Early Mechanistic Events Induced by Low Molecular Weight Polycyclic Aromatic Hydrocarbons in Mouse Lung Epithelial Cells: A Role for Eicosanoid Signaling.

Low molecular weight polycyclic aromatic hydrocarbons (LMW PAHs; < 206.3 g/mol) are under regulated environmental contaminants (eg, secondhand smoke) that lead to gap junction dysregulation, p38 MAPK activation, and increased mRNA production of inflammatory mediators, such as cytokines and cyclooxygenase (COX2), in lung epithelial cells. However, the early mechanisms involving lipid signaling through the arachidonic acid pathway and subsequent eicosanoid production leading to these downstream events are not known. Common human exposures are to mixtures of LMW PAHs, thus C10 cells (a mouse lung epithelial cell line) were exposed to a representative binary PAH mixture, 1-methylanthracene (1-MeA) and fluoranthene (Flthn), for 30 min-24 h with and without p38 and cytosolic phospholipase A2 (cPLA2) inhibitors. Cytosolic phospholipase A2 inhibition reversed PAH-induced phospho-p38 MAPK activation and gap junction dysregulation at 30 min. A significant biphasic increase in cPLA2 protein was observed at 30 min, 2, and 4 h, as well as COX2 protein at 2 and 8 h. Untargeted metabolomics demonstrated a similar trend with significantly changing metabolites at 30 min and 4 h of exposure relative to 1 h; a "cPLA2-like" subset of metabolites within the biphasic response were predominately phospholipids. Targeted metabolomics showed several eicosanoids (eg, prostaglandin D2 (PGD2), PGE2α) were significantly increased at 4, 8, and 12 h following exposure to the binary PAH mixture and this effect was p38-dependent. Finally, PAH metabolism was not observed until after 8 h. These results indicate an early lipid signaling mechanism of LMW PAH toxicity in lung epithelial cells due to parent PAH compounds.

Determining the presence of asthma-related molecules and salivary contamination in exhaled breath condensate.

BACKGROUND: Researchers investigating lung diseases, such as asthma, have questioned whether certain compounds previously reported in exhaled breath condensate (EBC) originate from saliva contamination. Moreover, despite its increasing use in 'omics profiling studies, the constituents of EBC remain largely uncharacterized. The present study aims to define the usefulness of EBC in investigating lung disease by comparing EBC, saliva, and saliva-contaminated EBC using targeted and untargeted mass spectrometry and the potential of metabolite loss from adsorption to EBC sample collection tubes. METHODS: Liquid chromatography mass spectrometry (LC-MS) was used to analyze samples from 133 individuals from three different cohorts. Levels of amino acids and eicosanoids, two classes of molecules previously reported in EBC and saliva, were measured using targeted LC-MS. Cohort 1 was used to examine contamination of EBC by saliva. Samples from Cohort 1 consisted of clean EBC, saliva-contaminated EBC, and clean saliva from 13 healthy volunteers; samples were analyzed using untargeted LC-MS. Cohort 2 was used to compare eicosanoid levels from matched EBC and saliva collected from 107 asthmatic subjects. Samples were analyzed using both targeted and untargeted LC-MS. Cohort 3 samples consisted of clean-EBC collected from 13 subjects, including smokers and non-smokers, and were used to independently confirm findings; samples were analyzed using targeted LC-MS, untargeted LC-MS, and proteomics. In addition to human samples, an in-house developed nebulizing system was used to determine the potential for EBC samples to be contaminated by saliva. RESULTS: Out of the 400 metabolites detected in both EBC and saliva, 77 were specific to EBC; however, EBC samples were concentrated 20-fold to achieve this level of sensitivity. Amino acid concentrations ranged from 196 pg/mL - 4 µg/mL (clean EBC), 1.98 ng/mL - 6 µg/mL (saliva-contaminated EBC), and 13.84 ng/mL - 1256 mg/mL (saliva). Eicosanoid concentration ranges were an order of magnitude lower; 10 pg/mL - 76.5 ng/mL (clean EBC), 10 pg/mL - 898 ng/mL (saliva-contaminated EBC), and 2.54 ng/mL - 272.9 mg/mL (saliva). Although the sample size of the replication cohort (Cohort 3) did not allow for statistical comparisons, two proteins and 19 eicosanoids were detected in smoker vs. non-smoker clean-EBC. CONCLUSIONS: We conclude that metabolites are present and detectable in EBC using LC-MS; however, a large starting volume of sample is required.

