* Institute of Pharmacology and Toxicology, Tzu Chi University No 701, Section 3, Hualien 970, Taiwan; Institute of Medical Sciences, Tzu Chi University No 701, Section 3, Hualien 970, Taiwan; and
Department of Pharmacology, Tzu Chi University No 701, Section 3, Hualien 970, Taiwan
1 To whom correspondence should be addressed at Department of Pharmacology, Tzu Chi University, No 701, Section 3, Chung Yang Road, Hualien 970, Taiwan. Fax: 886-3-856-1465. E-mail: ming{at}mail.tcu.edu.tw.
Received May 20, 2004; accepted September 6, 2004
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ABSTRACT |
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Key Words: tricholoroethylene; perchloroethylene; trachea; swine; prostaglandin; acetylcholinesterase.
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INTRODUCTION |
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Since inhalation is the most important route of TCE and PERC exposure, concerns over potential adverse effects on human health, particularly the respiratory tract, have been heightened (Burg and Gist, 1999; Langworth et al., 2001
). TCE affects cilia activity (Tomenius et al., 1979
) and also produces a thinner tracheal epithelial layer along with intraluminal hemorrhage as well as inflammatory cell infiltration into the underlying connective tissue (Koptagel and Bulut, 1998
). PERC can cause acute symptoms of cough and dyspnea in normal subject and a severe bronchospasm in asthmatic subject following an intense short-term exposure, indicating the potential to cause or aggravate asthma (Boulet, 1988
). With a mild hyperresponsiveness to methacholine, the syndrome related to PERC fits the diagnostic criteria of reactive airways dysfunction syndrome and irritant-induced asthma. These two pulmonary disorders are clinically very similar; both are non-immunogenic forms of airway injury that may be associated with industrial inhalation exposure. In this situation, the direct toxic effect on the airways causes persistent airway inflammation and bronchial hyperreactivity.
The regulation of muscle tone is related to the induction of airway obstruction or hyper-responsiveness. In addition, the impaired epithelial production and/or release of inflammatory factors may contribute to airway inflammation and hyperresponsiveness (Goldie et al., 1988). The proinflammatory mediators prostaglandins have been implicated in the inflammatory cascade that occurs in asthmatic airways (Wenzel, 1997
). Epithelium-derived AChE also plays an important role in cholinergic regulation in airways (Koga et al., 1992). Thus, we studied the effects of TCE and PERC on the basal and stimulant-induced contractile responses and epithelial prostaglandin release as well as acetylcholinesterase (AChE) activity in swine trachea in vitro. These endpoints may provide more inclusive toxicological profiles of these two chlorinated organic solvents on trachea to predict their potential to produce hyper-responsiveness and reactive airways dysfunction syndrome.
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MATERIALS AND METHODS |
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Measurement of isometric tension. Muscle tension was measured using an isometric force transducer interfaced to the computer via A/D converter. The smooth muscle strips were mounted vertically in a 10-ml water-jacketed organ bath filled with Krebs-bicarbonate buffer and gassed with a mixture of 95% O2 and 5% CO2 at 37°C. The bottom end of the strip was fixed to an L-shaped hook, and the top end was tied to a stainless-steel wire attached to a force displacement transducer (Grass FT.03) for monitoring changes in isometric force. Each strip was subjected to a load of 3 g and was allowed to equilibrate for at least 4060 min before the drugs were applied to the organ bath. This was determined to be optimal for force generation. Muscle contraction is expressed as percentage of the contractile response initially contracted by KCl (40 mM) or acetylcholine (ACh, 1 µM), which is taken as 100%.
