| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Guest Access | Sign In via User Name/Password
|
|
|
|||||||
Guest Access | Sign In via User Name/Password |
|||||||||
* From the Mount Sinai School of Medicine (Drs. Schachter, Rienzi, and Goswami), New York, NY; the Andrija Stampar School of Public Health (Dr. Zuskin) Zagreb, Croatia; and the National Institute for Occupational Safety and Health (Drs. Castranova, Whitmer, and Siegel), Morgantown, WV.
Correspondence to: E. Neil Schachter, MD, FCCP, The Mount Sinai School of Medicine, One Gustave L. Levy Pl, New York, NY 10029-6574;
 |
Abstract |
|---|
 TOP Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Key Words: airway smooth muscle • brewery dust extract • occupational asthma
|
Introduction |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
A number of reports suggest that dusts found in the brewery industry may be associated with occupational lung disease. Cernelc2 described allergic manifestations (asthma and allergic rhinitis) in school children who were helping in the cultivation of hops. Zuskin et al3 reported frequent respiratory symptoms as well as positive skin prick reactions to yeast dust in 22.7% of the workers employed in an animal food-processing plant. Other investigators4 have also documented the adverse airway effects of grain dust, including hops and barley. A case of occupational asthma due to barley grain dust was described by Yap et al.5 Exposure to grain dust is a common cause of respiratory symptoms, but the pathogenesis of these symptoms remains obscure. Respiratory complaints and changes in FEV1 are more frequent and more often are attributed to work by grain handlers than to that performed by nonexposed subjects.6 A single-inhalation challenge7 with corn dust extract resulted in airflow obstruction and lower respiratory tract inflammation in human subjects and in mice.
Borm et al8 have suggested that inflammatory events can be used to monitor the adverse respiratory effects of grain dust exposure. The results of a study by Schwartz et al9 suggested that the concentration of endotoxin in grain dust bioaerosols may be particularly important in the development of grain dust-induced airway disease. This inflammatory reaction appears to be immediate and does not require sensitization. Blaski et al10 suggested that atopy plays a minor role in the development of grain dust-induced airway disease. Reviews by Chan-Yeung and colleagues11 12 demonstrate acute and chronic respiratory effects of grain dust exposure in grain elevator workers documenting adverse respiratory effects. Grain processing also may be associated with respiratory impairment that continues into the retirement years and has been linked with chronic respiratory disability.13 It is, therefore, plausible that brewery workers exposed to hops, barley, and yeast also may develop acute and possibly chronic respiratory problems.
Both airway inflammation and constriction of the airway smooth muscle are known to be part of the pathophysiologic events that accompany occupational airway disease. In vitro studies using tracheal smooth muscle offer a convenient model for studying the airway effects of occupational irritants. We have previously investigated14 15 16 17 18 19 the effects of different organic dusts obtained from different industries in an in vitro model using guinea pig tracheal rings. These studies have demonstrated the potential for airway constriction by a number of organic dusts that are used in these industries. The current pharmacologic study was undertaken to further elucidate the mechanisms by which organic dusts, particularly those found in the brewery industry, may cause airway disease.
|
Materials and Methods |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Dust extracts were prepared from individually collected samples obtained in production areas. Separate sacks of hops and barley grain are shipped to the brewery from agricultural sources. The grains have already been ground before shipping, so that these two cereals arrive as a powder. Brewery yeast also is delivered separately as a powder to the brewery plant. An initial step in the manufacturing process consists of mixing barley and hops. This is performed in a vat in which water is added to the mixture and is allowed to settle over several days. The brewery yeast powder is then added and allowed to react with the hops/barley/water mixture for an additional several days. The contents are subsequently circulated through filters to remove particles and organisms. The filtrate is the final product, beer.
We collected the hops, barley, and brewery yeast powder directly from the shipped sacks. For the purpose of preparing extracts, the powders were filtered through fine sieves to remove large particulate matter that would be difficult to extract. These extracts represent dusts that workers would be likely to breathe during the initial part of the brewing process. The rationales for using water-soluble extracts from these dusts are multiple. Water-soluble extracts are the standard form of test antigens. Previous work by our laboratory has shown that many organic dusts, including textile dusts,18 20 food dusts,3 15 17 and animal confinement area dusts,16 19 contain active, water-soluble ingredients that cause airway constriction both in vitro and in vivo. Finally, the processing in the brewery plant makes it likely that workers inhale droplets containing water-soluble compounds from the original dusts.
The method of Sheldon et al21 was used to prepare sterile water-soluble extracts. All materials were processed at the Institute for Immunology in Zagreb.
Endotoxin and n-Formyl-Methionyl-Leucyl-Phenylalanine Assay
The amount of endotoxin in hops, barley, and brewery yeast was
determined by using the limulus amebocyte lysate assay.22
In addition, the concentration of a bacterial chemotactic peptide
(n-formyl-methionyl-leucyl-phenylalanine [FMLP]) was determined by a
modification of a high-performance liquid chromatography protocol
described by Fedan and colleagues.23
24
For the FMLP
assays, extracts were processed in methanol (50 mg/0.5 mL) for 4 h
at 20°C. The methanol extracts were passed through a 0.2-µm filter.
