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~ RE, LEV,%".;CE OF A~IIM,%L BIO-ASSAY TESTS TO L~~3.:T E[~EE!E!TCE Charles Ellis It has beenagreed by the T.R.C. that the prima-~j task of the Harrogate laboratories is the.~ setting up of a battery of bi0-ass~-y tests for tobacco products and establishing their relevance for th~ various diseases in humans which are associated with smoking. It is then for the individual companie~ to use these tests in their o~,.m laboratories to ~aide them towards their o',a~ version of a 'safer' cigarette. It will be noticed that the task given to Hsrrogate is twofold. Firstly, they have to establish a number of tests that can be carried out quantitatively, and -,. secondly, they have to try to get informed medical opinion to a .dmit the relevance of such bio-assay results to the human ca~e. This note discusses some aspects of this second task. Relevance could only be establishei on a logical as opposed to a persuasive, basis if the processes occurring both in the human being and in the test ea~-imal were understood at the cellular level and could bc related by acceptable arguments since i~ is only at cellular level that the physiological processes in man and ,~-uimals become comparable. It follows therefore that the overall st~-ategic pl~ for establishing ~ ...... r .... ~31 ~e mush be to endeavour to ~mderst~d the procezzes ooeurx'inG at cellular level in both m-~_n ~m,/ the test animal. BAT Industries document for Province of British Columbia 9 November 2000 BAT INDUSTRIES 00290365

We first review briefly the .~uJ.:::~l bio-assay tests as they are practiced today in order to assess the chances of interpreting them in the above way. Lens-term mouse painting, is now a reasonably quantitative exercise with a technique appreciated in zany laboratories. ~nere sme points of detail that require clarification but t~e total body of knowledse is probably adequate for the moment. The only serious atteupt to relate the results of mouse painti~z to what happens to the cells in the mouse epidermis arises out of the many studies of the immediate effect of a single paiatia.g. " Recent work at Harrogate sugEests the possibility of progress. " Measurements are made of changi~g cell size, changing cell population per unit volume and al~o of the effects on the mitotic cycle, ~nd these observations may be " related to the carciuogenic process siuce i~itiators and pr.omoters appear to Eive different effects. Considerable effort is being expended in several laboratories to force animals to inhale smoke. The resulting irritation and bronchitic effects can be described in terms of cell phenomena "and cam probably be related to the human case because in many animals the structure and behaviour of mucus cells and serous glands parallel the human case. The importance of inhalation e_k[periments is because the whole smoke is used and there is the possibility of distin~uishing between the effects of the vapour phase and the particulate phase. Inhalation exp_eriments, however, seem unlikely PULLINGER, B.D.: The first effects on raouse skin of some polycyclic hydrocarbons. J.Path.Ba~t. 50 ~;65-~-7~ (~9-'!.0) 2. IVE~O~[ O.H. & ~VEESO[[ A. : E:~erinentsl Skin C~:cinodeu~siz . Hice. Acta.nath.zicrob.Scan. ~ S,~or, l,~e:~t,.~,:~ (~6~) ~. Ei~JO : Acta.Path.H~.crobiol. Scm~d., q~65 ~ ~q~. BAT Industries document for Province of British Columbia 9 November 2000 BAT INDUSTRIES 00290366

So cent. ever to produce sufficient lung ~azou_rs in any experimental animals to m~_ke it a viable either in basic ~ork on establizhinG relevance or as a practical test for product development. ~ze cardiovascular effects alleged to be potentiated by the presence of nicotine in the system seem to be describable in terms of the formation of thrombi arisinG from an alteration in th~ behaviour of the platelets and also an alteration in the propc~'ties of the walls of the arteries. Hedical scientists in this case admit the relevance of the results of exp. eriments on large animals such as pigs ~ud, in fact, carry them out themselves. " THUS, in broad outline, we can reasonably expect informed medical opinion to accept the relevance of ~imal experiments in the fields of iryitative effects and cardiovascular effects providcd always we us~ whatever influence we have to get the ani:~al experiments interpreted at the cellular level. It is, however, in the field of lumz cancer that we can foresee difficulties. Even if we knew how carcinoma is caused'in experimental -~uimals by smoke condensate ~e should by that alone be little further fo~-;ard. Such an achievement would be sterile unless at the same time we had some knowledge how smokLug causes a basal cell in the huma~ lung to go carcinogenic. To find this out ought to be the tazk of the medical research scientists but we must face up to the fact that they are most unlikely to attc:~t anythinz serious on their o~.m initiative~ This is because lung c~ucor is a mos~ unpromising ca~e of carcinogenesis to on from the research standpoint, and fu--ther there is no urge to do it to relieve suffering since the medical. profeszion believe the cure is already ]mo~.m, just to stop smoking. BAT Industries document for Province of British Columbia 9 November 2000 BAT INDUSTRIES 00290367

