Sex differences in the development of airway epithelial tolerance to naphthalene

Exposure to air pollution has been linked to pulmonary diseases. Naphthalene (NA), an abundant polycyclic aromatic hydrocarbon in tobacco smoke and urban air, is a model toxicant for air pollution effects in the lung. Repeated exposures to NA in male mice result in tolerance, defined as the emergence of a resistant cell phenotype after prior exposure. Tolerance has not been studied in females. Females have sex differences in airway epithelial responses and in the prevalence of certain airway diseases. Male and female mice were exposed to a tolerance-inducing regimen of NA, and lungs were examined by airway level to characterize the cellular changes associated with repeated NA exposure and to assess the expression of genes and proteins involved in NA bioactivation and detoxification. The airway epithelium in treated males resembled that of controls. Females in the tolerant state were characterized by dense populations of ciliated cells in midlevel, distal, and bifurcating airways and a lower abundance of Clara cells at all airway levels. Cytotoxicity following a secondary challenge dose was also greater in females than males. Furthermore, females had decreased gene/protein expression of CYP2F2, a P-450 that metabolizes NA to a toxic epoxide, and glutamate-cysteine ligase, the rate-limiting enzyme in glutathione synthesis, than NA-tolerant males at all airway levels examined. We conclude that, while females develop tolerance, sex differences exist in the tolerant state by airway level, and females remain more susceptible than males to repeated exposures to NA.     

p53 codon 271 CGT to CAT mutant fraction does not increase in nasal respiratory and olfactory epithelia of rats exposed to inhaled naphthalene

A 2-year rat tumor bioassay testing whole body exposure to naphthalene (NA) vapor found a significant increase in nasal respiratory epithelial adenomas in male rats and in olfactory epithelial neuroblastomas in female rats. To obtain mechanistic insight into NA-induced nasal carcinogenesis, NA dose-response was characterized in nasal epithelium using a tumor-relevant endpoint. Specifically, levels of p53 codon 271 CGT to CAT mutation were measured in nasal respiratory and olfactory epithelium of NA-exposed male and female rats by allele-specific competitive blocker-PCR (ACB-PCR). Male and female, 8-9 week-old F344 rats (5 rats/group) were exposed to 0, 0.1, 1.0, 10, and 30ppm NA vapor for 13 weeks (6h/day, 5 days/week). The geometric mean p53 mutant fraction (MF) levels in nasal epithelium of control treatment groups ranged between 2.05 × 10(-5) and 3.05 × 10(-5). No significant dose-related changes in p53 mutant fraction (MF) were observed in the olfactory or respiratory epithelia of female rats. However, statistically significant treatment-related differences were observed in male respiratory and olfactory epithelium, with the p53 MF in the respiratory epithelium of male rats exposed to 30ppm NA significantly lower than that in controls. Further, a significant trend of decreasing p53 MF with increasing dose was observed in the male respiratory epithelium. Of the tissue types analyzed, respiratory epithelium is the most sensitive to the cytotoxic effects of NA, suggesting cytotoxicity may be responsible for the loss of p53 mutation. Because ACB-PCR has been used successfully to detect the effects of known mutagenic carcinogens, the absence of any significant increases in p53 MF associated with NA exposure adds to the weight of evidence that NA does not operate through a directly mutagenic mode of action.     

Proteomic profiling of acrolein adducts in human lung epithelial cells

Acrolein (2,3-propenal) is a major indoor and outdoor air pollutant originating largely from tobacco smoke or organic combustion. Given its high reactivity, the adverse effects of inhaled acrolein are likely due to direct interactions with the airway epithelium, resulting in altered epithelial function, but only limited information exists to date regarding the primary direct cellular targets for acrolein. Here, we describe a global proteomics approach to characterize the spectrum of airway epithelial protein targets for Michael adduction in acrolein-exposed bronchial epithelial (HBE1) cells, based on biotin hydrazide labeling and avidin purification of biotinylated proteins or peptides for analysis by LC-MS/MS. Identified protein targets included a number of stress proteins, cytoskeletal proteins, and several key proteins involved in redox signaling, including thioredoxin reductase, thioredoxin, peroxiredoxins, and glutathione S-transferase π. Because of the central role of thioredoxin reductase in cellular redox regulation, additional LC-MS/MS characterization was performed on purified mitochondrial thioredoxin reductase to identify the specific site of acrolein adduction, revealing the catalytic selenocysteine residue as the target responsible for enzyme inactivation. Our findings indicate that these approaches are useful in characterizing major protein targets for acrolein, and will enhance mechanistic understanding of the impact of acrolein on cell biology.     

