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SCIENTIFIC REPORT THE BIOLOGIC EFFECTS OF TOBACCO SMOKE OM'THE ALVEOLAR,MACROPHAGE HOST DEFENSE SYSTEM 0 0 8 0 ®

4% SCIENTIFIC REPORT THE BIOLOGIC EFFECTS OF TOBACCO SMOKE ON THE ALVEOLAR MACROPHAGE'HOST DEFENSE SYSTEM

REPORT ON' COMPARATIVE EVALUATION OF PRODUCT'MODIFICATION BY EXPERIMENTAL CIGARETTE FILTERS Prepared by: Gary L. Huber, M.D. G. Clinton Sornberger, Ph.D,. Vijay K. Mahajan, M.D. William Hinds, Ph.D. John Shea, M.S. Tobacco and Health Research Program Harvard Medical School Boston, Massachusetts 02215 January 1977

\ TABLE OF CONTENTS Page I. Introductioman&Background 1 A. The Pulmonary Alveolar Macrophage - the Cornerstone of Lung Defense 1 B. The Pulmonary Alveolar Macrophage and Inhaled Bacteria 4 C. Tobacco Smoke and Alveolar Macrophage Defenses 4 II. In Vivo Studies 5 A. Methodology of In Vivo Studies 8 B. Results of In Vivo Studies 10 C. Interpretation of Results of In Vivo Studies 12 III. In Vitro Studies 13 A. Methodology and Results of In Vitro Studies 19 B. Interpretation~of Results of In Vitro Studies 22 IV. Particle Size Analyses 24 A. Methodology of Particle Size Analysis 24 B. Results of Particle Size Analysis 24 C. Interpretation of Particle Size Analysis 26 V. Conclusions and Recommendations 27 VI. Selected References 28 VII. VIII. IX. X. Appendix A - Smoke Inhalation Apparatus Appendix B - Cigarette Tracers Appendix C - Cigarette Filter Modification Appendix D - In Vtitro Bioassay Systems

THE BIOLOGIC EFFECTS OF TOBACCO SMOKE ON THE ALVEOLAR MACROPHAGE HOST DEFENSE SYSTEM INTRODUCTION AND BACKGROUND x

\ INTRODUCTION AND BACKGROUND Several epidemiologic studies have linked tobacco cigarette consumption, in a dose-dependent manner, to the prevalence of the chronic obstructive airway diseases. Although the means by which tobacco smoke potentially might cause bronchitis, emphysema, or lung injury of any other nature, is poorly understood, the role of the pulmonary host defense system in the pathogenesis of these diseases seems of utmost importance. In many reports, it appears that bacterial multiplication in the bronchiali tree is either a cause of the anatomic andifunctional disturbances associated epidemiol~ogically withitobacco cigarette smoking, or is a major contributor to these disturbances. If smoking is a contributing cause of chronic bronc_hitisb em_phvsema, and related lung abnormalities, t en it ie ke wit t ese diseases wou ve acterial ' ions in their airwa s. Although a equate e i emiolo ic investi ations are still needed it a ears tl-Tat ~' t is indee a e case. I we are to un erstand the relationship etween~tobacco consumption and the potential development of these lung diseases, we must quantify the interaction of tobacco smoke with the defense system of the lung, especially as tobacco smoke relates to the antibacterial function of the key cellular component of the defense network, the pulmonary alveol'ar macrophage. ire nuanor,.a.ry AZ'VeoZar Macro~h.aGe - The Cornerstone of L:.cng, Defense: The lung has perhaps the largest surface area of the body that is in continuous contact with the externali environment. This vast pulmonary surface is highly susceptible to injury from many different potential toxins in the environment, including voluntarily inhaled materials such as tobacco smoke. This internal surface of tho lung ranges in magnitude from:50 to 1101or more square meters and is, in part, a function of body build and size. When people smoke, they do so with a uniquely individual and generalily reproducible pattern of inhalation. For most tobacco smokers, approximately 70-95 percent of the tobacco aerosol or partilculate matter, related to the so-called "tar," and.essentiallly all, of the water soluble components of the gas or vapor phase of smoke are retained by the lung and must be cleared or inactivated by the pulmonary defense network. It is clear that the pulmonary alveolar macrophage, shown in a three-dimensional scanning electron micrograph in Figure 1, is the cornerstone of defense for the extensive alveolar surface. Although still in the process of evaluation, it has been reporte& that there are probably 3,000 or more individual chemical components in tobacco cigarette smoke. The potential health hazards of each such com- ponent, or the almost unlimited combinations for potentiation or inter- action of one or more components, is far beyond the scope of this report, although we have undertaken some partitional studies of tobacco smoke in order to place potential toxins in various chemical, categories. However, we do know that the aerosol size of the particulate matter of tobacco smoke

' - 2' - in such that most of these particles will, deposit on the alveolar surface, with perhaps a lesser number impacting at the bifurcation of conducting airways as inertia maintains their downward course during deep inhalation. Many of the gas phase components, especially the highly water soluble ones, may be lost in the respiratory tree by absorption, with the poten, tial existing for reentry into the lung at the alveolar surface via the circulation and'passage across the thin blood-air barrier. or r , Figure 1. The pulmonary alveolar macrophage, the key cellular component of the pulmonary host defense system, on the alveolar surface (18). All but a fraction of the surface area of the lung is comprised by the walls and associated structures of the 300 million or more alveoli of the lung. This surface is "protected!" by an acellular surface active layer ~ and by the alveolar macrophage,. L~s40i#itse "eal . with ..*-Ufaw.- f4~t3, i3~~ ttitss~ts~~__emntiroA~.aL, suWh_w_. LOp init- -~ ..:Z.-~... . ,'~ ~ - tQL_ I