Transport stability of pesticides and PAHs sequestered in polyethylene passive sampling devices.

Research using low-density polyethylene (LDPE) passive samplers has steadily increased over the past two decades. However, such research efforts remain hampered because of strict guidelines, requiring that these samplers be quickly transported in airtight metal or glass containers or foil wrapped on ice. We investigate the transport stability of model pesticides and polycyclic aromatic hydrocarbons (PAHs) with varying physicochemical properties using polytetrafluoroethylene (PTFE) bags instead. Transport scenarios were simulated with transport times up to 14 days with temperatures ranging between -20 and 35 °C. Our findings show that concentrations of all model compounds examined were stable for all transport conditions tested, with mean recoveries ranging from 88 to 113 %. Furthermore, PTFE bags proved beneficial as reusable, lightweight, low-volume, low-cost alternatives to conventional containers. This documentation of stability will allow for more flexible transportation of LDPE passive samplers in an expanding range of research applications while maintaining experimental rigor.

Primary adrenal lymphoma causing hypoaldosteronism in a cat.

CASE SUMMARY: A 10-year-old, 5.1 kg (11.2 lb), male castrated cat was presented with signs of lethargy and decreased appetite at home after being previously healthy. Serum biochemical analysis identified normokalemia (5.1 mmol/l; reference interval [RI] 3.4-5.6 mmol/l) and severe hyponatremia (123 mmol/l; RI 145-158 mmol/l), with an Na/K ratio of 24 (RI 32-41). Baseline serum cortisol was low to normal, but serum aldosterone was markedly decreased with a pre-adrenocorticotropic hormone stimulation concentration of 13 pmol/l (RI 194-388 pmol/l) and post-adrenocorticotropic hormone stimulation concentration of 21 pmol/l (RI 277-721 pmol/l). Hematologic and biochemical analyses were otherwise unremarkable. Abdominal ultrasound revealed bilaterally enlarged adrenal glands with no other abnormalities noted; thoracic radiographs also did not identify any signs of metastasis. Fine-needle aspiration was strongly suggestive of lymphoma of the adrenal glands, and PCR for antigen receptor rearrangement was positive for B-cell clonal expansion; based on these findings, a diagnosis of primary adrenal B-cell lymphoma was made. Stable disease was achieved for a short period of time following vincristine, cyclophosphamide, prednisolone and fludrocortisone therapy, followed by progressive adrenal enlargement and electrolyte derangements that responded to neither doxorubicin nor adjustments in fludrocortisone dosage. Ultrasonographic metastasis was not identified at any time, and other organ derangements were not noted on hematologic or biochemical analyses. The cat was euthanized 55 days after initial presentation. RELEVANCE AND NOVEL INFORMATION: This is the first report of primary adrenal lymphoma in a cat, with presenting signs compatible with hypoaldosteronism. Lymphoma should be a differential for cats presenting with adrenal enlargement or clinical signs and biochemical changes consistent with hypoaldosteronism or hypoadrenocorticism.

Metabolomic analysis to define and compare the effects of PAHs and oxygenated PAHs in developing zebrafish.