Use of Nuclear Magnetic Resonance-Based Metabolomics to Characterize the Biochemical Effects of Naphthalene on Various Organs of Tolerant Mice

Naphthalene, the most common polycyclic aromatic hydrocarbon, causes airway epithelium injury in mice. Repeated exposure of mice to naphthalene induces airway epithelia that are resistant to further injury. Previous studies revealed that alterations in bioactivation enzymes and increased levels of gamma-glutamylcysteine synthase in the bronchioles protect tolerant mice from naphthalene and its reactive metabolites. In our current study, tolerance was induced in male ICR mice using a total of 7 daily intraperitoneal injections of naphthalene (200 mg/kg). Both naphthalene-tolerant and non-tolerant mice were challenged with a dose of 300 mg/kg naphthalene on day 8 to investigate metabolite differences. The lungs, liver, and kidneys were collected for histopathology 24 h after the challenge dose. Bronchial alveolar lavage fluid (BALF) and both hydrophilic and hydrophobic extracts from each organ were analyzed using nuclear magnetic resonance (NMR)-based metabolomics. The histological results showed no observable injuries to the airway epithelium of naphthalene-tolerant mice when compared with the control. In contrast, airway injuries were observed in mice given a single challenge dose (injury mice). The metabolomics analysis revealed that the energy metabolism in the lungs of tolerant and injury mice was significantly perturbed. However, antioxidant metabolites, such as glutathione and succinate, were significantly increased in the lungs of tolerant mice, suggesting a role for these compounds in the protection of organs from naphthalene-induced electrophilic metabolites and free radicals. Damage to the airway cellular membrane, as shown by histopathological results and increased acetone in the BALF and perturbation of hydrophobic lung extracts, including cholesterol, phosphorylcholine-containing lipids, and fatty acyl chains, were observed in injury mice. Consistent with our histopathological results, fewer metabolic effects were observed in the liver and kidney of mice after naphthalene treatments. In conclusion, NMR-based metabolomics reveals possible mechanisms of naphthalene tolerance and naphthalene-induced toxicity in the respiratory system of mice.     

Novel approaches to identify protein adducts produced by lipid peroxidation

Abstract

Lipid peroxidation is responsible for the generation of chemically reactive, diffusible lipid-derived electrophiles (LDEs) that covalently modify cellular protein targets. These protein modifications modulate protein activity and macromolecular interactions and induce adaptive and toxic cell signaling. Protein modifications induced by LDEs can be identified and quantified by affinity enrichment and liquid chromatography–tandem mass spectrometry (LC–MS/MS)-based techniques. Tagged LDE analog probes with different electrophilic groups can be covalently captured by click chemistry for LC–MS/MS analyses, thereby enabling in-depth studies of proteome damage at the protein and peptide sequence levels. Conversely, click-reactive, thiol-directed probes can be used to evaluate thiol damage caused by LDE by difference. These analytical approaches permit systematic study of the dynamics of protein damage caused by LDE and mechanisms by which oxidative stress contribute to toxicity and diseases.     

The role of macrophages in B cell tolerance. I. Antigen-specific failure of Thy-1, Ly-2 negative adherent cells from tolerant mice to reconstitute immunocompetence in adherent cell deficient spleen cells

T-Cell-independent B-cell tolerance to the hapten derivatives of carboxymethyl cellulose (CMC) or methyl cellulose (MC) appears to be controlled by Thy-1-, Ly-2- adherent (A) cells contained in the spleen or peritoneal fluid. Immunocompetence in nonadherent (NA) normal spleen cells could be restored in vitro by irradiated A cells from normal mice. However, NA cells reconstituted with irradiated A cells derived from hapten specifically tolerant mice failed to respond to the same hapten, but responded normally to an immunogenic challenge with another unrelated antigen. A cells that had been preincubated at 4 degrees C with hapten derivatized MC also failed to restore immunocompetence. While preincubation of unfractionated spleen cells with the tolerogen under the same conditions resulted in B-cell unresponsiveness, such treatment of NA cells failed to render B cells tolerant. Treatment of A cells from tolerant mice with the reducing agent potassium iodide (KI) in vitro restored their capacity to render cultures of NA cells immunocompetent to the relevant hapten. Moreover, treatment with KI of spleen cells from mice injected with the tolerogen was shown to render them responsive. We suggest that B-cell tolerance induced by hapten derivatives of CMC and MC is mediated by suppressive macrophages contained among A cells. Certain subpopulations of macrophages are known to exert cytotoxic effects upon target cells by the release at close range of oxidating agents. We postulate that hapten derivatized CMC and MC, through unique properties of the carrier, bind to and possibly activate macrophages rendering them specifically suppressive for hapten binding B cells.     