This material then was placed on a 10-µm, 3.9 mm x 15 cm uBondapak
column (Waters Corporation; Milford, MA). FMLP and oxidized FMLP were
eluted from the column with 25:75 (vol/vol) acetonitrile/0.1 M
phosphoric acid (pH, 3) at 1 mL/min. Absorbance was read at 190 nm.
Guinea Pig Trachea Preparation
We used the tracheas of young albino Hartley male guinea pigs
(weight, 300 to 390 g; Charles River Laboratories; Wilmington,
MA). The animals were killed by CO2 asphyxiation
for 3 min, and the tracheas were removed within 3 min of death. The
animal tissues were manually trimmed to remove connective and other
tissues. Four segments (rings, each 4 to 6 mm wide) were cut
from a single trachea, and each was suspended between two L-shaped
stainless steel hooks mounted in a 20-mL organ chamber containing
Krebs-Hanseliet buffer of the following composition in distilled water:
NaCl, 110.0 mmol/L; KCl, 4.80 mmol/L;
CaCl2, 2.35 mmol/L; MgSO4,
10.20 mmol/L; KHPO4, 10.20 mmol/L;
NaHCO3, 25.0 mmol/L; dextrose, 110.0 mmol/L; and
Na2 ethylenediamine tetra-acetic acid, 0.03
mmol/L. Organ chambers were maintained at a mean (±SD) temperature of
36.5 ± 0.5°C and were continuously aerated with 95%
O2 and 5% CO2 to maintain
a mean pH of 7.5 ± 0.l. The tissue segments were initially set to
2 g of tension and were allowed to stabilize for
approximately 1.5 h before the experiment began. During that
period, the tissue was washed at 15-min intervals. After the relaxation
period, the tension in each tissue segment was adjusted to 2 g for
all subsequent assays. Isometric contractions were recorded using a
force displacement transducer (model FTO3C; Grass; Quincy, MA)
attached to a polygraph recorder. Before the contraction-response assay
with hops, barley, and yeast dust extracts was undertaken, a challenge
with carbachol (10-4 mol/L) was performed to
evaluate maximal carbachol constriction. A dose-response curve with
dust extracts was obtained by adding increasing volumes of extract or
Krebs solution (used as a control) into the tissue bath in
progressive aliquots of 10, 30, 100, 300, and 1,000 µL. The potency
of the dust extract was determined by comparing the biological activity
of the extract with the maximal contraction induced by carbachol
(10-4 mol/L) on the same tissue. The data were
expressed as a percentage of the initial maximal carbachol contraction.
Steady-State Characterization of the Brewery Dust Extract
Dose-Response Curves
After equilibration, each tissue segment was maximally
contracted with carbachol (10-4 mol/L). This
response was measured in grams of tension and was designated as the
maximal carbachol response for that tissue (100%). All subsequent
contractions of the segment were normalized to this maximal carbachol
response and were expressed as a percentage of the initial maximal
carbachol-induced contraction. Isometric contractions in response to
hops, barley, and brewery yeast dust extracts were measured as a
function of the sequential cumulative dose increments administered in
1/2 log-unit dose steps, as outlined above. Concentration response
curves were plotted using computer software (Kaleidagraph, version 3.0;
Synergy Software; Reading, PA) on a desktop computer (Power Macintosh
8200; Apple Computer; Cupertino, CA). Data points were fit by iteration
of the following logistic function:
 |
Drug Treatment
As in a typical drug experiment, the tissue was washed and the
baseline was reestablished after an initial contraction with carbachol
(10-4 mol/L). A specific blocking agent or a
control solution (Krebs buffer) was added to the organ bath and was
incubated with the tissue for 20 min. A dust extract dose-response
challenge then was performed. After the dose response, the tissue was
washed again and carbachol (10-4 mol/L) was
added to verify the viability of the tissue. In these drug experiments,
different drugs, such as atropine (10-6 mol/L,
anticholinergic), pyrilamine (10-6 mol/L,
antihistamine H1 blocking agent), indomethacin
(10-6 mol/L, prostaglandin synthesis inhibitor),
verapamil (10-5 mol/L, calcium channel
blocker), TMB8 (10-5 mol/L, inhibitor of
intracellular calcium mobilization), nordihydroguaiaretic acid (NDGA)
(10-5 mol/L, arachidonic acid pathway
inhibitor), LY17L1883 (10-5 mol/L, leukotriene
synthesis inhibitor), captopril (10-5 mol/L,
angiotensin-converting enzyme [ACE] inhibitor), and thiorphan
(10-5 mol/L, endopeptidase inhibitor) were added
into the organ bath.