Polycyclic aromatic hydrocarbons (PAHs) and their oxygenated derivatives are ubiquitously present in diesel exhaust, atmospheric particulate matter and soils sampled in urban areas. Therefore, inhalation or non-dietary ingestion of both PAHs and oxy-PAHs are major routes of exposure for people; especially young children living in these localities. While there has been extensive research on the parent PAHs, limited studies exist on the biological effects of oxy-PAHs which have been shown to be more soluble and more mobile in the environment. Additionally, investigations comparing the metabolic responses resulting from parent PAHs and oxy-PAHs exposures have not been reported. To address these current gaps, an untargeted metabolomics approach was conducted to examine the in vivo metabolomic profiles of developing zebrafish (Danio rerio) exposed to 4 µM of benz[a]anthracene (BAA) or benz[a]anthracene-7,12-dione (BAQ). By integrating multivariate, univariate and pathway analyses, a total of 63 metabolites were significantly altered after 5 days of exposure. The marked perturbations revealed that both BAA and BAQ affect protein biosynthesis, mitochondrial function, neural development, vascular development and cardiac function. Our previous transcriptomic and genomic data were incorporated in this metabolomics study to provide a more comprehensive view of the relationship between PAH and oxy-PAH exposures on vertebrate development.

Application of a magnesium/co-solvent system for the degradation of polycyclic aromatic hydrocarbons and their oxygenated derivatives in a spiked soil.

This study evaluates the capability, efficacy and practicality of a combined approach based on solvent extraction and chemical reduction to simultaneously degrade polycyclic aromatic hydrocarbons (PAHs) and their oxygenated derivatives (OPAHs) in spiked soil. The spiked soil was washed using a composite organic solvent consisting of ethanol and ethyl lactate (1:1, v/v) and then degradation of the extracted contaminants using zero-valent magnesium. The extraction conditions were optimized at 25 °C with solvent-soil ratio of 2:1 (v/w) and the ensuing degradation efficiency ranged from 79% to 88% for the OPAHs, and 66% to 87% for the PAHs after 24 h of reaction at pH of 6.1. The reductive degradation of the spiked contaminants followed pseudo-first-order kinetics; however, comparing the kinetic results of this study to soil-free studies, the degradation rates are significantly reduced. It can be inferred that extracted organic or inorganic components from the soil medium hinder the degradation process, possibly by reducing the reactivity of the activated metal. Furthermore, to our understanding, this study is the first report on the simultaneous degradation of these priority pollutants and their oxygenated derivatives. The experimental results encourage the application of this magnesium/co-solvent system for future pilot-scale remediation studies.

Reductive degradation of oxygenated polycyclic aromatic hydrocarbons using an activated magnesium/co-solvent system.

This study evaluates the capability of zero-valent magnesium and a protic co-solvent to promote the degradation of oxygenated polycyclic aromatic hydrocarbons compounds, specifically 9-fluorenone, 9,10-anthraquinone, 7,12-benz(a)anthraquionone, and 7H-benz(de)anthracene-7-one. At room temperature conditions, greater than 86% degradation efficiency is observed after 24h of reaction time for a mixture containing 0.05 g of magnesium and four selected oxygenated aromatic hydrocarbons with 250 mg L(-1) concentrations. It is noted that glacial acetic acid is needed as an activator for the degradation reaction to proceed. It is also presumed that the acid removes oxide and hydroxide species from the magnesium surface. With the GC-MS analysis of the reaction products, possible reductive pathways are suggested. Furthermore, this study is the first report on the degradation of these emerging contaminants and it is proposed that the magnesium-powder/protic-solvent system is a promising low-cost reagent and may allow for the future development of an economic and environmentally-friendly remediation application.

Multivariate evaluation and optimization of an activated-magnesium/co-solvent system for the reductive degradation of polycyclic aromatic hydrocarbons.

The present study evaluates the capability of an activated-magnesium metal and protic co-solvents to promote the reductive degradation of three different polycyclic aromatic hydrocarbons, specifically pyrene, benzo[k]fluoranthene and benzo[g,h,i]perylene. Multivariate analyses demonstrated that the kinetics of degradation was affected by several experimental factors such as magnesium loading, acid addition and solubility of the compounds. It was determined that an acid activator is needed for the degradation reaction to proceed and it is also noted that the use of a 1:1 ethanol/ethyl lactate co-solvent is ideal for the complete dissolution of all three compounds with concentrations varying from 200 to 275mgL(-1). The experimental results also indicate that, at room temperature conditions, only 0.05-0.1g of magnesium is required in order to achieve greater than 93% degradation efficiency after 24h of reaction. This methodology is attractive and may allow for the development of an economic and environmentally friendly field application for the remediation of other polycyclic aromatic hydrocarbons.