Copper stress‐induced changes in leaf soluble proteome of Cu‐sensitive and tolerant Agrostis capillaris L. populations

Changes in leaf soluble proteome were explored in 3‐month‐old plants of metallicolous (M) and nonmetallicolous (NM) Agrostis capillaris L. populations exposed to increasing Cu concentrations (1–50 μM) to investigate molecular mechanisms underlying plant responses to Cu excess and tolerance of M plants. Plants were cultivated on perlite (CuSO₄ spiked‐nutrient solution). Soluble proteins, extracted by the trichloroacetic acid/acetone procedure, were separated with 2‐DE (linear 4–7 pH gradient). Analysis of CCB‐stained gels (PDQuest) reproducibly detected 214 spots, and 64 proteins differentially expressed were identified using LC‐MS/MS. In both populations, Cu excess impacted both light‐dependent (OEE, cytochrome b6‐f complex, and chlorophyll a‐b binding protein), and ‐independent (RuBisCO) photosynthesis reactions, more intensively in NM leaves (ferredoxin‐NADP reductase and metalloprotease FTSH2). In both populations, upregulation of isocitrate dehydrogenase and cysteine/methionine synthases respectively suggested increased isocitrate oxidation and enhanced need for S‐containing amino‐acids, likely for chelation and detoxification. In NM leaves, an increasing need for energetic compounds was indicated by the stimulation of ATPases, glycolysis, pentose phosphate pathway, and Calvin cycle enzymes; impacts on protein metabolism and oxidative stress increase were respectively suggested by the rise of chaperones and redox enzymes. Overexpression of a HSP70 may be pivotal for M Cu tolerance by protecting protein metabolism. All MS data have been deposited in the ProteomeXchange with the dataset identifier PXD001930 ( http//proteomecentral.proteomexchange.org/dataset/PXD001930 ).     

The proteome response of salt-resistant and salt-sensitive barley genotypes to long-term salinity stress

Responses of plants to salinity stress and the development of salt tolerance are extremely complex. Proteomics is a powerful technique to identify proteins associated with a particular environmental or developmental signal. We employed a proteomic approach to further understand the mechanism of plant responses to salinity in a salt-tolerant (Afzal) and a salt-sensitive (Line 527) genotype of barley. At the 4-leaf stage, plants were exposed to 0 (control) or 300 mM NaCl. Salt treatment was maintained for 3 weeks. Total proteins of leaf 4 were extracted and separated by two-dimensional gel electrophoresis. More than 500 protein spots were reproducibly detected. Of these, 44 spots showed significant changes to salt treatment compared to the control: 43 spots were upregulated and 1 spot was downregulated. Using MALDI-TOF-TOF MS, we identified 44 cellular proteins have been identified, which represented 18 different proteins and were classified into seven categories and a group with unknown biological function. These proteins were involved in various many cellular functions. Up regulation of proteins which involved in reactive oxygen species scavenging, signal transduction, protein processing and cell wall may increase plant adaptation to salt stress. The upregulation of the three of four antioxidant proteins (thioredoxin, methionine sulfoxide reductase and dehydroascorbate reductase) in susceptible genotype Line 527 suggesting a different tolerance mechanism (such as tissue tolerance) to tolerate a salinity condition in comparison with the salt sensitive genotype.     

Comparing isogenic strains of Beijing genotype Mycobacterium tuberculosis after acquisition of Isoniazid resistance: A proteomics approach

We determined differences in the protein abundance among two isogenic strains of Mycobacterium tuberculosis (Mtb) with different Isoniazid (INH) susceptibility profiles. The strains were isolated from a pulmonary tuberculosis patient before and after drug treatment. LC‐MS/MS analysis identified 46 Mtb proteins with altered abundance after INH resistance acquisition. Protein abundance comparisons were done evaluating the different bacterial cellular fractions (membrane, cytosol, cell wall and secreted proteins). MS data have been deposited to the ProteomeXchange with identifier PXD002986.     

Augmentation of arginase 1 expression by exposure to air pollution exacerbates the airways hyperresponsiveness in murine models of asthma

Background

Arginase overexpression contributes to airways hyperresponsiveness (AHR) in asthma. Arginase expression is further augmented in cigarette smoking asthmatics, suggesting that it may be upregulated by environmental pollution. Thus, we hypothesize that arginase contributes to the exacerbation of respiratory symptoms following exposure to air pollution, and that pharmacologic inhibition of arginase would abrogate the pollution-induced AHR.

