Fiber biodurability and biopersistence: historical toxicological perspective of synthetic vitreous fibers (SVFs), the long fiber paradigm, and implications for advanced materials.

Extensive toxicology studies of synthetic vitreous fibers (SVFs) demonstrated that fiber dimension, durability/dissolution, and biopersistence are critical factors for risk of fibrogenesis and carcinogenesis. Lessons learned from the SVF experience provide useful context for predicting hazards and risk of nano-enabled advanced materials. This review provides (1) a historical toxicological overview of animal and in vitro toxicology studies of SVFs, (2) key findings that long durable fibers pose a risk of fibrogenic and tumorigenic responses and not short fibers or long soluble fibers, (3) in vitro and in vivo test methods for biodurability and biopersistence and associated predictive thresholds for fibrosis or tumors, and (4) recommendations for testing of advanced materials. Generally, SVFs (fiber lengths >20 µm) with in vitro fiber dissolution rates greater than 100 ng/cm2/hr (glass fibers in pH 7 and stone fibers in pH 4.5) and in vivo fiber clearance less than WT1/2 40 or 50 days were not associated with fibrosis or tumors. Long biodurable and biopersistent fibers exceeding these fiber dissolution and clearance thresholds may pose a risk of fibrosis and cancer. Fiber length-, durability-, and biopersistent-dependent factors that influence pathogenicity of mineral fibers are also expected to affect the biological effects of high aspect ratio nanomaterials (HARN). Only with studies aimed to correlate in vitro durability, in vivo biopersistence, and biological outcomes will it be determined whether similar or different in vitro fiber dissolution and in vivo half-life thresholds, which exempt carcinogenicity classification of SVFs, can also apply to HARNs.

The Intergenerational Transmission of Developmental Nicotine Exposure-Induced Neurodevelopmental Disorder-Like Phenotypes is Modulated by the Chrna5 D397N Polymorphism in Adolescent Mice.

Maternal tobacco smoking during pregnancy constitutes developmental nicotine exposure (DNE) and is associated with nicotine dependence and neurodevelopmental disorders in both children and grandchildren as well as animal models thereof. Genetic variants such as the CHRNA5 single nucleotide polymorphism (SNP) rs16969968, which leads to an aspartic acid to asparagine substitution at amino acid position 398 (D398N) in the alpha-5 nicotinic acetylcholine receptor subunit, can also confer risk for nicotine dependence and neurodevelopmental disorders in the absence of DNE. However, the degrees to which, the consequences of maternal smoking on offspring outcomes are influenced by genetic variants and interactions therewith are not well understood. Addressing this void in the literature, the present study utilizes a DNE mouse model engineered to possess the equivalent of the human D398N SNP in CHRNA5 (D397N SNP in mice) to assess how the N397 risk allele impacts the induction and intergenerational transmission of a range of neurodevelopmental disorder-related behavioral phenotypes in first- and second-generation DNE offspring. Results reveal that offspring possessing the N397 variant in the absence of DNE as well as DNE offspring and grand offspring possessing theD397 variant exhibit analogous neurodevelopmental disorder-like phenotypes including hyperactivity, risk-taking behaviors, aberrant rhythmicity of activity, and enhanced nicotine consumption. DNE amplified these behavioral anomalies in first-generation N397 progeny, but the severity of DNE-evoked behavioral perturbations did not significantly differ between first-generation D397 and N397 DNE mice for any measure. Remarkably, the behavioral profiles of second-generation N397 DNE progeny closely resembled DNE-naive D397 mice, suggesting that the N397 variant may protect against the intergenerational transmission of DNE-induced neurodevelopmental disorder-like behaviors.

Developmental nicotine exposure engenders intergenerational downregulation and aberrant posttranslational modification of cardinal epigenetic factors in the frontal cortices, striata, and hippocampi of adolescent mice.

