NO2− has been viewed primarily as a reservoir for NO and NO-modified species, activated by acids or metal catalysis. Isotopic labeling of NO and NO2− modifications in vitro and in vivo now demonstrates that NO2− also participates directly in these reactions through a symmetric N2O3 intermediate. The current perspective holds that the generation of secondary signaling mediators from nitrite (NO2−) requires acidification to nitrous acid (HNO2) or metal catalysis. Herein, the use of stable isotope–labeled NO2− and LC-MS/MS analysis of products reveals that NO2− also participates in fatty acid nitration and thiol S-nitrosation at neutral pH. These reactions occur in the absence of metal centers and are stimulated by autoxidation of nitric oxide (•NO) via the formation of symmetrical dinitrogen trioxide (nitrous anhydride, symN2O3). Although theoretical models have predicted physiological symN2O3 formation, its generation is now demonstrated in aqueous reaction systems, cell models and in vivo, with the concerted reactions of •NO and NO2− shown to be critical for symN2O3 formation. These results reveal new mechanisms underlying the NO2− propagation of •NO signaling and the regulation of both biomolecule function and signaling network activity via NO2−-dependent nitrosation and nitration reactions.

Alzheimer's disease (AD) is a major cause of dementia in the elderly. Although early-onset (familial) AD is attributed to gene mutations, the cause for late-onset (sporadic) AD, which accounts for 95% of AD cases, is unknown. In this study, we show that exposure of 6-week-old amyloid beta precursor protein (APP)/presenilin (PS1) overexpressing mice, a well-established animal model of AD, and nontransgenic littermates to a cyclic O-3 exposure protocol, which mimics environmental exposure episodes, accelerated learning/memory function loss in male APP/PS1 mice but not in female APP/PS1 mice or nontransgenic littermates. Female APP/PS1 mice had higher brain levels of amyloid beta peptide (A beta 42) and A beta 40, compared with male APP/PS1 mice; O-3 exposure, however, had no significant effect on brain A beta load in either male or female mice. Our results further show that male APP/PS1 mice had lower levels of antioxidants (glutathione and ascorbate) and experienced augmented induction of NADPH oxidases, lipid peroxidation, and neuronal apoptosis upon O-3 exposure, compared with female APP/PS1 mice. No significant effect of O-3 on any of these parameters was detected in nontransgenic littermates. In vitro studies further show that 4-hydroxynonenal, a lipid peroxidation product which was increased in the plasma and cortex/hippocampus of O-3-exposed male APP/PS1 mice, induced neuroblastoma cell apoptosis. Together, the results suggest that O-3 exposure per se may not cause AD but can synergize with genetic risk factors to accelerate the pathophysiology of AD in genetically predisposed populations. The results also suggest that males may be more sensitive to O-3-induced neuropathophysiology than females due to lower levels of antioxidants.

Exposure to ozone has been associated with increased incidence of respiratory morbidity in humans; however the mechanism(s) behind the enhancement of susceptibility are unclear. We have previously reported that exposure to episodic ozone during postnatal development results in an attenuated peripheral blood cytokine response to lipopolysaccharide (LPS) that persists with maturity. As the lung is closely interfaced with the external environment, we hypothesized that the conducting airway epithelium of neonates may also be a target of immunomodulation by ozone. To test this hypothesis, we evaluated primary airway epithelial cell cultures derived from juvenile rhesus macaque monkeys with a prior history of episodic postnatal ozone exposure. Innate immune function was measured by expression of the proinflammatory cytokines IL-6 and IL-8 in primary cultures established following in vivo LPS challenge or, in response to in vitro LPS treatment. Postnatal ozone exposure resulted in significantly attenuated IL-6 mRNA and protein expression in primary cultures from juvenile animals; IL-8 mRNA was also significantly reduced. The effect of antecedent ozone exposure was modulated by in vivo LPS challenge, as primary cultures exhibited enhanced cytokine expression upon secondary in vitro LPS treatment. Assessment of potential IL-6-targeting microRNAs miR-149, miR-202, and miR-410 showed differential expression in primary cultures based upon animal exposure history. Functional assays revealed that miR-149 is capable of binding to the IL-6 3' UTR and decreasing IL-6 protein synthesis in airway epithelial cell lines. Cumulatively, our findings suggest that episodic ozone during early life contributes to the molecular programming of airway epithelium, such that memory from prior exposures is retained in the form of a dysregulated IL-6 and IL-8 response to LPS; differentially expressed microRNAs such as miR-149 may play a role in the persistent modulation of the epithelial innate immune response towards microbes in the mature lung.

