Pharmacologic effects of tobacco dust extract on isolated Guinea pig trachea.

STUDY OBJECTIVE: To determine the effects of tobacco dust extract (TDE) on isolated guinea pig tracheal smooth muscle. DESIGN: A controlled, in vitro smooth-muscle study of the effect of pharmacologic agents on TDE. METHODS: The effect of TDE on isolated guinea pig tracheal smooth muscle was tested using water-soluble extracts of dust obtained from machines in a cigarette manufacturing plant. Dose-related contractions of nonsensitized guinea pig trachea were demonstrated using these extracts. The dust extracts contained significant quantities of bacterial components (eg, endotoxin). Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with drugs known to modulate smooth-muscle contraction: atropine, indomethacin, pyrilamine, nordihydroguaretic acid, acivicin, bromophenacyl bromide, 3,4,5-trimethoxybenzoic acid-8-(diethylamino)octyl ester, captopril, and capsaicin. RESULTS: Atropine strikingly reduced the contractile effects of these extracts. Inhibition of contraction by blocking other mediators was less complete. There was no inhibition of contraction by hexamethonium (10(-4) mol/L, 10(-5) mol/L, 10(-6) mol/L), suggesting that nicotine was not the major contractile mediator of TDE. A separate analysis using different molecular weight fractions of TDE indicated that the constrictor activity appears to be primarily in the fraction with a molecular weight < 10 kd. Additionally, the constrictor effect resided entirely in the nonlipid fraction of the extract. We suggest that TDE causes dose-related airway smooth-muscle constriction by nonimmunologic mechanisms involving a variety of airway mediators and possibly cholinergic receptors. CONCLUSIONS: The bronchoconstrictor activity of TDE resides primarily in its low molecular weight, nonlipid fraction, and hexamethonium studies suggest that this agent is not nicotine.

Pharmacologic effects of grain weevil extract on isolated guinea pig tracheal smooth muscle.

The grain weevil, an insect (pest) that infects grain, is a frequent contaminant of processed wheat, and its presence may contribute to respiratory abnormalities in grain workers. We studied the in vitro effects of an extract of grain weevil (GWE) on airway smooth muscle. Pharmacologic studies included in vitro challenge of guinea pig trachea with GWE, in parallel organ baths, pretreated with mediator-modifying agents or a control solution. Dose-related contractions of nonsensitized guinea pig trachea (GPT) were demonstrated using this extract. Pharmacologic studies were performed by pretreating guinea pig tracheal tissue with drugs known to modulate smooth muscle contraction: atropine, indomethacin, pyrilamine, acivicin, NDGA, BPB, TMB8, captopril, and capsaicin. Atropine, pyrilamine, BPB, and capsaicin significantly reduced the contractile effects of the extract at most of the challenge doses (p < 0.01 or p < 0.05). Inhibition of GWE-induced contraction by blocking of other mediators was less complete. We suggest that GWE causes dose-related airway smooth muscle constriction of the GPT by nonimmunologic mechanisms involving a variety of airway mediators and possibly cholinergic receptors.

Interlaboratory evaluation of endotoxin analyses in agricultural dusts--comparison of LAL assay and mass spectrometry.

Endotoxin exposure is associated with wheeze and asthma morbidity, while early life exposure may reduce risk of allergy and asthma. Unfortunately, it is difficult to compare endotoxin results from different laboratories and environments. We undertook this study to determine if lipopolysaccharide (LPS) extraction efficiency could account for differences among laboratories. We generated and collected aerosols from chicken and swine barns, and corn processing. We randomly allocated side-by-side filter samples to five laboratories for Limulus assay of endotoxin. Lyophilized aliquots of filter extracts were analyzed for 3-hydroxy fatty acids (3-OHFAs) as a marker of LPS using gas chromatography-mass spectrometry. There were significant differences in endotoxin assay and GC-MS (LPS) results between laboratories for all dust types (p < 0.01). Patterns of differences between labs varied by dust type. Relationships between assay and GC/MS results also depended on dust type. The percentages of individual 3-OHFA chain lengths varied across labs (p < 0.0001) suggesting that each lab recovered a different fraction of the LPS available. The presence of large amounts of particle associated LPS and absence of a freezing thawing cycle were associated with lower correlations between LPS and bioactivity, consistent with an absence of Limulus response to cell-bound endotoxin. These data suggest that extraction methods affect endotoxin measurements. The LAL methods may be most suitable when comparing exposures within similar environments; GC-MS offers additional information helpful in optimizing sample treatment and extraction. GC-MS may be of use when comparing across heterogeneous environments and should be considered for inclusion in future studies of human health outcomes.

Comparison of endotoxin assays using agricultural dusts.

Endotoxins from gram-negative bacteria pose a significant respiratory hazard. Establishing dose-response relationships is problematic because there are no standard procedures for sampling and analysis. The goal of this study was to compare endotoxin analyses in six laboratories using Limulus-based assays for analysis of organic dusts from three agricultural environments: chicken barns, swine barns, and corn processing facilities. For each dust generation experiment 14 side-by-side air samples were collected on 37-mm glass fiber filters at flows of 1.8 L/min. Each laboratory was randomly allocated two filters from each of seven experiments per dust type. Three laboratories used the QCL-1000 endpoint assay, and three used the kinetic-QCL method. To eliminate variability among different lots, a single lot of Limulus amebocyte lysate for endpoint assays and one similar lot for kinetic assays was provided. Precision of assays performed within labs was very good, with pooled coefficients of variation for replicate samples ranging from 1 to 11% over all labs and all dust types. There were significant differences between laboratories for all three dust types (p < 0.01). The pattern of differences between labs varied by dust type. For chicken dust, labs using the endpoint method reported higher results than those using kinetic methods. For swine and corn dusts, labs using the kinetic method reported the highest endotoxin values. For chicken dust, results from all labs except A and B were highly correlated (r = 0.86-1.00). For swine dust, only labs B and E, and C and D were correlated. For corn, A, B, and D were significantly correlated with most other labs. In conclusion, statistical differences in performance between laboratories were apparent and may be related to the extraction and analytical methods. The results of this study will be useful for standardization of sampling and analysis of airborne endotoxin in agriculture.