BACKGROUND: Maternal smoking of traditional or electronic cigarettes during pregnancy, which constitutes developmental nicotine exposure (DNE), heightens the risk of neurodevelopmental disorders including ADHD, autism, and schizophrenia in children. Modeling the intergenerationally transmissible impacts of smoking during pregnancy, we previously demonstrated that both the first- and second-generation adolescent offspring of nicotine-exposed female mice exhibit enhanced nicotine preference, hyperactivity and risk-taking behaviors, aberrant rhythmicity of home cage activity, nicotinic acetylcholine receptor and dopamine transporter dysfunction, impaired furin-mediated proBDNF proteolysis, hypocorticosteronemia-related glucocorticoid receptor hypoactivity, and global DNA hypomethylation in the frontal cortices and striata. This ensemble of multigenerational DNE-induced behavioral, neuropharmacological, neurotrophic, neuroendocrine, and DNA methylomic anomalies recapitulates the pathosymptomatology of neurodevelopmental disorders such as ADHD, autism, and schizophrenia. Further probing the epigenetic bases of DNE-induced multigenerational phenotypic aberrations, the present study examined the expression and phosphorylation of key epigenetic factors via an array of immunoblot experiments. RESULTS: Data indicate that DNE confers intergenerational deficits in corticostriatal DNA methyltransferase 3A (DNMT3A) expression accompanied by downregulation of methyl-CpG-binding protein 2 (MeCP2) and histone deacetylase 2 (HDAC2) in the frontal cortices and hippocampi, while the expression of ten-eleven translocase methylcytosine dioxygenase 2 (TET2) is unaltered. Moreover, DNE evokes multigenerational abnormalities in HDAC2 (Ser394) but not MeCP2 (Ser421) phosphorylation in the frontal cortices, striata, and hippocampi. CONCLUSIONS: In light of the extensive gene regulatory roles of DNMT3A, MeCP2, and HDAC2, the findings of this study that DNE elicits downregulation and aberrant posttranslational modification of these factors in both first- and second-generation DNE mice suggest that epigenetic perturbations may constitute a mechanistic hub for the intergenerational transmission of DNE-induced neurodevelopmental disorder-like phenotypes.

Habenular TCF7L2 links nicotine addiction to diabetes.

Diabetes is far more prevalent in smokers than non-smokers, but the underlying mechanisms of vulnerability are unknown. Here we show that the diabetes-associated gene Tcf7l2 is densely expressed in the medial habenula (mHb) region of the rodent brain, where it regulates the function of nicotinic acetylcholine receptors. Inhibition of TCF7L2 signalling in the mHb increases nicotine intake in mice and rats. Nicotine increases levels of blood glucose by TCF7L2-dependent stimulation of the mHb. Virus-tracing experiments identify a polysynaptic connection from the mHb to the pancreas, and wild-type rats with a history of nicotine consumption show increased circulating levels of glucagon and insulin, and diabetes-like dysregulation of blood glucose homeostasis. By contrast, mutant Tcf7l2 rats are resistant to these actions of nicotine. Our findings suggest that TCF7L2 regulates the stimulatory actions of nicotine on a habenula-pancreas axis that links the addictive properties of nicotine to its diabetes-promoting actions.

Developmental nicotine exposure elicits multigenerational disequilibria in proBDNF proteolysis and glucocorticoid signaling in the frontal cortices, striata, and hippocampi of adolescent mice.

Maternal smoking of conventional or vapor cigarettes during pregnancy, a form of developmental nicotine exposure (DNE), enhances the risk of neurodevelopmental disorders such as ADHD, autism, and schizophrenia in children. Modeling the multigenerational effects of smoking during pregnancy and nursing in the first- (F1) and second- (F2) generation adolescent offspring of oral nicotine-treated female C57BL/6J mice, we have previously reported that DNE precipitates intergenerational transmission of nicotine preference, hyperactivity and impulsivity-like behaviors, altered rhythmicity of home cage activity, corticostriatal nicotinic acetylcholine receptor and dopamine transporter dysfunction, and corticostriatal global DNA methylome deficits. In aggregate, these DNE-evoked behavioral, neuropharmacological, and epigenomic anomalies mirror fundamental etiological aspects of neurodevelopmental disorders including ADHD, autism, and schizophrenia. Expanding this line of research, the current study profiled the multigenerational neurotrophic and neuroendocrine consequences of DNE. Results reveal impaired proBDNF proteolysis as indicated by proBDNF-BDNF imbalance, downregulation of the proBDNF processing enzyme furin, atypical glucocorticoid receptor (GR) activity as implied by decreased relative nuclear GR localization, and deficient basal plasma corticosterone (CORT) levels in adolescent DNE offspring and grandoffspring. Collectively, these data recapitulate the BDNF deficits and HPA axis dysregulation characteristic of neurodevelopmental disorders such as ADHD, autism, and schizophrenia as well as the children of maternal smokers. Notably, as BDNF is a quintessential mediator of neurodevelopment, our prior findings of multigenerational DNE-induced behavioral and neuropharmacological abnormalities may stem from neurodevelopmental insults conferred by the proBDNF-BDNF imbalance detected in DNE mice. Similarly, our findings of multigenerational GR hypoactivity may contribute to the increased risk-taking behaviors and aberrant circadian rhythmicity of home cage activity that we previously documented in first- and second-generation DNE mice.

