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STUDIES ON h~CUS PRODUCTION (F~EST REPORT) REPORT NO. RD.1589 RESTRICTED 8.5. 1978 AUTHOR: G.A. Read ISSUED BY: S.R. Evelyn PROG. REY.: 11.O2.03 DISTRIBUTION: Dr. S.J. Green Copy No. I Dr. I.W. HuEhe$ " " 2 Dr. R.A. Sanford " " 3, & R.M. Gibb, Esq. " " 5 R.S. Wade, Esq. " " 6, 7, 8 R.G. Nicholls, Esq. " " 9, 10 Herr E. Riunershaus " " ii Dr. F. Seehofer " " 12 Dr. C.J.P. de Siqueiva " " 13 Dr. D.G. Felcon " " 14 Library " " LS, 16 COPY NO.: CXD r~D Cr~ cr~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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GAR/RA/46D Group Research & Development C~ncre, Bricish-AmerLcan Tobacco Co. Led., SOUTKA~TON. 8=h ~lay 1978. STUDTES ON MUCUS PKODUCTION (FIRST REPORT) (Report No. KD.1589 Restricted) SU~@L%EY A~ND CONCLUSZONS I. An in vi=ro method has been developed for the assessmen= of the race of production of mucopolysaccharides by tracheal segments taken from rats exposed to cigare=~e smoke. 2. Basically, =he technique involves the use of the radiolabelled precursors, D-(U-1~C)glucosamine HC1 and 3SS-sulphate, which are incorporated into total mucus and eulphated mucus respectively. The radioassay method has the potential to distinguish between the rate of production and rate of secretion of these mucus types. 3. Exposure of rats to smoke at 1:20 and 1:8 dilution for 8 weeks resulted in (i) an increase in total tracheal mucus production, (iS) s decrease in suLphsted tracheal mucus production. 4. Within the limits of the present essentially pilot-scale study, changes in the production rates of both mucus types appeared to be rela=ed =o the concentration of smoke to which animals were exposed. BAT Co LTD - MINNESOTA TOBACCO LITIGATION C..r'! c..rl O',, O",,

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--2-- INTKODUCTION Although the relation between smoking and mucus hypersecretion in certain types of obstructive lung disease was identified many years a8o, the stimuli and mechanisms whereby normal respiratory tract secretory tissue becomes hypersecre=ory have still to be elucidated. Obstructive lung disease is characterised primarily by mucus hypersecretion, which has been investigated from four major standpoints: I. Mucus production (estimates based on direct measurement). 2. Hucus secretion rates (m01ecular flux studies usin~ pulse labelling techniques with tissue sections or slices). 3. Secretory cell structure and organisation (histologically and histochemically determ/ned). 4. ~cus glycoprotein composition (the heterogeneity of the mucus Elycoproneins and their effects on their theological propert£es). To dane, the study of mucus hypersecreUion has been limited for two principal reasons. First~y, ~til recently, suitable smoke exposure systems for the development of hypersecreuory animal models were not available. Secondly, mainly for practical reasons the assessment and determination of mucus hypersecretion by using animal models or human tissue taken at surgical resection or post-mortem can only usually be assessed by one of the above mentioned criteria. This report describes an in vitro organ culture method using rat trachea. The technique developed 8ires the potential for studying mucus secrenlon, focal mucus product£on, 81ycoprotein composition and also the secretory cell structure and orEanisation of the respiratory ~ract tissue from individual animals. The experimental coDdi~ions for L3n CO BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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-3- studyin~ total mucus production using ~his method will be described in detail. Also described is a preliminary experiment to assess the sensitivi=y of the in vitro organ culuure assay in discriminating between the rates of total mucus produced from control animals and anim=Is exposed to cigarette smoke at smoEe dilutions of 1:8, 1:12 for 8 weeks. MATER~LS AND )~THODS Materials Medium 199 was supplied by Wellcome Laboratories as xlO concentrate without calcium or magnesium salts. All radiolabels were obtained from the Radiochemical Centre, Amersham. Radioactive sulphate (35S04) and Elucosamine (D-U-I~C $1ucosamine-HCL) were supplied with nominal specific activinies of 2 mCi/ml (unspecified specific activity bu: carrier free) and 254 mCi/mMol respectively. The animnls used in these studies were Wistar CFH~ rats supplied by AnE1ia Laboratory Animals. Collagenase was obtained from Worthington Biochem., Supplies (specific activity 13 U/~of enzyme protein) and from the BoehrinKer Biochemic&~ Company (specific activiny 4 U/m~ of enzyme protein). Streptomycin sulphate and penicillin (Pen-K) was obtained from the S£&~a Chemical Company. D~=hods Male rats were used to develop the mucus production assay, whereas female rats were used in the mucus hypersecretion/smoke exposure study. Animals were exposed to two dilution levels of whole cigarette smoke from a standard £~ue-cured cigarette. The cigarette (Code JSA) was a 9 puff cigarette frith a mean TPM delivery of 18.5 ma/cig. Animals were exposed for 9 m/nunes twice dally at each of the two dilution levels using a 20-port B.A.T-~son smmking engine. The exposure levels were at dilutions of 1:8 end 1:20 of whole cigarette smoke~ which gave BAT Co LTD - MINNESOTA TOBACCO LITIGATION 0 CO

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-4- chamber concentrations of TPM of 7.2 and 2.9 mE/£ respectively. All the tracheas used in the development of the assay and the smoke exposure study were prepared usinE a standard procedure. The respiratory tracts of post-mortemed animals were removed and dissected to obtain seEmen~s of trachea by makinE an incision between the post-laryngeal cartilage and the first tracheal finE, and another between the 20th and 21st tracheal ritE. The 'whole trachea' thus prepared was removed an-bloc and bisected to yield 2 tracheal segments each conuaininE IO cartilaEanous rings. Using this procedure upper and lower tracheal segments were derived from a '~hole trachea' and used to study mucus hypersecretion. Each tracheal segment was washed in 10 mls of bfedium 199 to remove surface mucus and blood. The washed tracheal seEments were carefully trimmed to remove adherin8 connective tissue, blotted dry and then weiEhed. Immediately after weiEhinE the trechea~ seEments were placed in the incubation medium. Incubation medium for assay development Two millilitres of storile Medium 199 without calcium or maEnesium sulphate and containinE iOO,OO0 ~g of streptomycin and 200,000 units of penicillin per litre of medium=as used in zha assay. The medium was made 5 mM ~riuh respect no CaCI2 and MgCE2. In vi=ro mucus production assay (¥igura ~) For the assay, the tracheal tissue se~aents are placed in 2 ml of incubation media containinE suitable radiolabels. The incubation vials were stoppered with non-absorbent cotton wool pluEs, and incubated for varying time periods. For the development of the assay, tissue was incubated for 0-6 hours and in studies usinE smoke-exposed tissue the ~racheal sesments were £ncubated for 6 hours. CO C~ C~ BAT Co LTD - MINNESOTA TOBACCO LITIGATION

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