and reached adult levels at 14 and 28 DPN, respectively. CYP2F2 expression appeared earlier in proximal airways, but did not reach adult levels of expression until after 28 DPN. CYP activity, measured by naphthalene metabolism, increased with age and corresponded to CYP2F2 protein expression. We conclude that in the mouse, (1) Clara cell maturation is a postnatal event, (2) Clara cell differentiation is complete at the same age in proximal and distal airways, (3) CYP reductase protein expression occurs at the same time as CC10 expression, but CYP2B and CYP2F2 lag behind, and (4) stereoselective naphthalene monooxygenase activity corresponds with CYP2F2 protein expression.

Cytochrome P450 2E1 catalyzes the metabolic activation of nitrosamines and haloalkenes to carcinogenic and cytotoxic derivatives; however, the regulation of this P450 isozyme is not well understood. Hydroxylation of p-nitrophenol to p-nitrocatechol has been used as a marker of CYP2E1 activity, but currently available methodologies are not sufficiently sensitive to allow measurements in small tissue samples or in tissues with low activity such as lung. We describe here a method for measuring p-nitrocatechol formation using HPLC with electrochemical detection which is rapid and specific. It has a level of sensitivity (pmol) sufficient to monitor CYP2E1 activities in incubations containing as little as 10 micrograms microsomal protein prepared from airway subcompartments of the lung, a tissue with low and varying CYP2E1 activities among different parts of the airways.

Selective Clara cell injury produced by many bioactivated lung toxicants is thought to result from high levels of activating enzymes found in differentiated Clara cells. A recent study found an elevated susceptibility to the Clara cell toxicant 4-ipomeanol in neonatal rabbits when Clara cell P450 activity is low. To determine whether differentiating Clara cells in another species (mouse) are more susceptible to injury by a different bioactivated Clara cell toxicant (naphthalene), adult, 14-day postnatal (DPN) and 7DPN male mice were given a single intraperitoneal dose (25, 50, or 100 mg/kg) of naphthalene and killed 24 hr later. Epithelial damage, as assessed by quantitative histopathology, included cellular swelling, vacuolization, and exfoliation. In 7DPN mice, bronchiolar epithelium was severely injured at the lowest dose of naphthalene tested, 25 mg/kg. Bronchiolar epithelium in 14DPN mice was moderately injured at 25 mg/kg; injury severity was greatest at 50 and 100 mg/kg. Minimal bronchiolar epithelial injury occurred in adult mice at 50 mg/kg and moderate injury at 100 mg/kg. In proximal bronchi, epithelium of 7DPN mice showed signs of injury only at 100 mg/kg. Bronchial epithelium of adult mice was not injured at any dose. Isolated distal airways from 7DPN and 14DPN mice were more sensitive to naphthalene exposure than isolated distal airways from adult mice. Despite the low levels of P450 activity, differentiating Clara cells in neonatal mice are more susceptible to injury by the bioactivated cytotoxicant naphthalene than are differentiated Clara cells in adult mice.

Glutathione (GSH) and glutathione disulfide (GSSG) are biologically important intracellular thiols; alterations in the GSH/GSSG ratio are often used to assess exposure of cells to oxidative stress. Although several methods are available for measuring GSH and GSSG, all have some disadvantages including the need to generate derivatives, the inability to conveniently measure both GSH and GSSG, and a lack of sufficient sensitivity to allow detection in very small samples/cells of extrahepatic tissue. These studies present a rapid, validated HPLC-electro-chemical method for determining GSH and GSSG in small samples such as those from microdissected airways of the mouse containing 50-200 micrograms protein which is suitable for routine use. GSH and GSSG can be measured at levels of 1 and 2 pmol on column, respectively, with acceptable accuracy and precision and without the need to generate derivatives. In microdissected airways from the mouse, the intraday assay coefficient of variation for GSH varied from 4.7 to 5.9% and for GSSG was 4.4 to 5.7%. The interday assay coefficient of variation ranged from 6.0 to 7.6% for GSH and 5.5 to 23% for GSSG. The effects of repeated freezing and thawing on the concentrations of GSH and GSSG indicate that multiple cycles do not significantly alter the GSH or GSSG concentration as the number of cycles increases. Addition of GSH or GSSG to samples increased the peak areas appropriately, without altering the peak shape, retention time, or peak area of the corresponding reduced (oxidized) thiol. The ratio of GSH/GSSG in freeze-clamped liver ranged from 46 to 248, while liver tissue which was homogenized fresh had GSH/GSSG ratios of 62-150. The technique appears to be capable of reproducibly measuring GSH and GSSG in small quantities of nonhepatic tissue.