Developmental nicotine exposure precipitates multigenerational maternal transmission of nicotine preference and ADHD-like behavioral, rhythmometric, neuropharmacological, and epigenetic anomalies in adolescent mice.

Maternal smoking during pregnancy, a form of developmental nicotine exposure (DNE), is associated with increased nicotine use and neurodevelopmental disorders such as ADHD in children. Here, we characterize the behavioral, rhythmometric, neuropharmacological, and epigenetic consequences of DNE in the F1 (first) and F2 (second) generation adolescent offspring of mice exposed to nicotine prior to and throughout breeding. We assessed the effects of passive oral methylphenidate (MPH) administration and voluntary nicotine consumption on home cage activity rhythms and activity and risk-taking behaviors in the open field. Results imply a multigenerational predisposition to nicotine consumption in DNE mice and demonstrate ADHD-like diurnal and nocturnal hyperactivity and anomalies in the rhythmicity of home cage activity that are reversibly rescued by MPH and modulated by voluntary nicotine consumption. DNE mice are hyperactive in the open field and display increased risk-taking behaviors that are normalized by MPH. Pharmacological characterization of nicotinic and dopaminergic systems in striatum and frontal cortex reveals altered expression and dysfunction of nicotinic acetylcholine receptors (nAChRs), hypersensitivity to nicotine-induced nAChR-mediated dopamine release, and impaired dopamine transporter (DAT) function in DNE mice. Global DNA methylation assays indicate DNA methylome deficits in striatum and frontal cortex of DNE mice. Collectively, our data demonstrate that DNE enhances nicotine preference, elicits hyperactivity and risk-taking behaviors, perturbs the rhythmicity of activity, alters nAChR expression and function, impairs DAT function, and causes DNA hypomethylation in striatum and frontal cortex of both first and second-generation adolescent offspring. These findings recapitulate multiple domains of ADHD symptomatology.

A novel genetic marker of decreased inflammation and improved survival after acute myocardial infarction.

The CHRNA5 gene encodes a neurotransmitter receptor subunit involved in multiple processes, including cholinergic autonomic nerve activity and inflammation. Common variants in CHRNA5 have been linked with atherosclerotic cardiovascular disease. Association of variation in CHRNA5 and specific haplotypes with cardiovascular outcomes has not been described. The aim of this study was to examine the association of CHRNA5 haplotypes with gene expression and mortality among patients with acute myocardial infarction (AMI) and explore potential mechanisms of this association. Patients (N = 2054) hospitalized with AMI were genotyped for two common variants in CHRNA5. Proportional hazard models were used to estimate independent association of CHRNA5 haplotype with 1-year mortality. Both individual variants were associated with mortality (p = 0.0096 and 0.0004, respectively) and were in tight LD (D' = 0.99). One haplotype, HAP3, was associated with decreased mortality one year after AMI (adjusted HR = 0.42, 95% CI 0.26, 0.68; p = 0.0004). This association was validated in an independent cohort (N = 637) of post-MI patients (adjusted HR = 0.23, 95% CI 0.07, 0.79; p = 0.019). Differences in CHRNA5 expression by haplotype were investigated in human heart samples (n = 28). Compared with non-carriers, HAP3 carriers had threefold lower cardiac CHRNA5 mRNA expression (p = 0.023). Circulating levels of the inflammatory marker hsCRP were significantly lower in HAP3 carriers versus non-carriers (3.43 ± 4.2 versus 3.91 ± 5.1; p = 0.0379). Activation of the inflammasome, an important inflammatory complex involved in cardiovascular disease that is necessary for release of the pro-inflammatory cytokine IL-1 ß, was assessed in bone marrow-derived macrophages (BMDM) from CHRNA5 knockout mice and wild-type controls. In BMDM from CHRNA5 knockout mice, IL-1ß secretion was reduced by 50% compared to wild-type controls (p = 0.004). Therefore, a common haplotype of CHRNA5 that results in reduced cardiac expression of CHRNA5 and attenuated macrophage inflammasome activation is associated with lower mortality after AMI. These results implicate CHRNA5 and the cholinergic anti-inflammatory pathway in survival following AMI.

