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Ii KO1 ©36:5g7 Page l. PROGRESS REPORT CTR Grant No. 1143-R1 Progress Report No. 4 '(June 4, 1979 - Dec. 3, 1Q79) Aaron Janoff, Ph.D. S.U.N.Y. at Stony Brook , Dept. of Pathology Health Sciences Center S. U.N. Y. Storq.Brook, B.Y. 11794 Title: "Suppression of Protease Inhibition,by Cigarette Smoke"

H ~O'1 0'6 S9$ Page 2. Contents: (i) Preliminary assays of rat BMPi. (5) (2) Effects of cigarette smoke on human Bldi'i in vitro. (3) Effects of cigarette smoke on hu®an BHPi in vivo;, (4) Time course of lung«1Pi inactivation in rats after achte exposure to inhaled cigarette smoke. Effects of snti-oxidant pretreatment on lung.clPi inactivation in rats after acute exposure to inhel.led cigarette smoke. (1) Preliminary assays of rat BAPi: In preparation for projected studies an the effects of inhaled cigarette smoke on activity of rat lung BMPL (acid-stable, iotr MH, bronchial mucous inhibitor), we analyzed rat lung secretions obtained by bronehopulmonary lavage for the presence and quantity of acid- stable, low MW trypsin-inhibitor. Eareriment 1: Lung fluids were collected by saline lavage through an eudotracheal cannula in anesthetized rats, after saline perfusion of the lung circulation to remove serum inhib- itors. The ldvage fluids were freed of cells by centrifugation and concentrated 50-fold by ultrafiltration `s an Amicon UM-2 membrane (85x retention above 1400 MFI). A portion of the reten- tate was clarified by high-speed centrifugation and was then treated by the method of Schiessler (Methods in Enzymology, 7Q+Y: 847-859, 1976) to isolate the cationic-protein,trypsin- inhibitory fraction. Acid-stable, low MW trypsin inhibitors (BNPi) are contained in this fraction whereas «e iPi is not (acidic glycoprotein). Briefly, this involved extraction of

110;0~~6399 Page 3. cationic proteins by SP - Sephadex at pH 5.4 (0.05M ammonium acetate buffer) folloved by stepvise elution of these proteins using a salt gradient between 0.05M and 1.OM NaCL. All fractions eluted from the gel containing UP - absorbing (A280) material were then tested for trypsin-inhibiting aetSvity using benzoyl- arginine - p - nitroanilide as substrate. So eluted fraction was found to possess trypsin - inhibitory activity. Experiment 2: A second portion of the concentrated rat lung lav- age fluid (vida su~ra) was analyzed for lov W (acid-stable) trypsin-inhibitory activity, after treatment of samples with varyin,g concentrations of perchloric acid ot trichloroacetic acid to eliminate inhibitory activity of ae 1Pi. Rat serum vas treated in parallel for comparison. As expected, treatment of rat serum with 5% perchloric or trichloroacetic acid eliminated all tryp- sin inhibitory capacity (TIC), since only high NW proteinase inhibitors are present in serum (Bld'i is not). The same result, could be obtained using lotrer concentrations of the acids (as low as 1.5%). When samples of rat lung lavage concentrate were treated vith either perchloric or trichloroacetic acid over concentrationsranging between 1.5% and 5.0% (to eliminate activity of high !6i inhibitors such as rtiPi), the residual TIC varied between 0% and 30% of the initial TIC of the untreated lavage- concentrate. In the case of human bronchial mucous, this value has consistently been found to approximate 80% of starting TIC values (Tegner, Acta OtolaTTngol. 85: 282, 19T8). It thus appeared that either (a) the rat has much lover levels of BNPi

, 0 3 0" 4 0.0 Page 4. in its lung secretions than does man, aWor (b) that rat BNPi is not acid-stable. In either case, the rat becomes an unsuitable animal model for studies of the effects of cigarette smoke,i:ihalation on lung Btd'i activity. Eneritaent 3: Before abandoning the rat model, we treated a group of animals vith two drugs that reportedly (Boldac, Brit. J. Sza. Path. 59: 311, 1978) stimulate bronchial mucous gland byperplasia and mucous secretion, in an effort to increase the content and activity of BMPi in rat lung. Twelve animals'mre given daily subcutaneous injections of isoprenaline (25 mgs.) or of pilocarpine (10 hgs.) for 12 days. Tventy-four hours after the last injection, histologic examination confirmed that considerable bronchial mucous gland hyperplasia had taken place, but biochemical analysis failed to show a significant increase in acid-stable TIC of bronchopulmonary washings. (2) Effects of ciaarette smoke on human BXPi - In vitro study: We next turned to hudoan models for our study of the effects of cig- arette smoke on lung Blei activity.' In the first phase of this work, in vitro experiments were carried out, as follows. Partially purified BM vas prepared from human sputum by (a) treatment with 5.0%perchloric acid (v/v, final concentration) followed by cen- trifugation to remove acid-denaturable, high MiW proteins. (b) The supernate was neutralized, treated vith 1% cetylpyridinium chloride (w/v, final concentration) for 6 hours in the cold and re-centri- fuged to eliminate acid-Mucopolyaaccharides. (c) The final supernate