Methods

To investigate the role of arginase in the air pollution-induced exacerbation of airways responsiveness, we employed two murine models of allergic airways inflammation. Mice were sensitized to ovalbumin (OVA) and challenged with nebulized PBS (OVA/PBS) or OVA (OVA/OVA) for three consecutive days (sub-acute model) or 12 weeks (chronic model), which exhibit inflammatory cell influx and remodeling/AHR, respectively. Twenty-four hours after the final challenge, mice were exposed to concentrated ambient fine particles plus ozone (CAP+O₃), or HEPA-filtered air (FA), for 4 hours. After the CAP+O₃ exposures, mice underwent tracheal cannulation and were treated with an aerosolized arginase inhibitor (S-boronoethyl-L-cysteine; BEC) or vehicle, immediately before determination of respiratory function and methacholine-responsiveness using the flexiVent^®. Lungs were then collected for comparison of arginase activity, protein expression, and immunohistochemical localization.

Results

Compared to FA, arginase activity was significantly augmented in the lungs of CAP+O₃-exposed OVA/OVA mice in both the sub-acute and chronic models. Western blotting and immunohistochemical staining revealed that the increased activity was due to arginase 1 expression in the area surrounding the airways in both models. Arginase inhibition significantly reduced the CAP+O₃-induced increase in AHR in both models.

Conclusions

This study demonstrates that arginase is upregulated following environmental exposures in murine models of asthma, and contributes to the pollution-induced exacerbation of airways responsiveness. Thus arginase may be a therapeutic target to protect susceptible populations against the adverse health effects of air pollution, such as fine particles and ozone, which are two of the major contributors to smog.     

The toxicity of styrene to the nasal epithelium of mice and rats: studies on the mode of action and relevance to humans

Inhaled styrene is known to be toxic to the nasal olfactory epithelium of both mice and rats, although mice are markedly more sensitive. In this study, the nasal tissues of mice exposed to 40 and 160 ppm styrene 6 h/day for 3 days had a number of degenerative changes including atrophy of the olfactory mucosa and loss of normal cellular organisation. Pretreatment of mice with 5-phenyl-1-pentyne, an inhibitor of both CYP2F2 and CYP2E1 completely prevented the development of a nasal lesion on exposure to styrene establishing that a metabolite of styrene, probably styrene oxide, is responsible for the observed nasal toxicity. Comparisons of the cytochrome P-450 mediated metabolism of styrene to its oxide, and subsequent metabolism of the oxide by epoxide hydrolases and glutathione S-transferases in nasal tissues in vitro, have provided an explanation for the increased sensitivity of the mouse to styrene. Whereas cytochrome P-450 metabolism of styrene is similar in rats and mice, the rat is able to metabolise styrene oxide at higher rates than the mouse thus rapidly detoxifying this electrophilic metabolite. Metabolism of styrene to its oxide could not be detected in human nasal tissues in vitro, but the same tissues did have epoxide hydrolase and glutathione S-transferase activities, and were able to metabolise styrene oxide efficiently, indicating that styrene is unlikely to be toxic to the human nasal epithelium.     

Quantitative trait analysis of the development of pulmonary tolerance to inhaled zinc oxide in mice

Background

Individuals may develop tolerance to the induction of adverse pulmonary effects following repeated exposures to inhaled toxicants. Previously, we demonstrated that genetic background plays an important role in the development of pulmonary tolerance to inhaled zinc oxide (ZnO) in inbred mouse strains, as assessed by polymorphonuclear leukocytes (PMNs), macrophages, and total protein in bronchoalveolar lavage (BAL) phenotypes. The BALB/cByJ (CBy) and DBA/2J (D2) strains were identified as tolerant and non-tolerant, respectively. The present study was designed to identify candidate genes that control the development of pulmonary tolerance to inhaled ZnO.

Methods

Genome-wide linkage analyses were performed on a CByD2F2 mouse cohort phenotyped for BAL protein, PMNs, and macrophages following 5 consecutive days of exposure to 1.0 mg/m³ inhaled ZnO for 3 hours/day. A haplotype analysis was carried out to determine the contribution of each quantitative trait locus (QTL) and QTL combination to the overall BAL protein phenotype. Candidate genes were identified within each QTL interval using the positional candidate gene approach.