[PDF] [Full Text] [Abstract] , August 1, 2001; 91 (2): 725-732. J Appl Physiol Lizzie Y. Santiago, Matthew C. Hann, Abdellaziz Ben-Jebria and James S. Ultman Ozone absorption in the human nose during unidirectional airflow [PDF] [Full Text] [Abstract] , March 26, 2010; 285 (13): 9716-9728. J. Biol. Chem. Giuseppe L. Squadrito and Sadis Matalon Weifeng Song, Shipeng Wei, Yongjian Zhou, Ahmed Lazrak, Gang Liu, James D. Londino, Acid, and Chloramines Channels by Chlorine, Hypochlorous + Inhibition of Lung Fluid Clearance and Epithelial Na [PDF] [Full Text] [Abstract] , September , 2010; 299 (3): L289-L300. Am J Physiol Lung Cell Mol Physiol Giuseppe L. Squadrito, Edward M. Postlethwait and Sadis Matalon physiological implications Elucidating mechanisms of chlorine toxicity: reaction kinetics, thermodynamics, and [PDF] [Full Text] [Abstract] , June , 2011; 110 (6): 1664-1673. J Appl Physiol Hu and Marcel Filoche Talita Felipe de Vasconcelos, Bernard Sapoval, José S. Andrade, Jr., James B. Grotberg, Yingying Particle capture into the lung made simple?

Ozone (O3) is a serious public health concern. Recent findings indicate that the damaging health effects of O3 extend to multiple systemic organ systems. Herein, we hypothesize that O3 inhalation will cause downstream alterations to the liver. To test this, male Sprague-Dawley rats were exposed to 0.5 ppm O3 for 8 h/day for 5 days. Plasma liver enzyme measurements showed that 5 day O3 exposure did not cause liver cell death. Proteomic and mass spectrometry analysis identified 10 proteins in the liver that were significantly altered in abundance following short-term O3 exposure and these included several stress responsive proteins. Glucose-regulated protein 78 and protein disulfide isomerase increased, whereas glutathione S-transferase M1 was significantly decreased by O3 inhalation. In contrast, no significant changes were detected for the stress response protein heme oxygenase-1 or cytochrome P450 2E1 and 2B in liver of O3 exposed rats compared to controls. In summary, these results show that an environmentally-relevant exposure to inhaled O3 can alter the expression of select proteins in the liver. We propose that O3 inhalation may represent an important unrecognized factor that can modulate hepatic metabolic functions.

The immune mechanisms for neonatal susceptibility to respiratory pathogens are poorly understood. Given that mucosal surfaces serve as a first line of host defense, we hypothesized that the innate immune response to infectious agents may be developmentally regulated in airway epithelium. To test this hypothesis, we determined whether the expression of IL-8 and IL-6 in airway epithelium after LPS exposure is dependent on chronological age. Tracheas from infant, juvenile, and adult rhesus monkeys were first exposed to LPS ex vivo, and then processed for air-liquid interface primary airway epithelial cell cultures and secondary LPS treatment in vitro. Compared with adult cultures, infant and juvenile cultures expressed significantly reduced concentrations of IL-8 after LPS treatment. IL-8 protein in cultures increased with animal age, whereas LPS-induced IL-6 protein was predominantly associated with juvenile cultures. Toll-like receptor (TLR) pathway RT-PCR arrays showed differential expressions of multiple mRNAs in infant cultures relative to adult cultures, including IL-1α, TLR10, and the peptidoglycan recognition protein PGLYRP2. To determine whether the age-dependent cytokine response to LPS is reflective of antecedent exposures, we assessed primary airway epithelial cell cultures established from juvenile monkeys housed in filtered air since birth. Filtered air-housed animal cultures exhibited LPS-induced IL-8 and IL-6 expression that was discordant with age-matched ambient air-housed animals. A single LPS aerosol in vivo also affected this cytokine profile. Cumulatively, our findings demonstrate that the innate immune response to LPS in airway epithelium is variable with age, and may be modulated by previous environmental exposures.