Deletion of lynx1 reduces the function of α6* nicotinic receptors.

The α6 nicotinic acetylcholine receptor (nAChR) subunit is an attractive drug target for treating nicotine addiction because it is present at limited sites in the brain including the reward pathway. Lynx1 modulates several nAChR subtypes; lynx1-nAChR interaction sites could possibly provide drug targets. We found that dopaminergic cells from the substantia nigra pars compacta (SNc) express lynx1 mRNA transcripts and, as assessed by co-immunoprecipitation, α6 receptors form stable complexes with lynx1 protein, although co-transfection with lynx1 did not affect nicotine-induced currents from cell lines transfected with α6 and ß2. To test whether lynx1 is important for the function of α6 nAChRs in vivo, we bred transgenic mice carrying a hypersensitive mutation in the α6 nAChR subunit (α6L9'S) with lynx1 knockout mice, providing a selective probe of the effects of lynx1 on α6* nAChRs. Lynx1 removal reduced the α6 component of nicotine-mediated rubidium efflux and dopamine (DA) release from synaptosomal preparations with no effect on numbers of α6ß2 binding sites, indicating that lynx1 is functionally important for α6* nAChR activity. No effects of lynx1 removal were detected on nicotine-induced currents in slices from SNc, suggesting that lynx1 affects presynaptic α6* nAChR function more than somatic function. In the absence of agonist, lynx1 removal did not alter DA release in dorsal striatum as measured by fast scan cyclic voltammetry. Lynx1 removal affected some behaviors, including a novel-environment assay and nicotine-stimulated locomotion. Trends in 24-hour home-cage behavior were also suggestive of an effect of lynx1 removal. Conditioned place preference for nicotine was not affected by lynx1 removal. The results show that some functional and behavioral aspects of α6-nAChRs are modulated by lynx1.

Nicotine reverses hypofrontality in animal models of addiction and schizophrenia.

The prefrontal cortex (PFC) underlies higher cognitive processes that are modulated by nicotinic acetylcholine receptor (nAChR) activation by cholinergic inputs. PFC spontaneous default activity is altered in neuropsychiatric disorders, including schizophrenia-a disorder that can be accompanied by heavy smoking. Recently, genome-wide association studies (GWAS) identified single-nucleotide polymorphisms (SNPs) in the human CHRNA5 gene, encoding the α5 nAChR subunit, that increase the risks for both smoking and schizophrenia. Mice with altered nAChR gene function exhibit PFC-dependent behavioral deficits, but it is unknown how the corresponding human polymorphisms alter the cellular and circuit mechanisms underlying behavior. Here we show that mice expressing a human α5 SNP exhibit neurocognitive behavioral deficits in social interaction and sensorimotor gating tasks. Two-photon calcium imaging in awake mouse models showed that nicotine can differentially influence PFC pyramidal cell activity by nAChR modulation of layer II/III hierarchical inhibitory circuits. In α5-SNP-expressing and α5-knockout mice, lower activity of vasoactive intestinal polypeptide (VIP) interneurons resulted in an increased somatostatin (SOM) interneuron inhibitory drive over layer II/III pyramidal neurons. The decreased activity observed in α5-SNP-expressing mice resembles the hypofrontality observed in patients with psychiatric disorders, including schizophrenia and addiction. Chronic nicotine administration reversed this hypofrontality, suggesting that administration of nicotine may represent a therapeutic strategy for the treatment of schizophrenia, and a physiological basis for the tendency of patients with schizophrenia to self-medicate by smoking.

Chronic treatment with varenicline changes expression of four nAChR binding sites in mice.

