Tobacco Documents Online

smoke exposure on rat tracheal Thorax 1981 ;'361 ~6"~2.-624 Effect of tobacco ,submucosN glands: an ulti-astmcturaI study From the D~artment of Pathology, Huntingdon ~eseamh Centre, Huntln.gdon, CarabddgesMre .. ~~-s is .d~es~ffbedf 'W~ff~,J.r~-~tlae m~cous cells ma~ny o~ the ro~agh' er~do- plasmic retieulum cisternae were grossly dilated with an accumulation of amorphous, electron- lucent material. The Golgi zones were prominent, and the secretion granules often contained dense cores, and appeared to have coalesced. Histochemically, increased amounts ofsulphated mucus were present in exposed rats. Serous, eiliated, and myoepithelial cells were unaffected by smoke exposure. Exposure of rats to irritants, by inhalation, produces charlges in the mucus-secreting submucosa] glands of the ai~ys, S~lpkur dioxide .exposure for thr~ w~ks resuIeed in significant increases in the length ~a;nd deptk ~t' ~r:aeh:ea! submu~osal glands) lnet~ease~d ,cell: size,, aej~a:a~ ~ameter, an6 lumen diamet~ of gl-a~-ds were re~bYl:e2, m rats exposed to tob~'cco* smoke for six weeks.~ Exposure of rats to tobacco smoke for 30 days increased the relative proporgon of the tracheal wall occupied by glands, and also increased their mucin content,s These animal studies have been used as models for chronio bronchitis, which is eharacterlsed primarily by hypersecretlon of mucus~ and has been linked with tobacco smoking.~ Ult-rastructural studie~ of tobacco sm0ke-induced mucus hypersecretion-in animal modds have not been traced. The present study therefore investigated the uitrastructural intracellular changes in sub- mucosal #ands from rats exposed to tobacco smoke for up to two years. Methods One hundred and ten male and 110 female CFHB Wistar, specific pathogen-free rats (Anglia Labom-. tory Animals, Huntingdon) were exposed to 6~ v/v fresh tobacco smoke for two 20-minute periods per day, for up to two years. The rats were exposed to smoke from a conventional tobacco cigarette with a 12 mg tar yield, in HP, C rodent smoking machines. An equal number of rats was exposed to fresh air. as Address for reprint requests: Dr DJ Lewis, Department of Pathology, Huntingdon Research Centre, Huntingdon, Cambridgcshk¢ PEI8 6ES. 622 controls. The expcrimeni was performed under strict barrier conditions: .All. the surviving male rats in the tobacco smoke groui~, were killed after 20 months because of a high smo'ke-induced mortality. Samples of" trachea from all r, at~ ~ere fi~ed in 10% neural buffered formalin,. embedded' in paraffin wax, anti stained with hacma- toxylin and e-osin. In addition, sections from five male and five female, rats per group were stained by the high iron diamine technique.~ Ultrastruetural investigations'were limited tO five male and five female rats from both the tobacco smoke exposed and control groups killed after 20 months, and five females from each group after two years' 6xposure~ All rats were killed after overnight ~'¢e0very from the last smok~exposurc~ The rats were killed by an intraperitbneal injection of sodium pentobarbitone. Samples of trachea (l mm rings) were fixed by immersion in 4~o glutaraldehyde. in 0.15 M cacodylale buffer, pH 7.3, at ~-°C, for two hours. After washing in buffer the rings were post- fixed in 1% osmium tetroxide, dehydrated in alcohol, and embedded in cpon. On~ micron survey sections were stained with toluidine blue and silver/" gold ultrathin sections, of areas containing sub- mucosal glands, were stained with uranyl acetate and "le~ad citrate. The sections were examined with a Philips EM300 at 60 kV. Before investigaiion of possible smoke-induced changes, all rats were screened by light microscopy for the I~resence of chronic respiratory disease to ensure their suitability for assessment. R~sults By light microscopy the tracheal submucosp. ! glands t,/OTICE: THIS MATERIAL IM,,~Y BE PROTECTED B.~ COPYRIGHT. LAW (TITLE 17. U. S. CODE,) -1305280061