I suggest the conclusion is inescapable, that if we are to make shy progress towards establishLnS relevance of animal experiments in the field of lumz c~ucer then the Industr~j itself must take the initiative iu settinS up models for how smoking mi~h% cause the disease ia human beinss. It is easy to shelter behind the excuse that we are not qualified to do this, but we c~ at least try to set up a mechanism ~nd it will be an important step for~-~ard if we c~_u support it by enough experimental material to m~:e the medical szicntists take us seriously and sit do~n% with us to show how we are wrens. The situation is not hopeless. T.R.C. has already " adopted one provisional workin~ hypothesis and Kotin has at least reco~nized the problem a~nd has indicated his 5eneral views. In the symposium on st~cture, function and measurem=.nt of respiratory cilia held in Febr.,ary ~965 (~ne .~merican Reviews of Respiratory Disea'~es vol.93 No.3 March ~96~) he is reported as saying in the discussion, page "The electron micrograph sho~in~ the macropha~es emphasizes that there are two ways to get at a basal cell - knock off the surface p~otection, or sneak in from the back: and we certainly recognize the possibility of sneaking in from the bsck." I understand Kotin's model to bc that first irritm~t a~ents in the smoke attack the cilial~ ~.nd mucus pro~uc{ng cells lying on top of the basal cells. Although Kotin does not ssy so in terms his h~othesis must be that smoke constituents dissolve in the mu, ouz blanket covering BAT Industries document for Province of British Columbia 9 November 2000 BAT INDUSTRIES 00290368

such desqu.~--mated areas and may penetrate to ~d react with the basal cells. ~-~o lines of invcstisation de sussested by this hypothesis. Firstly, to find out what constituents are taken up by the mucus, information incidentally that is also demanded by .the T.R.C. model that will be referred to later. Secondly, to f~nd out whether in healthy smokers ~;ithou~ luoz cancer there are iu fact areas of the lung denuded of cilia and ~oblet cells. Such en investigation is essentially medical and would involve access to autopsies at hospitals on, say, road casualties. We could, however, stimulate ~d support such an • investigation. The need £or such research is emphasized by Carson et al..(Am.N.Y.Acad.Sc. ~966 ~30 (~) 9~5) where they repo~'t their observations in animals on the~econstruction of the mucociliary apparatus from undifferentiated basal epithelium while under a chronic e~ozure regimen. They believe that their studies suzcested the capacity for complete recovery and repair in the healthy organism even while the insult continued. I have not been able to find any detaile~ account of Koti~'s macrophase hypothesis other than the rem~.vk already quoted, and it may be rcadin~ too u~ch into this to take it seriQusly~ but he seems to envisnge "macrophages engulfinG smoke materials deposited.in the alvcoli and transporting them back into contiguity with the basal cells. A significant £ea~re of. such a hypothesis would be the implication that it is the smokc which is trapped in the alvcoli ~hich is to bl,~ne. BAT Industries document for Province of British Columbia 9 November 2000 BAT INDUSTRIES 00290369

o cont. T.R.C. has also contributed a model which may be compared with Kotin's "Exposed Area" hypothesis. Like Kotin, T.R.C. assumes the mucous bla~l~et of the lung of a smoker contains some smoke coastituents but suggests a different mechanism by which these might penetrate into the basal cells. They propose that the-cilia, continuously moving in the m.ueus, act through their tips as the point of ent~j constituents into the body of the ciliated cell. By normal diffusion proccsse~ they ~.~ill zpreod throughout the cytoplasm and therefore will be conti~.~ous to, ~nd separated by Just t~.:o touzhing membranes, from the target, the interior of the basal cell. It has bec~ suggested that the irritative properties of smoke may play a role also in this model in so far as these may damage the tips of the cilia and provide a~ easier mode of entry for smoke constituents. One immediate result of considering models such as these ~s that they suggest ek~eriments which must in any case deepen our ]~nowledge of ~.~hat is happeninz in the human lung. Both Kotin's hypothesis and that of T.R.C~ focus attention on the interaction of smoke with human mucus. The need for more knowledge in this field has been accepted by T.R.C. and the chemistry department at H~rrosate is starting up a ma~or pro~ect. Initially, they will work with sheep mucus ~ich has been exposed to smoke by tracheotomy. T.R.C. have also under considecation a proposal by Huntingdon Reseawch Ccutre to attempt to t~ace the movement o£ labelled compounds into the ciliated ~ells and even into the basal cells. So f~ T.R.C. h~.s made no progress in stimulating medical scientists to find out to ~hat extent the lungs of hum~_u smokers have vulnerable exposed area~. BAT Industries document for Province of British Columbia 9 November 2000 BAT INDUSTRIES 00290370

Q It ca_u be seen that there are two main lines of research that must be ofollo:-:ed if we are to be successful in establishJ.u~ relevance. Firstly, active steps must be taken to Luterpret our animal bio-assay tests in terms of whzt is happeninz to the sm.imal cell. Secondly, it consziously to assume the responsibility of trying to set up a model how inhaled smoke misht cause lung cancer in a human smoker. Even if our progress in this second research is .halting and slow the mere o fact that we are trying to do something and are thiuking about the problem is bottud to have its effect on medical scientists and ~tcrease the credibility of our views. CD~/?z BAT Industries document for Province of British Columbia 9 November 2000 BAT INDUSTRIES 00290371