Inhalation experiments using laboratory animals are performed under controlled conditions to assess the toxicity of and to investigate interventional strategies to ameliorate injury resulting from oxidant gas exposures. A variety of dynamic inhalation exposure systems that use whole-body or nose-only exposure chambers have been developed for rodents. In a whole-body exposure chamber, the animals are immersed in the test atmosphere, whereas in nose-only or head-only exposure systems, exposures are localized primarily to the head and/or nasal regions. There are advantages and disadvantages with both types of exposure approaches. Considerations such as animal number, exposure duration, end points of study, and availability of test material should influence the selection of a particular exposure system.

Cl2 gas toxicity is complex and occurs during and after exposure, leading to acute lung injury (ALI) and reactive airway syndrome (RAS). Moreover, Cl2 exposure can occur in diverse situations encompassing mass casualty scenarios, highlighting the need for postexposure therapies that are efficacious and amenable to rapid and easy administration. In this study, we assessed the efficacy of a single dose of nitrite (1mg/kg) to decrease ALI when administered to rats via intraperitoneal (ip) or intramuscular (im) injection 30min after Cl2 exposure. Exposure of rats to Cl2 gas (400ppm, 30min) significantly increased ALI and caused RAS 6–24h postexposure as indexed by BAL sampling of lung surface protein and polymorphonucleocytes (PMNs) and increased airway resistance and elastance before and after methacholine challenge. Intraperitoneal nitrite decreased Cl2-dependent increases in BAL protein but not PMNs. In contrast im nitrite decreased BAL PMN levels without decreasing BAL protein in a xanthine oxidoreductase-dependent manner. Histological evaluation of airways 6h postexposure showed significant bronchial epithelium exfoliation and inflammatory injury in Cl2-exposed rats. Both ip and im nitrite improved airway histology compared to Cl2 gas alone, but more coverage of the airway by cuboidal or columnar epithelium was observed with im compared to ip nitrite. Airways were rendered more sensitive to methacholine-induced resistance and elastance after Cl2 gas exposure. Interestingly, im nitrite, but not ip nitrite, significantly decreased airway sensitivity to methacholine challenge. Further evaluation and comparison of im and ip therapy showed a twofold increase in circulating nitrite levels with the former, which was associated with reversal of post-Cl2 exposure-dependent increases in circulating leukocytes. Halving the im nitrite dose resulted in no effect in PMN accumulation but significant reduction of BAL protein levels, indicating a distinct nitrite dose dependence for inhibition of Cl2-dependent lung permeability and inflammation. These data highlight the potential for nitrite as a postexposure therapeutic for Cl2 gas-induced lung injury and also suggest that administration modality is a key consideration in nitrite therapeutics.