Reduction of benzo[a]pyrene with acid-activated magnesium metal in ethanol: a possible application for environmental remediation.

Persistent organic pollutants (POPs) are a well-known threat to the environment. Substances such as polycyclic aromatic hydrocarbons (PAHs) in contaminated soils and sediments can have severe and long-term effects on human and environmental health. There is an urgent need for the development of safe technologies for their effective degradation. Here we present a new technique using ball-milled magnesium powder and ethanol solvent as a convenient electron transfer/proton source for the partial reduction of PAHs under ambient conditions. The rates of degradation were determined while evaluating the influences of acetic acid and type of ball-milled magnesium added to the reaction mixture. The results of these triplicate studies indicate that with the use of acetic acid as an activator and ball-milled magnesium carbon (Mg/C), this reducing system (Mg-EtOH) is able to achieve a 94% conversion of 250 µg/mL of toxic benzo[a]pyrene into a mixture of less toxic and partially hydrogenated polycyclic compounds within 24h. This methodology can be used as a combined process involving ethanol washing followed by reduction reaction and it can also be considered as an easy handling and efficient alternative process to the catalytic hydrogenation of PAHs.

Basics in canine and feline rhinoscopy.

Patients suffering from upper respiratory disease such as chronic nasal congestion, sneezing, nasal discharge, and epistaxis invite complete evaluation of their paired nasal cavities. Thorough assessment of these cavities employs sundry diagnostic procedures that enable the investigating clinician to characterize the internal structures of the nasal cavities. After the conscious patient undergoes a complete physical examination, a gross assessment of its external nasal structures is established and areas of physical asymmetry are noted. A working anatomic knowledge of these asymmetric foci helps to guide the next diagnostic steps. The patient is then placed under general anesthesia, during which, in list order, imaging studies, rhinoscopy, and nasal biopsy or foreign body retrieval, are performed.

Clinicopathologic features of suspected brevetoxicosis in double-crested cormorants (Phalacrocorax auritus) along the Florida Gulf Coast.

Outbreaks of morbidity and mortality in double-crested cormorants (Phalacrocorax auritus) along Florida's Gulf Coast have occurred sporadically for at least 30 yr. During these outbreaks, the Clinic for the Rehabilitation of Wildlife, located on Sanibel Island in Florida, has admitted a substantial number of cormorants with consistent presentation of primarily neurologic clinical signs. In order to investigate the association of these outbreaks in cormorants with exposure to brevetoxin, we compared the timing of admittance of cormorants with outbreak-specific clinical signs to blooms of the brevetoxin-producing marine algae, Karenia brevis (formerly Gymnodinium breve), around Sanibel Island from 1995 through 1999. The clinic admitted 360 out of 613 cormorants with the common clinical sign of severe cerebellar ataxia in six outbreaks occurring during this period. The ataxia was characterized by a broad-based stance, truncal incoordination, hypermetric gait, and intention tremors of the head. The histopathologic findings in 10 cormorants euthanized in 1997 were mild and nonspecific. An immunohistochemical staining technique for the detection of brevetoxin in cormorants documented the uptake of brevetoxin in tissues from four cormorants admitted during an outbreak in 1997, but a modified technique used on samples from 11 cormorants admitted during a K. brevis bloom in 2000 produced indeterminate results. Admittance of cormorants with outbreak-specific clinical signs was positively correlated (P < 0.05) with concurrent concentrations of K. brevis in local water. The cross-correlation coefficient was also significant when increased K. brevis levels preceded cormorant admittances by 2, 4, 6, and 8 wk. This delay in time between K. brevis blooms and cormorant admittance and our clinical finding of neurologic abnormalities in cormorants without overt histopathologic features suggest an association between K. brevis blooms and local cormorant morbidity.