Enzyme immunoassays (EIA) of prostaglandins. After treatment of tracheal epithelial segments with TCE (100 ppm), PERC (100 ppm) or other agents for 2 h, HEPES supernatants from the epithelial segments incubated at 37°C were collected and initially stored at 20°C until assayed for prostaglandins (PGs) and acetylcholinesterase (AChE) activity. For PGD2 and PGE2 measurements, an enzyme immunoassay (EIA) technique was used. In briefly, samples from previous supernatants were thawed slowly at 4°C and then centrifuged at 1200 g for 10 min. After centrifugation, 50 µl supernatant and 50 µl prostaglandin tracer were added to each well of a 96-well plate coated with anti-mouse IgG antibody, followed by prostaglandin monoclonal antibody (50 µl), and then the plate was incubated at 4°C for 18 hr. Next, each well of the plate was washed with the wash buffer 5 times and added with 200 µl Ellman's reagent. Then the plate was shaken in the dark for 60 min and absorbance at 405 nm was determined using a microplate reader (Molecular Devices Corp., Sunnyvale, CA). The amount of PG was calculated from a standard curve of PGs.
Measurement of acetylcholinesterase activity. Acetylcholinesterase activity was measured by a modified method of Ellman and coworkers (Ellman and Callaway, 1961). Before assay, samples were thawed slowly at 4°C and then centrifuged at 1200 g for 15 min. The supernatant (200 µl) was then diluted with 600 µl phosphate buffer (pH 8), added with 100 µl dithiobisnitrobenzoic acid (DTNB, 0.01 M) and 100 µl acetylthiocholine (0.075 M) for assaying AChE activity, which was read at the wavelength of 412 nm using a spectrophotometer (Beckman Instruments, Irvine, CA). One unit (U) of enzyme activity is defined as 1 micromoles of acetylthiocholine hydrolyzed per minute at pH 8 and 25°C.
Materials. All dilutions of drugs were prepared on the day of the experiments. Stock solutions were made by dissolving TCE and PERC (Ridel-de Haen) in dimethylsulfoxide (DMSO, J. T. Baker) at 1% (v/v) or 10% (v/v). Acetylcholine (Sigma) was dissolved in distilled water. The PGD2 and PGE2 EIA kit was purchased from Cayman Chemical (Ann Arbor, MI). All other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).
The HEPES buffer consisted of the following (mM): NaCl 140, KCl 5, CaCl2 2, glucose 5.5, HEPES 10, and pH was adjusted to 7.4. The Krebs-bicarbonate buffer was composed of (mM): NaCl 113, KCl 4.8, CaCl2 2.5, NaHCO3 18, KH2PO4 1.2, MgSO4 1.2, glucose 5.5, mannitol 30, and pH was adjusted to 7.4.
Statistical analysis. Results were expressed as means ± S.E.M. Statistical significance of difference between groups was determined by ANOVA followed by Tukey's multiple comparison test. Values of p < 0.05 indicate significant difference.
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RESULTS |
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DISCUSSION |
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As the applied concentrations of TCE and PERC were raised to 1000 ppm, TCE and PERC transiently induced the relaxant response on smooth muscle precontracted by KCl and ACh. The relaxing action of TCE and PERC on muscle tension is consistent to that of other aliphatic chlorinated hydrocarbons in our previous study (Chan et al., 2002). The mechanism of their actions may not involve stimulation of beta-adrenergic receptors, since TCE and other volatile anesthetics were demonstrated to cause a marked positive chronotropic effect, which was unaltered in the presence of beta-adrenergic blocker propranolol in rat atrial preparations (Krisna and Paradise, 1977
). Alternatively, TCE and PERC have been reported to decrease the amplitude of electrically induced intracellular free Ca2+ transients in rat cardiac myocytes (Hoffmann et al., 1994
). The question is raised whether the relaxation response in airways exposed to chlorinated hydrocarbons is due to the decrease in intracellular Ca2+ mobilization. Assessing the degree of Ca2+ mobilization following treatment with TCE and PERC would help to address the question of mechanisms here.