Low concentrations of inhaled hydrogen sulfide (H(2)S) induce hypometabolism in mice. Biological effects of H(2)S in in vitro systems are augmented by lowering O(2) tension. Based on this, we hypothesized that reduced O(2) tension would increase H(2)S-mediated hypometabolism in vivo. To test this, male Sprague-Dawley rats were exposed to 80 ppm H(2)S at 21% O(2) or 10.5% O(2) for 6 h followed by 1 h recovery at room air. Rats exposed to H(2)S in 10.5% O(2) had significantly decreased body temperature and respiration compared with preexposure levels. Heart rate was decreased by H(2)S administered under both O(2) levels and did not return to preexposure levels after 1 h recovery. Inhaled H(2)S caused epithelial exfoliation in the lungs and increased plasma creatine kinase-MB activity. The effect of inhaled H(2)S on prosurvival signaling was also measured in heart and liver. H(2)S in 21% O(2) increased Akt-P(Ser473) and GSK-3β-P(Ser9) in the heart whereas phosphorylation was decreased by H(2)S in 10.5% O(2), indicating O(2) dependence in regulating cardiac signaling pathways. Inhaled H(2)S and low O(2) had no effect on liver Akt. In summary, we found that lower O(2) was needed for H(2)S-dependent hypometabolism in rats compared with previous findings in mice. This highlights the possibility of species differences in physiological responses to H(2)S. Inhaled H(2)S exposure also caused tissue injury to the lung and heart, which raises concerns about the therapeutic safety of inhaled H(2)S. In conclusion, these findings demonstrate the importance of O(2) in influencing physiological and signaling effects of H(2)S in mammalian systems.

We assessed the safety and efficacy of combined intravenous and aerosolized antioxidant administration to attenuate chlorine gas-induced airway alterations when administered after exposure. Adult male Sprague-Dawley rats were exposed to air or 400 parts per million (ppm) chlorine (a concentration likely to be encountered in the vicinity of industrial accidents) in environmental chambers for 30 minutes, and returned to room air, and they then received a single intravenous injection of ascorbic acid and deferoxamine or saline. At 1 hour and 15 hours after chlorine exposure, the rats were treated with aerosolized ascorbate and deferoxamine or vehicle. Lung antioxidant profiles, plasma ascorbate concentrations, airway morphology, and airway reactivity were evaluated at 24 hours and 7 days after chlorine exposure. At 24 hours after exposure, chlorine-exposed rats had significantly lower pulmonary ascorbate and reduced glutathione concentrations. Treatment with antioxidants restored depleted ascorbate in lungs and plasma. At 7 days after exposure, in chlorine-exposed, vehicle-treated rats, the thickness of the proximal airways was 60% greater than in control rats, with twice the amount of mucosubstances. Airway resistance in response to methacholine challenge was also significantly elevated. Combined treatment with intravenous and aerosolized antioxidants restored airway morphology, the amount of airway mucosubstances, and airway reactivity to control levels by 7 days after chlorine exposure. Our results demonstrate for the first time, to the best of our knowledge, that severe injury to major airways in rats exposed to chlorine, as characterized by epithelial hyperplasia, mucus accumulation, and airway hyperreactivity, can be reversed in a safe and efficacious manner by the post-exposure administration of ascorbate and deferoxamine.

The concentration of glutathione (GSH), the most abundant intracellular free thiol and an important antioxidant, is decreased in the lung in both fibrotic diseases and experimental fibrosis models. The underlying mechanisms and biological significance of GSH depletion, however, remain unclear. Transforming growth factor β (TGF-β) is the most potent and ubiquitous profibrogenic cytokine and its expression is increased in almost all fibrotic diseases. In this study, we show that increasing TGF-β1 expression in mouse lung to a level comparable to those found in lung fibrotic diseases by intranasal instillation of AdTGF-β1223/225, an adenovirus expressing constitutively active TGF-β1, suppressed the expression of both catalytic and modifier subunits of glutamate–cysteine ligase (GCL), the rate-limiting enzyme in de novo GSH synthesis, decreased GSH concentration, and increased protein and lipid peroxidation in mouse lung. Furthermore, we show that increasing TGF-β1 expression activated JNK and induced activating transcription factor 3, a transcriptional repressor involved in the regulation of the catalytic subunit of GCL, in mouse lung. Control virus (AdDL70-3) had no significant effect on any of these parameters, compared to saline-treated control. Concurrent with GSH depletion, TGF-β1 induced lung epithelial apoptosis and robust pulmonary fibrosis. Importantly, lung GSH levels returned to normal, whereas fibrosis persisted at least 21 days after TGF-β1 instillation. Together, the data suggest that increased TGF-β1 expression may contribute to the GSH depletion observed in pulmonary fibrosis diseases and that GSH depletion may be an early event in, rather than a consequence of, fibrosis development.