INTRODUCTION: Chronic treatment with nicotine is known to increase the α4ß2-nAChR sites in brain, to decrease α6ß2-nAChR sites and to have minimal effect on α3ß4-and α7-nAChR populations. Varenicline is now used as a smoking cessation treatment, with and without continued smoking or nicotine replacement therapy. Varenicline, like nicotine, upregulates the α4ß2-nAChR sites; however, it is not known whether varenicline treatment changes expression of the other nAChR subtypes. METHODS: Using a mouse model, chronic treatments (10 days) with varenicline (0.12  mg/kg/h) and/or nicotine (1 mg/kg/hr), alone or in combination, were compared for plasma and brain levels of drugs, tolerance to subsequent acute nicotine and expression of four subtypes of nAChR using autoradiography. RESULTS: The upregulation of α4ß2-nAChR sites elicited by chronic varenicline was very similar to that elicited by chronic nicotine. Treatment with both drugs somewhat increased up-regulation, indicating that these doses were not quite at maximum effect. Similar down-regulation was seen for α6ß2-nAChR sites. Varenicline significantly increased both α3ß4-and α7-nAChR sites while nicotine had less effect on these sites. The drug combination was similar to varenicline alone for α3ß4-nAChR sites, while for α7 sites the drug combination was less effective than varenicline alone. Varenicline had small but significant effects on tolerance to acute nicotine. CONCLUSIONS: Effects of varenicline in vivo may not be limited to the α4ß2*-nAChR subtype. In addition, smoking cessation treatment with varenicline may not allow receptor numbers to be restored to baseline and may, in addition, change expression of other receptor subtypes.

Mice expressing the ADNFLE valine 287 leucine mutation of the Β2 nicotinic acetylcholine receptor subunit display increased sensitivity to acute nicotine administration and altered presynaptic nicotinic receptor function.

Several mutations in α4 or ß2 nicotinic receptor subunits are linked to autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). One such missense mutation in the gene encoding the ß2 neuronal nicotinic acetylcholine receptor (nAChR) subunit (CHRNB2) is a valine-to-leucine substitution in the second transmembrane domain at position 287 (ß2VL). Previous studies indicated that the ß2VL mutation in mice alters circadian rhythm consistent with sleep alterations observed in ADNFLE patients (Xu et al., 2011). The current study investigates changes in nicotinic receptor function and expression that may explain the behavioral phenotype of ß2VL mice. No differences in ß2 mRNA expression were found between wild-type (WT) and heterozygous (HT) or homozygous mutant (MT) mice. However, antibody and ligand binding indicated that the mutation resulted in a reduction in receptor protein. Functional consequences of the ß2VL mutation were assessed biochemically using crude synaptosomes. A gene-dose dependent increase in sensitivity to activation by acetylcholine and decrease in maximal nAChR-mediated [(3)H]-dopamine release and (86)Rb efflux were observed. Maximal nAChR-mediated [(3)H]-GABA release in the cortex was also decreased in the MT, but maximal [(3)H]-GABA release was retained in the hippocampus. Behaviorally both HT and MT mice demonstrated increased sensitivity to nicotine-induced hypolocomotion and hypothermia. Furthermore, WT mice display only a tonic-clonic seizure (EEG recordable) 3 min after injection of a high dose of nicotine, while MT mice also display a dystonic arousal complex (non-EEG recordable) event 30s after nicotine injection. Data indicate decreases in maximal response for certain measures are larger than expected given the decrease in receptor expression.

α6* nicotinic acetylcholine receptor expression and function in a visual salience circuit.

Nicotinic acetylcholine receptors (nAChRs) containing α6 subunits are expressed in only a few brain areas, including midbrain dopamine (DA) neurons, noradrenergic neurons of the locus ceruleus, and retinal ganglion cells. To better understand the regional and subcellular expression pattern of α6-containing nAChRs, we created and studied transgenic mice expressing a variant α6 subunit with green fluorescent protein (GFP) fused in-frame in the M3-M4 intracellular loop. In α6-GFP transgenic mice, α6-dependent synaptosomal DA release and radioligand binding experiments confirmed correct expression and function in vivo. In addition to strong α6* nAChR expression in glutamatergic retinal axons, which terminate in superficial superior colliculus (sSC), we also found α6 subunit expression in a subset of GABAergic cell bodies in this brain area. In patch-clamp recordings from sSC neurons in brain slices from mice expressing hypersensitive α6* nAChRs, we confirmed functional, postsynaptic α6* nAChR expression. Further, sSC GABAergic neurons expressing α6* nAChRs exhibit a tonic conductance mediated by standing activation of hypersensitive α6* nAChRs by ACh. α6* nAChRs also appear in a subpopulation of SC neurons in output layers. Finally, selective activation of α6* nAChRs in vivo induced sSC neuronal activation as measured with c-Fos expression. Together, these results demonstrate that α6* nAChRs are uniquely situated to mediate cholinergic modulation of glutamate and GABA release in SC. The SC has emerged as a potential key brain area responsible for transmitting short-latency salience signals to thalamus and midbrain DA neurons, and these results suggest that α6* nAChRs may be important for nicotinic cholinergic sensitization of this pathway.