Effect of tobacco smo'l~e e.~posure on rat tracheal subrnt¢cosal glands: an ultrastructural stu@ 623 smoke~¢xposed~ rats. When compared with submucosaI glands from control animals, the mucous cells from smoke- exposed rats illustrated marked ultrastructura! changes. The most prominent change was the presence of grossly dilated cisternae of the rough endoplasmic reticulum (R.ER), which were filled with an amorphous ele~troo-luccnt mucus-like mate~ia! (fig I)~. These .dilated eister~_ae were usually Fig I M~cous cell.with dilated rough endoplasmic retlculum ( D). Original magnification x 10000. located in the basal region of the cell, but occasionally examples were seen in the supranuclear region, Not all RER cistcrnae were dilated; even in severely affected cells large numbers of parallel cisternae were often present (fig 2). Fig 2 Mucous cell with grossly dilated rottgh endoplasm~c retlculurn ( D) containing amorpho#s mucus-hke material. Note adjacent not~-dilated retieuh#n (arrows). Original mogn~icaHo~ x 11800. secretion granules appeared to have coalesced and contained especially prominent electron-dense cor~s. In some cases when the core was large the granules resembled those found in serous ceils (fig 3). The Golgi complexes of th~ cells appeared enlarged, with numerous vesicles and vacuoles, and occupied a large proportion of the supranuclear cytoplasm (fig 3). All other organelles within th~ mucous cells appeared normal and unaffected by tobacco smoke exposure. , ]Fig 3 Mucous cells with bul~m'~s apical projections (P) filled with secretion granules containing prominent electron-dcose cares. F.xtensive supranuc~ear Go~gi complex (G) with dilaled vesicles. Origino! magn~cation × 10000.. Serous, myoepithelial, and ciliated cells of the acini and ducts appeared unaffected by tobacco smoke exposure and were indistinguishable from those of control rats. Discussion Tobacco sn~oke exposure is known to produce hyper- trophy of, and hypersecretion by, the submucosal glands.~ z Coles found that exposure of rats to tobaccosmoke for six weeks caused an increase, both in slze of glands (hypertrophy of the mucous tubules) and in the secretory rate of mucous c¢llsY He considered it likely that the rate of glycoprotein TI052'80062

synthesis increased to match the higher discharge rate of mucous cells. The results of the pr~ent study indicate that these changes are associated with ultmstructural mani- festations of hyp¢ractivlty within the mucous cells. Using ultrastructural autoradiography Meyrick and l~eid~ investigated the patkv~ay by which mucus is l~vodUced, and ~staMfshgg the ~ole~..of individual was initially |ocalised over the rough eudoplasmic reticulum (RER,) in mucous cells and later migrated t~.e G~o]~ ¢omp,, lex, ~[n compadsoB~ membol~tes of addition to hyperactivity of RER, hypertrophy of the Golgi complex (illustrated by increases in the extent -and in the number of stacks, vacuoles, and vesicles) is usually accompanied by an increased secretory • activity. As the rats were killed after overnight recovery from smoke exposure,, the synthesis and bui, ld,up ,of pol~pcptides within ~thc R~R is probably a ~,esu]:t of diseharg~ of secre~t~o'~ gra'~t~'es d~a~rfng exposure, This stimulation of mucus secrctfon during irritation would bc expected to stimulate rapid mucus synthegis to. ~eata~ the'~eMase~.: gramdes, However, i~t the ~ to .l~¢to~w.