Statistical Methods
Mean values were compared between different treatment protocols
using matched tracheal rings (from the same animal) by paired
t test using appropriate software (Statview; Brain Power;
Calabasas, CA).
|
Results |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
|
|
Endotoxin and FMLP Assay
The endotoxin content of the abstracts was 230.55 endotoxin units
(EU)/mg for hops, 16,872.55 EU/mg for barley, and 5,811.15 EU/mg for
brewery yeast. The FMLP content was 36.57 µg/g for hops, 8.27 µg/g
for barley, and 0.95 µg/g for brewery yeast. There was no correlation
between the endotoxin and FMLP contents in these brewery dust extracts.
For reference, average laboratory measurements of cotton dust reveal
values of 5,000 EU/mg for endotoxin and 10.5 µg/g for FMLP
content.23
24
|
Discussion |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
By analyzing the patterns of response to the different drugs used in our experiments with brewery dust extracts, we concluded that the blockade of specific receptors has a modifying effect on the induced constriction of all three brewery dust extracts that were studied. In particular, the muscarinic blocking agent atropine had a striking effect on the maximal (plateau) response to the dust extract and on the dose-response curves for all extracts, while the antihistamine pyrilamine only minimally suppressed the effect of brewery dust extracts. For agents affecting the arachidonic acid pathway (eg, indomethacin and LY171883), only small effects were noted, primarily for brewery yeast and barley.
Modulation of the autonomic nervous system has been shown to effect bronchoconstriction resulting from many organic aerosols. For example, Peters et al25 have shown that FMLP causes bronchoconstriction in humans, while Fedan et al23 reported that FMLP induced the constriction of excised guinea pig tracheal smooth muscle. The bronchoconstrictor response in vivo was, in part, prevented by the anticholinergic agent ipratropium bromide. Similar findings were noted by Fuller et al26 in clinical challenge studies using bradykinin.
Thiorphan, a neutral endopeptidase inhibitor, and captopril, an ACE inhibitor, have been associated with the enhancement of kinin-induced (eg, bradykinin) contraction of airway smooth muscle.27 28 Paradoxically, in our system these enzyme inhibitors reduced the contractile effect of the brewery dust extracts. One possible interpretation of this finding is that the extracts may contain peptides that mediate relaxation and are sensitive to ACE and endopeptidase inhibitors. Alternatively, it may be possible that some normally inactive dust extract ingredient that is cleaved to an active constrictor by these enzymes is not formed in the presence of the inhibitors.
Both verapamil, a calcium channel blocker, and TMB8, an inhibitor of calcium mobilization, considerably suppressed the effects of the brewery dust extract. Both agents limit free intracellular calcium levels. An increase in intracellular Ca++ occurs in many smooth muscle preparations that are induced to constrict by receptor and nonreceptor mechanisms.29 30 Calcium mobilization for the contractile mechanism may originate from intracellular or extracellular stores. Because the elevation of cytosolic calcium is involved in the sequence leading to smooth muscle constriction, the effects of the calcium-modifying agents are not unexpected. The possible role for intracellular and extracellular calcium-blocking agents in the prevention of dust-related airway obstruction remains to be explored.
The effects of LY171833 and indomethacin on the constriction caused by brewery dust were variable. These studies suggest that in addition to cholinergic mechanisms, there are at least some other mediators involved in these events.
Brewery dust extracts contain measurable amounts of endotoxin and FMLP. Fedan et al24 have shown that endotoxin alone does not cause the constriction of guinea pig tracheal smooth muscle in vitro. In addition, although FMLP is a tracheal smooth muscle agonist with a concentration of 91 nm eliciting one half of the maximal response, the delivered doses of FMLP in the current study were too low to contribute substantially to the contractile responses observed. Therefore, the constrictor effects of brewery dusts that were seen in this model are believed to be the result of components other than these bacterially derived contaminants.
Our experimental data from brewery dust extracts suggest that the short-term clinical effects of brewery dust in workers may, in part, be related to nonimmunologic (ie, non-IgE) mechanisms that are similar to those seen with other organic dusts extracts, such as spices,17 swine confinement agents,16 and animal food.3 The contribution of airway inflammation, and possibly of allergic mechanisms, to the development of chronic respiratory disease in workers will require further study using this model. The effect of atropine suggests that cholinergic nerves and or receptors may be involved. The roles of other mediators appear to vary with different extracts. Such findings may have clinical and therapeutic implications for the acute and chronic respiratory symptoms and lung function changes experienced by brewery workers.
|
|
|
Footnotes |
|---|
This research was supported in part by grant No. RO1-OHO-2593–01A1 from the National Institute for Occupational Safety and Health, the Centers for Disease Control and Prevention, Atlanta, GA; and the Gaisman Foundation, Mount Sinai Medical School, New York, NY.
Received for publication July 20, 2000. Accepted for publication November 20, 2000.
|
References |
|---|
|
TOPAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
-adrenoreceptor agonist and KCl induced-contractions in isolated rabbit aorta. Gen Pharmacol 24,921-928[ISI][Medline]
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