Varenicline blocks ß2*-nAChR-mediated response and activates ß4*-nAChR-mediated responses in mice in vivo.

INTRODUCTION: The smoking cessation aid, varenicline, has higher affinity for the alpha4beta2-subtype of the nicotinic acetylcholine receptor (α4ß2*-nAChR) than for other subtypes of nAChRs by in vitro assays. The mechanism of action of acute varenicline was studied in vivo to determine (a) subtype activation associated with physiological effects and (b) dose relationship as an antagonist of nicotine. METHODS: Acute doses of saline, nicotine, and varenicline were given to mice, and locomotor depression and hypothermia were measured. Subunit null mutant mice as well as selective antagonists were used to study mode of action of varenicline as an agonist. Varenicline as an antagonist of nicotine was also investigated. RESULTS: Varenicline evokes locomotor depression and hypothermia at higher doses than necessary for nicotine. Null mutation of the α7- or ß2-nAChR subunit did not decrease the effectiveness of varenicline; however, null mutation of the ß4 subunit significantly decreased the magnitude of the varenicline effect. Effects of the highest dose studied were blocked by mecamylamine (general nAChR antagonist) and partially antagonized by hexamethonium (largely peripheral nAChR antagonist). No significant block was seen with ondansetron antagonist of 5-hydroxytryptamine 3 receptor. Using a dose of nicotine selective for ß2*-nAChR subtype effects with these tests, dose-dependent antagonism by varenicline was seen. Effective inhibitory doses were determined and appear to be in a range consistent with binding affinity or desensitization of ß2*-nAChRs. CONCLUSIONS: Varenicline acts as a functional antagonist of ß2*-nAChRs, blocking certain effects of nicotine. At higher doses, varenicline is an agonist of ß4*-nAChRs producing physiological changes in mice.

Role of reactive oxygen and nitrogen species in olfactory epithelial injury by the sulfur mustard analogue 2-chloroethyl ethyl sulfide.

The inhalation of sulfur mustard (SM) causes substantial deposition in the nasal region. However, specific injury has not been characterized. 2-chloroethyl ethyl sulfide (CEES) is an SM analogue used to model injury and screen potential therapeutics. After the inhalation of CEES, damage to the olfactory epithelium (OE) was extensive. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling-positive cells were present by 4 hours, and maximal at 18-72 hours. Cleaved caspase 3 immunohistochemistry (IHC) was maximal at 18 hours after the inhalation of 5% CEES. Olfactory marker protein (OMP)-positive olfactory neurons were markedly decreased at 18 hours. IHC-positive cells for 3-nitrotyrosine (3-NT) within epithelium were elevated by 8 hours, waning by 18 hours, and absent by 72 hours. AEOL 10150, a catalytic manganoporphyrin antioxidant, administered both subcutaneously (5 mg/kg) and intranasally (50 µM, "combined treatment"), decreased OE injury. CEES-induced increases in markers of cell death were decreased by combined treatment involving AEOL 10150. CEES-induced changes in OMP and 3-NT immunostaining were markedly improved by combined treatment involving AEOL 10150. The selective inducible nitric oxide synthase inhibitor 1400W (5 mg/kg, subcutaneous), administered 1 hour after inhalation and thereafter every 4 hours (five doses), also reduced OE damage with improved OMP and 3-NT staining. Taken together, these data indicate that reactive oxygen and nitrogen species are important mediators in CEES-induced nasal injury.

Airway obstruction due to bronchial vascular injury after sulfur mustard analog inhalation.