Results

A significant quantitative trait locus (QTL) on chromosome 1, as well as suggestive QTLs on chromosomes 4 and 5, for the BAL protein phenotype, was established. Suggestive QTLs for the BAL PMN and macrophage phenotypes were also identified on chromosomes 1 and 5, respectively. Analysis of specific haplotypes supports the combined effect of three QTLs in the overall protein phenotype. Toll-like receptor 5 (Tlr5) was identified as an interesting candidate gene within the significant QTL for BAL protein on chromosome 1. Wild-derived Tlr5-mutant MOLF/Ei mice were tolerant to BAL protein following repeated ZnO exposure.

Conclusion

Genetic background is an important influence in the acquisition of pulmonary tolerance to BAL protein, PMNs, and macrophages following ZnO exposure. Promising candidate genes exist within the identified QTL intervals that would be good targets for additional studies, including Tlr5. The implications of tolerance to health risks in humans are numerous, and this study furthers the understanding of gene-environment interactions that are likely to be important factors from person-to-person in regulating the development of pulmonary tolerance to inhaled toxicants.     

Proteomic and phosphoproteomic analyses of yeast reveal the global cellular response to sphingolipid depletion

Sphingolipids are essential components of eukaryotic cells with important functions in membrane biology and cellular signaling. Their levels are tightly controlled and coordinated with the abundance of other membrane lipids. How sphingolipid homeostasis is achieved is not yet well understood. Studies performed primarily in yeast showed that the phosphorylation states of several enzymes and regulators of sphingolipid synthesis are important, although a global understanding for such regulation is lacking. Here, we used high‐resolution MS‐based proteomics and phosphoproteomics to analyze the cellular response to sphingolipid synthesis inhibition. Our dataset reveals that changes in protein phosphorylation, rather than protein abundance, dominate the response to blocking sphingolipid synthesis. We identified Ypk signaling as a pathway likely to be activated under these conditions, and we identified potential Ypk1 target proteins. Our data provide a rich resource for on‐going mechanistic studies of key elements of the cellular response to the depletion of sphingolipid levels and the maintenance of sphingolipid homeostasis. All MS data have been deposited in the ProteomeXchange with identifier PXD003854 ( http://proteomecentral.proteomexchange.org/dataset/PXD003854 ).     

Prioritizing targets for structural biology through the lens of proteomics: The archaeal protein TGAM_1934 from Thermococcus gammatolerans

ORFans are hypothetical proteins lacking any significant sequence similarity with other proteins. Here, we highlighted by quantitative proteomics the TGAM_1934 ORFan from the hyperradioresistant Thermococcus gammatolerans archaeon as one of the most abundant hypothetical proteins. This protein has been selected as a priority target for structure determination on the basis of its abundance in three cellular conditions. Its solution structure has been determined using multidimensional heteronuclear NMR spectroscopy. TGAM_1934 displays an original fold, although sharing some similarities with the 3D structure of the bacterial ortholog of frataxin, CyaY, a protein conserved in bacteria and eukaryotes and involved in iron–sulfur cluster biogenesis. These results highlight the potential of structural proteomics in prioritizing ORFan targets for structure determination based on quantitative proteomics data. The proteomic data and structure coordinates have been deposited to the ProteomeXchange with identifier PXD000402 ( http://proteomecentral.proteomexchange.org/dataset/PXD000402 ) and Protein Data Bank under the accession number 2mcf, respectively.     

WCS120 protein family and proteins soluble upon boiling in cold-acclimated winter wheat

The amount of proteins soluble upon boiling (especially WCS120 proteins) and the ability to develop frost tolerance (FT) after cold acclimation was studied in two frost-tolerant winter wheat cultivars, Mironovskaya 808 and Bezostaya 1. Protein gel blot analysis, mass spectrometry (MS) and image analysis of two-dimensional gel electrophoresis (2-DE) gels were used to identify and/or quantify the differences in protein patterns before (non-acclimated, NA) and after 3 weeks of cold acclimation (CA) of the wheats, when FT increased from -4 degrees C (lethal temperature (LT(50)), for both cultivars) to -18.6 degrees C in Bezostaya 1 and -20.8 degrees C in Mironovskaya 808. Only WCS120 protein was visible in NA leaves while all five WCS120 proteins were induced in the CA leaves. Mironovskaya 808 had higher accumulation of three members of WCS120 proteins (WCS120, WCS66 and WCS40) than Bezostaya 1. MS analysis of total sample of proteins soluble upon boiling showed seven COR proteins in the CA samples and only three COR proteins in the NA samples of cultivar Mironovskaya 808 (MIR). In conclusion, the level of the accumulation of WCS120, WCS66 and WCS40 distinguished our two frost-tolerant winter wheat cultivars. Moreover, the differences of CA and NA samples of the MIR were shown by liquid chromatography (LC)-tandem mass spectrometry (MS/MS).