~l~¢~fia:tcl~ f~aeea:/~ulate a,~d ~harge their s~retion g~nules, Meyrick and Reid~ concluded secretion granule,v from these findings that th¢ polypeptid¢ core of the ~¢ ultrastructural app~rance of the mucus mucus is synthesised in ~¢ REK and the oligo- ~cretion granul~ was also altered in mucous ~lls sa~harid= of the glycoprotein were incorporated by from tobacco smoke-¢x~osed rats. The el~tron- the Golgi complex, dense cores of the~ granules were ~pecially promi- Several possible sites of action for compounds nent, which possibly correlat~ with the change which alter the rate o~ mucus secretion, its compo- towards increased sulphated mucus. This finding is in sition, or its rate of s~th~is have been sugg~t~.~ agreem,nt with the reported incr~sed ~tlo of Thee sit= include the g~nom~ r=po~ibl¢ for the sulphated to sialic acid mucus in human~ smokers,s information for ~ucus synth~is; the REK in which prima~ polypc~tid's synth~is occurs; the Golgi Referents complex; the s~rc~y a~paratus, which r~gul~tes ~e ~I~ b'f mu~ff~om, ~he eells~'~he lysosom~% ~ ~mb D. Mucus secretion in hypersecretory, stat~. which are able to decade excess mucus; and the Branches 1969;18:453-65. mucus itself, by mucolysis, t Jones R, Boldue P, Reid L. Goblet cell glycoprotein and ~ tracheal gland hypertrophy in rat aimays: the eff~t-of Thus, the ultrastructural changes obse~ed in tobacco smoke with or without th* ant[-inflammato~ mucous cells of the present study occUrr~ in those agent phenylmelhyloxadiazole. Brl~p Pathol 1973;54: organell~ which have been shown to be involved in 229-39. mucus synthesis,~ and have also ~en considered * Hayashi M, S0rnberger GC, Huber GL. Morphomctrlc anal~s¢~ 0f t~cheal gland secretion and hypertrophy possible sit~ a[ which exogcnous-comffounds could - male and female ~ts aft~ experimental exposure to aff~t mucus productionP ~egross dilation of the tobacco smoke. AmRev ResMr D~ 1979;119:67-73. RER s~g~ests ~at the amount of polypcptide core of " i R~id L. Patholo~ of chronic bronchitis, ~ncet 195411 the mucus within the cell was increased considerably, 275.9. ~ Kollerstrom N, Lord PW, Whimster WF. A difference and was stored at the site of synth~is, the composition of bronchial mucus between smokers Dilatation of RER with an unusual amount of and non-smoke~. Thorax 1977;32:1~5-9. polypcptide did not provoke a lysosomal r~ponse ~ Spiccr SS. Diamine methods for differentiating mueo- which would ~ expired to occur to remove ~cess ~ubstanccs h[stochcmically. J Histochem Cytochem 1965; 13:211-34. mucus from the cytoplasm. ~is suggests that the ~ Col~ s. Regulation of the secreto~ cycles of mucous and RER involved is not damaged; if it were, auto- serous cell~inthehumanbronehial gland.ln: Elstein M. phagie dig~tion by lysosom~ would occur. Thus, Parkc DV. Mucus in health and d~sease. New York and the dilatation of the RER is probab~ a physiologi~l • London: Plenum, 1977:!55-68. Meyr[ck B, Reid L. In v~tro incorpomtlon of [*H] thrconinc cellular event repr~enting hyperactivity, ~ther than . and PHi glucose b~ the mucous and serous cells of the a degeneratiw one. Similar'dilatation of the RER a~ " " human bronchial submucosal gland, d Cell Bio11975 an intracellul~ storage site for cell products is known 3~0~. to o~ur in plasma edls and fibroblasts; and tPark*DV. Pharmacology of mucus. BrMedBull1978;M: 89-94. Ghadially sugg~ted that there may be a def~t in the a, Ghadially FN. Ultra*tructural ~athology of the cell. transport of s~rcto~ material in these ~s~.x° In London and Boston: Butterworths, 1975. T1052~