RATIONALE: Sulfur mustard (SM) is a frequently used chemical warfare agent, even in modern history. SM inhalation causes significant respiratory tract injury, with early complications due to airway obstructive bronchial casts, akin to those seen after smoke inhalation and in single-ventricle physiology. This process with SM is poorly understood because animal models are unavailable. OBJECTIVES: To develop a rat inhalation model for airway obstruction with the SM analog 2-chloroethyl ethyl sulfide (CEES), and to investigate the pathogenesis of bronchial cast formation. METHODS: Adult rats were exposed to 0, 5, or 7.5% CEES in ethanol via nose-only aerosol inhalation (15 min). Airway microdissection and confocal microscopy were used to assess cast formation (4 and 18 h after exposure). Bronchoalveolar lavage fluid (BALF) retrieval and intravascular dye injection were done to evaluate vascular permeability. MEASUREMENTS AND MAIN RESULTS: Bronchial casts, composed of abundant fibrin and lacking mucus, occluded dependent lobar bronchi within 18 hours of CEES exposure. BALF contained elevated concentrations of IgM, protein, and fibrin. Accumulation of fibrin-rich fluid in peribronchovascular regions (4 h) preceded cast formation. Monastral blue dye leakage identified bronchial vessels as the site of leakage. CONCLUSIONS: After CEES inhalation, increased permeability from damaged bronchial vessels underlying damaged airway epithelium leads to the appearance of plasma proteins in both peribronchovascular regions and BALF. The subsequent formation of fibrin-rich casts within the airways then leads to airways obstruction, causing significant morbidity and mortality acutely after exposure.

Treatment with the catalytic metalloporphyrin AEOL 10150 reduces inflammation and oxidative stress due to inhalation of the sulfur mustard analog 2-chloroethyl ethyl sulfide.

Sulfur mustard (bis-2-(chloroethyl) sulfide; SM) is a highly reactive vesicating and alkylating chemical warfare agent. A SM analog, 2-chloroethyl ethyl sulfide (CEES), has been utilized to elucidate mechanisms of toxicity and as a screen for therapeutics. Previous studies with SM and CEES have demonstrated a role for oxidative stress as well as decreased injury with antioxidant treatment. We tested whether posttreatment with the metalloporphyrin catalytic antioxidant AEOL 10150 would improve outcome in CEES-induced lung injury. Anesthetized rats inhaled 5% CEES for 15 min via a nose-only inhalation system. At 1 and 9 h after CEES exposure, rats were given AEOL 10150 (5 mg/kg, sc). At 18 h post-CEES exposure BALF lactate dehydrogenase activity, protein, IgM, red blood cells, and neutrophils were elevated but were decreased by AEOL 10150 treatment. Lung myeloperoxidase activity was increased after CEES inhalation and was ameliorated by AEOL 10150. The lung oxidative stress markers 8-OHdG and 4-HNE were elevated after CEES exposure and significantly decreased by AEOL 10150 treatment. These findings demonstrate that CEES inhalation increased lung injury, inflammation, and oxidative stress, and AEOL 10150 was an effective rescue agent. Further investigation utilizing catalytic antioxidants as treatment for SM inhalation injury is warranted.

Alpha-lipoic acid induces elevated S-adenosylhomocysteine and depletes S-adenosylmethionine.

Lipoic acid is a disulfhydryl-containing compound used in clinical medicine and in experimental models as an antioxidant. We developed a stable isotope dilution capillary gas chromatography/mass spectrometry assay for lipoic acid. We assayed a panel of the metabolites of transmethylation and transsulfuration 30 min after injecting 100 mg/kg lipoic acid in a rat model. Lipoic acid values rose 1000-fold in serum and 10-fold in liver. A methylated metabolite of lipoic acid was also detected but not quantitated. Lipoic acid injection caused a massive increase in serum S-adenosylhomocysteine and marked depletion of liver S-adenosylmethionine. Serum total cysteine was depleted but liver cysteine and glutathione were maintained. Serum total homocysteine doubled, with increases also in cystathionine, N,N-dimethylglycine, and alpha-aminobutyric acid. In contrast, after injection of 2-mercaptoethane sulfonic acid, serum total cysteine and homocysteine were markedly depleted and there were no effects on serum S-adenosylmethionine or S-adenosylhomocysteine. We conclude that large doses of lipoic acid displace sulfhydryls from binding sites, resulting in depletion of serum cysteine, but also pose a methylation burden with severe depletion of liver S-adenosylmethionine and massive release of S-adenosylhomocysteine. These changes may have previously unrecognized deleterious effects that should be investigated in both human disease and experimental models.

Lipoic acid suppression of neutrophil respiratory burst: effect of NADPH.