Our results furthermore demonstrate that pretreatment of high concentrations of PERC enhanced ACh- and histamine-evoked muscle contraction and decreased the relaxant effects of beta-adrenergic activation on muscle precontracted by ACh. Since activation of muscarinic M2 receptors has inhibitory effect on beta-adrenoceptor agonists effects in tracheal smooth muscle (Zhang et al., 1996), it is possible that the reduction of the beta-adrenergic effect by PERC may be through, at least in part, activation of muscarinic M2 receptors. Alternatively, it cannot be excluded the possibility that beta-2 receptor activation is less effective under the larger muscle contraction potentiated by PERC. In contrast, TCE pretreatment did not alter the spasmogen-stimulated muscle contraction and beta adrenoceptor activation. It seems that PERC pretreatment is more effective than TCE particularly in smooth muscle contractile responses. Our previous study also demonstrated that the aliphatic chlorinated hydrocarbons such as dichloromethane, dichloroethane, and trichloromethane exerted differential effects on the enhancement of tracheal muscle contractions induced by stimulants (Chan et al., 2002
). Furthermore, these aliphatic chlorinated hydrocarbons directly provoke muscle contractile responses, but TCE and PERC did not. Thus, it is important to note that exposure of distinct chlorinated hydrocarbons to airways may produce differential effects on muscle contractile responses in trachea.
Airway epithelial injury and inflammation are involved in airway diseases such as asthma (Goldie et al., 1988). Prostaglandins, the endogenous proinflammatory mediators, have been implicated in the inflammatory cascade that occurs in airway allergic asthma (Raskovic et al., 1998
; Wenzel, 1997
). Our present results showed that TCE and PERC increased the prostaglandin release from tracheal epithelium, particular PGE2. This augmentation suggested to be mediated by the influence of enzyme cyclooxygenase or prostaglandin biosynthesis, since indomethacin inhibited TCE- and PERC-induced prostaglandin release from epithelium. Besides the increase in epithelial prostaglandin secretion, TCE was reported to induce inflammatory cell infiltration into the underlying connective tissue in rat tracheal mucosa (Koptagel and Bulut, 1998
). Thus TCE and PERC appear to enhance the release of metabolites of arachidonic acid, resulting in promoting inflammation. Further study is required to elucidate whether the prostaglandin production from airway epithelia exposed to TCE and PERC results in airway inflammation.
Chlorinated aliphatic hydrocarbons including TCE and PERC have been shown to diminish human erythrocyte membrane AChE activity (Korpela and Tahti, 1986). TCE and PERC significantly decreased the AChE activity in swine tracheal epithelium. Since neostigmine, an AChE inhibitor, enhanced the muscle contractile response to ACh (1 µM) in the presence of epithelia (preliminary data), it is reasonable to speculate that the reduction in AChE activity from airway epithelium exposed to TCE and PERC may lead to the decrease in hydrolysis of ACh, resulting in augmenting cholinergic stimulation in airways. It is noteworthy that the effect of organic solvents on muscle contractility in the present study is not due to epithelial release of PGs or AChE, since the epithelia were denuded. Thus, the influence of epithelia exposed to chlorinated hydrocarbons on muscle contraction required further investigation.
In conclusion, the current findings demonstrate that exposure of smooth muscle to TCE and PERC enhanced the precontracted muscle tension in swine trachea. Pretreatment with TCE and PERC also potentiated the muscle contractile responses stimulated by spasmogens and suppressed beta-adrenoceptor induced relaxation. Furthermore, TCE and PERC increased PGE2 release from tracheal epithelium but decreased epithelial AChE activity. Thus, inhalation of these two chlorinated hydrocarbons during occupational exposure may induce airway hyper-responsiveness triggered by spasmogens, mediate beta-adreonceptor hypofunction, and lead to airway inflammation. Therefore, high concentrations of exposure to TCE and PCE should be prevented, especially in the subjects exhibiting airway hyperresponsiveness, such as asthmatics.
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ACKNOWLEDGMENTS |
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REFERENCES |
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