BACKGROUND Chlorine (Cl(2))-induced lung injury is a serious public health threat that may result from industrial and household accidents. Post-Cl(2) administration of aerosolized ascorbate in rodents decreased lung injury and mortality. However, the extent to which aerosolized ascorbate augments depleted ascorbate stores in distal lung compartments has not been assessed. METHODS We exposed rats to Cl(2) (300 ppm for 30 min) and returned them to room air. Within 15-30 min postexposure, rats breathed aerosolized ascorbate and desferal or vehicle (mean particle size 3.3 μm) through a nose-only exposure system for 60 min and were euthanized. We measured the concentrations of reduced ascorbate in the bronchoalveolar lavage (BAL), plasma, and lung tissues with high-pressure liquid chromatography, protein plasma concentration in the BAL, and the volume of the epithelia lining fluid (ELF). RESULTS Cl(2)-exposed rats that breathed aerosolized vehicle had lower values of ascorbate in their BAL, ELF, and lung tissues compared to air-breathing rats. Delivery of aerosolized ascorbate increased reduced ascorbate in BAL, ELF, lung tissues, and plasma of both Cl(2) and air-exposed rats without causing lung injury. Based on mean diameter of aerosolized particles and airway sizes we calculated that approximately 5% and 1% of inhaled ascorbate was deposited in distal lung regions of air and Cl(2)-exposed rats, respectively. Significantly higher ascorbate levels were present in the BAL of Cl(2)-exposed rats when aerosol delivery was initiated 1 h post-Cl(2). CONCLUSIONS Aerosol administration is an effective, safe, and noninvasive method for the delivery of low molecular weight antioxidants to the lungs of Cl(2)-exposed individuals for the purpose of decreasing morbidity and mortality. Delivery is most effective when initiated 1 h postexposure when the effects of Cl(2) on minute ventilation subside.

Computational fluid dynamics (CFD) models are useful for predicting site-specific dosimetry of airborne materials in the respiratory tract and elucidating the importance of species differences in anatomy, physiology, and breathing patterns. We improved the imaging and model development methods to the point where CFD models for the rat, monkey, and human now encompass airways from the nose or mouth to the lung. A total of 1272, 2172, and 135 pulmonary airways representing 17±7, 19±9, or 9±2 airway generations were included in the rat, monkey and human models, respectively. A CFD/physiologically based pharmacokinetic model previously developed for acrolein was adapted for these anatomically correct extended airway models. Model parameters were obtained from the literature or measured directly. Airflow and acrolein uptake patterns were determined under steady-state inhalation conditions to provide direct comparisons with prior data and nasal-only simulations. Results confirmed that regional uptake was sensitive to airway geometry, airflow rates, acrolein concentrations, air:tissue partition coefficients, tissue thickness, and the maximum rate of metabolism. Nasal extraction efficiencies were predicted to be greatest in the rat, followed by the monkey, and then the human. For both nasal and oral breathing modes in humans, higher uptake rates were predicted for lower tracheobronchial tissues than either the rat or monkey. These extended airway models provide a unique foundation for comparing material transport and site-specific tissue uptake across a significantly greater range of conducting airways in the rat, monkey, and human than prior CFD models.

We examine a previously published branch‐based approach for modeling airway diameters that is predicated on the assumption of self‐consistency across all levels of the tree. We mathematically formulate this assumption, propose a method to test it and develop a more general model to be used when the assumption is violated. We discuss the effect of measurement error on the estimated models and propose methods that take account of error. The methods are illustrated on data from MRI and CT images of silicone casts of two rats, two normal monkeys, and one ozone‐exposed monkey. Our results showed substantial departures from self‐consistency in all five subjects. When departures from self‐consistency exist, we do not recommend using the self‐consistency model, even as an approximation, as we have shown that it may likely lead to an incorrect representation of the diameter geometry. The new variance model can be used instead. Measurement error has an important impact on the estimated morphometry models and needs to be addressed in the analysis. Anat Rec,, 2012. © 2012 Wiley Periodicals, Inc.