Lipoic acid (LA) and its reduced product dihydrolipoic acid (DHLA) are potent antioxidants with capacity to scavenge reactive oxygen species (ROS) and recycle endogenous antioxidants. LA may increase cellular glutathione (GSH), an antioxidant lacking in the lung's epithelial lining fluid in lung disorders such as idiopathic pulmonary fibrosis (IPF). Neutrophils (PMN) are key innate responders and are pivotal in clearing bacterial infection, therefore it is crucial to understand the impact LA may have on their function. Circulating neutrophils were isolated from healthy volunteers and pretreated with LA or diluent. Cells were subsequently activated with phorbol 12-myristate 13-acetate (PMA, 100 ng/ml) to induce ROS production. SOD-inhibitable reduction of acetylated cytochrome c demonstrated the PMA-dependent respiratory burst was suppressed by LA. Oxygen consumption also was diminished when PMA-stimulated cells were pretreated with LA. PMN respiratory burst was partially restored by addition of NADPH but not other pyridine nucleotides. LA did not inhibit glucose-6-phosphate dehydrogenase activity of PMN. These data together suggest that the reduction of LA to DHLA using cellular NADPH may limit the capacity of the PMN NADPH oxidase to produce superoxide. Further studies will be required to determine if LA can diminish excessive superoxide produced by PMN and/or alveolar macrophages in IPF or relevant disease models in vivo.

Cigarette smoke extract-induced suppression of caspase-3-like activity impairs human neutrophil phagocytosis.

Neutrophils are the primary inflammatory cell in smokers' lungs, but little is known about the ability of cigarette smoke to modulate neutrophil function. Neutrophils undergo caspase-3-dependent spontaneous, as well as phagocytosis-induced, apoptosis. This study investigated the ability of cigarette smoke extract (CSE) to alter neutrophil caspase-3 activity, apoptosis, and phagocytosis. CSE treatment resulted in a dramatic suppression of neutrophil caspase-3-like activity, which correlated with reduced cleavage of glutamate-L-cysteine ligase catalytic subunit, a known target of active caspase-3. CSE did not affect procaspase-3 processing to its active fragment, suggesting a direct effect of CSE on active caspase-3. Consistent with this, CSE inhibited active recombinant caspase-3 activity, which was abolished by dithiothreitol, suggesting a redox-sensitive mechanism. CSE-induced suppression of caspase-3 activity did not alter spontaneous apoptosis but did impair phagocytic activity. Since CSE treatment resulted in profound suppression of caspase-3 activity but did not alter apoptosis, the possibility of a threshold level of caspase-3 activity was investigated. CSE reduced caspase-3 activity in a concentration-dependent manner. Despite near complete suppression of caspase-3 activity, spontaneous apoptosis was not altered. Conversely, treatment with the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, reduced spontaneous apoptosis. These data demonstrate that CSE does not suppress caspase-3 activity below a threshold level to prevent spontaneous apoptosis, but the level of inhibition is sufficient to impair neutrophil phagocytic activity. These divergent functions of caspase-3 may contribute to the persistence of neutrophils in the lungs of smokers, as well as be a factor in their higher incidence of community-acquired pneumonia.

Lithium alters measures of auditory gating in two strains of mice.

BACKGROUND: There are similarities between schizophrenia and bipolar disorder, especially during the psychotic phase. Auditory gating deficits are common in both schizophrenia (does not remit postpsychotic event) and bipolar disorder (only during the manic phase). Lithium has been used to treat psychosis acutely in both bipolar disorder and schizophrenia. An animal model was used to assess the effects of lithium treatment on normal and deficient auditory gating. METHODS: Mice of the DBA/2 (deficient gating) and C3H (normal gating) strains were treated for 6 weeks with either standard rodent chow or rodent chow supplemented with 2.55g/kg lithium carbonate. After 6 weeks of treatment, auditory evoked potentials were recorded under anesthesia. Differences between the groups and treatments were determined using analysis of variance. RESULTS: The normally impaired DBA/2 mice showed improved auditory gating following lithium treatment, while the C3H mice, the benchmark "normal" mouse strain, were impaired after lithium treatment. CONCLUSIONS: C3H mice treated with lithium had significantly impaired auditory gating as a result of treatment. This may be due to norepinephrine facilitation, through a blockade of presynaptic alpha(2) autoreceptors. DBA/2 mice had improved gating as a result of treatment with lithium, likely due to improved functioning of the gamma-aminobutyric acid system.