K ~41 03 6 4 0 1 Page 5. was dialyzed vs 0.2M Tris - KC1 (pH 8.0) containing 0.25bt NaCl and concentrated by ultrafiltkation through an Amicon UM-2 membrane. Fresh;, aqueous solutions of cigarette smoke were pre- pared by bubbling smoke from 3 cigarettes through 3.0 ml of Tris- HC1 at 0°C (final pH = 8.0). Smoke solutions were then mixed v.its partly purified BMPi (0.5 Vg protein/ul) for 30 min. at 3T°C. The protehse inhibitory activity of treated and control BMPi fractions tnas then assayed using human leukocyte elastase as enzyme and particulate elastin as substrate. It was found that the leukocyte elastase inhibitory capacity (EIC) of smoke-treated BHPi iasolated from human sputum was decreased 50% compared to untreated controls. Control and smoke- treated BMPi: elastase mixtures were also analyzed by thin-layer gel filtration followed by immuno diffusion vs antisera to human leukocyte elastase and to human BMPi (anti-HUSI-l, kindly provided by Dr. H. Frits, University of t4unich). Smoke-treated samples showed reduced elastaee: BMPi complexes together with free elas- tase, whereas controls showed only elastase: BMPi complexes. Introduction of anti-oxidants (2.6nA-4M thymol or hydroquinone) into smoke solutions 15 min. prior to addition of BMPi prevented both the suppression of EIC and the appearance of free elastase following iasnuno-gel-filtration analysis. Moreover, the model oxidant 1P-chlorosuccinimide, at 10'3M concentration, also suppressed EIC of BMPi as did an oxidizing system composed of

HKO'10'<402 Page 6. canine myeloperoxidase (20mU/ml), H20Z (10uM) and 0.15M Cl- (at pH 7.k) These results strongly suggest that, as vithwlPi, the inactivation of BMPi by cigarette smoke is due to oxidation of the inhibitor. Identical experiments were also carried out with purified HUSI-1 (human seminal plasma inhibitor) - a low W, rscid-stable protegnase inhibitor present in human seminal plasma, vhich is antigenically, physically and chemically indistinguishable from human BMPi. B,USI-1 (Schiessler, et al.; Methods in Enzymoloesr, igls 847-859, 1976) vas kindly provided by Dr. H. Fritz, University of Munich. Similar results vere obtained after inoubating HU5I-1 with smoke solution as are described above for BMPi isolated by us from human sputum. (3) Effects of cigarette smoke on hv®an B2+Pi - In vivo study: Theforegoing results suggest that oxidative inactivation of BldPi by cigarette smoke (and leukocarte-derived oxidants such as Byelo- perozidase + 8202 + Cl-) may play a role in development of chronic bronchitis in cigarette smokers. To explore this possibility further, ve are currently undertaking measurements of BIdQi activ- ity in tracheal aspirates obtained from human smokers and non- smokers. The aspirates, along with medical and smoking~~resprovned to us by Dr. Jack Chalon of the Dept. of Anesthesiology at fl.Y.U. Medical Center. Our analysis will consist of the folloving procedures: , (a) B?Pi will be isolated as described in section (2) above for sputum. (b) The isolated inhibitor vill be quantitated immunochem- ically by radial immunodiffusion vs antiserum to HUBI-1. (c) The isolated inhibitor vill also be analyzed biochemically for protein- ase-inhibitory activity using human leukocyte elastase with partic••

H KP% i';':i5/'. 0 3 Page 7. ulate elastin as substrate, trypsin with benzoyl-arginine-p- aitroanilide as substrate, and ch;rmotrypsin with benzoyl-tyrosine- eth„}*1 ester as substrate. The data will be espressed as mg enzyme inhibited/mg antigenic BMPi. Comparisons will include heavy smokers ( 20 cig/day), moderate smokers (10-19 cig/day) and non+smokers. To date, 20 carefully screened specimens have been obtained (no specimens are included from individuals with acute bronchitic infection, pneumonia, hepatitis, tuberculosis or sarcoid), and the analysis of BMPi activity 1s to'begin shortly. (4) Time course of lung•rlPi inactivation in rats acutely exposed to inhaled cigarette smoke: The inactivation of alpha 1-proteinase inhibitor (K1Pi) in the lung by inhaled cigarette smoke was the subject of a previous report from this laboratory (Progress Report #3 and Science in press). These studies showed that lung <1Pi activity was decreased folloving acute inhalation of as little as 3 to 6 puffs of cigarette smoke in non-adapted rats, and suggesxed that such a mechanism might help to account for development of pulmonary empbysema in cigarette smokers. Oxidative mechanisms were implicated as being responsible for loss of.elPi activity after smoke inhala- tion. In these original experiments, the analysis of lung se1Pi activity was restricted to an interval between 1 and 2 hr after smoke exposure. We are currently expanding the analysis to include two other time periods; namely, 8 min, and 8 hr. after smoking. Lavage fluids are obtained at the shorter and longer time periods and are concentrated and analyzed as before (see Progress Report

0 .i6 ~ Page 8. # 3). These experiments are current]ly in progress. (5) Effects of pretreatment vith anti-oxidants on the inactibation of lung -t1Pi in rats acutely exposed to inhaled cigarette smoke: Rats,veighing 190-210g, are injected intiraperitonea2ly with 30mg (30 I. U. ) of vitamin E miaed with 36mg of vitamin C dissolved in 0.15M HaCl containing l% Trreen -85 (polyoxyet2ylene sorbitan tri- oleate). Doses of vitamins are given at -48 hr,~-2k hr, -16 hr, and -3 hr. Controls are injected with equal volumes of solvent alone (saline + Tween) at the foregoing times. At zero time, animals from both the treated and control groups are d.tvided into two sub- groups and exposed to sham-restraint or to cigarette smoke inhala.- tion in the Walton horizontal smoking machine. Analysis of lung .e ipi inactivation is carried out at 1-2 hr after smoke exposure, as described before (Progress Report # 3). These' experiments are currently in progress.