- 3 - ~ From the standpoint of monitoring biological systems, the alveolar macrophage provides a very sensitive bioassay for detecting the earliest possible lung pathology associated with inhalation of a potentially toxic agent, such as tobacco smoke. One special consideration of the role of the alveolar macrophage in the pathogenesis of chronic lung disease deserves clarification. If some of the diseases of man that have been associate& with tobacco consumption, such as chronic bronchitis, are primarily diseases of the airway, why study the effect of tobacco on a cell, the alveolar macrophage, that is primarily a cell' of the alveolus, not the airway? The answer lies, in part, in what happens to the macrophage after it fulfills its protective role in phagocytizing and removing particles, such as tobacco, deposited on the alveoli. Classically, as shown in Figure 2, it has generally been accepted that these cells originate from the bone marrow, arrive at the lung as monocytes via the blood stream or lymphatics, adapt to the high oxygen environment of the alveolar surface and reside there for a finite period before eventually leaving the lung via the mucociliary stream. It is during this exit pathway that an alveolar macrophage, altered by exposure to tobacco smoke, can potentially by itself initiate and mediate lung injury. This hypothesis, by which the macrophage serves as an "indirect" or "secondary" mediator of lung injury, as opposed to a "direct" or "primary" lung injury from inhaled smoking products ~er se, is currently the most acceptable explanation for the pathogenesis of chronic bronchitis an& emphysema. It is, therefore, of crucial importance to evaluate and ful!ly understand the effects of potential modification of smoking products directly on this important cell, especially as such effects might relate as a precursor to significant lung injury. Figure 2. An alveolar aacrophage entering the mucocilliary escalator, presumably exiting in transit from the lung (18)1.

The PuZmonary AZveobar Macrophaqe and InhaZ'ed &xcteria: The lung is generally free of bacteria in healthy individuals, despite the continuous inhalation of organisms from the surrounding environment. It was once thought that the primary host defense mechanismiin the lung against inhaled bacteria was the bronchial! mucociliary stream. However, many studies now have showniconclusively that the most important mechanism by whichithe lung remains sterile appears to involve the action of the pulmonary alveolar macrophage system. The bronchial mucosa and the mucociliary stream are also important, but much less so then the macro- phage host defense network. Since tobacco cigarette smoke consumption may be related to chronic obstructive lung disease in a!dose-dependent manner, and since people with chronic bronchitis have infected bronchial secretion, it is important to understand the direct effect of tobacco ~)n the pulmonary antibacterial defense network. Tobacco Shroke and AZveoZar Macrophage Defenses: _ Several reports on the effects of tobacco smoke on the antibacterial defenses of the lun and s eci ic = on the a eo ar macro age, have been controversial. Most of the controversv related to such stu ies evolves from what, for _~gn„arP rnnrrnl nfrnhaccosmoke ex osure the most part, have been j P ~evels and dosimetr and u h sioliogic delivery of smoke to experimental ani~~ n t e Tobacco and Healt' Research Programithat we have developed at Harvard University, we have placed a primary emphasis on providing the same kind of quantitative control of the delivery of our smoking products, both to experimental animals and to in vitro bioassay systems, that are generally more characteristic of the biologic monitoring of these systems. Tobacco smoke i~s a.complex aerosol, consisting of a particulate and a gas-phase. Tar is the total particulate matter of smoke collected and weighed on special filter pads, minus nicotine and minus the water content. Total particulate matter is the total solid components that can be collecte& on a filter. The gas-phase is chemically complex and unstable. Much interest has been given to carbon monoxide present in the gas-phase, both from the standpoint of its potential healthihazards and as a tracer of water insoluble components of smoke. Certain water soluble components of the gas-phase, especially acrolein and acetaldehyde, have been implicated as macrophage and cilia cytotoxins. In that these and other gas-phase components may exert their cytotoxi,city, under certain conditions, as oxidants, several: investigators have developed antioxidant filtration procedures to reduce the cytotoxicity of tobacco smoke. An evaluation of the effect of one such agent, N,N1-diphenyl-p-phenylenediamine or DPPD, on the biologic cytotoxicity properties of tobacco smoke was the purpose of the experimental studies reported herein.

\ IN VZVO STUDIES

- S - IN VIVO STUDIES The processes by which inhaled micro-organisms are inactivated within the lungs have been extensively investigated in our laboratory in controlled experimental studies using an aerosol generator system that can deliver a predictable inoculum of bacteria to the lungs of experimental animals. Imthis aerosol generator system, shown in Figure 3, small animals can be exposed to bacteria containe&in droplet nuclei. Following intrapulmonary bacterial inoculation with approximately 105 bacteria, the lungs of control and experimental animals can be removed under sterile conditions, homogenized, and the number of viable bacteria remaining in the lung quantified in pour- plate dilutions, as shown in Figure 4. Figure 3. Schemati~c representation of the aerosol generator exposure apparatus used for quantitative inoculation of the lungs of small animals with radiolabeled bacteria. Bacteria are delivered on droplet nuclei, evenly distributed by the baffle system.

6 VIABILIT Y EXCISE LUNGS Bact.ria Pour-plates HOAfOGEN/IE ISOTOPE CLEARANCE Figure 4. Schematic representation showing removal, homogenization, and quantification of intrapulmonary bacterial vi~abil_ty by pour-plate dilutions and bacterial culture. Independently, physicaL removal of inhaled bacteria can be determined by quantitating the amount of isotope remaining in an aliquot of lung homogenate. The use of radiolabeled bacteria in this system permits the simultaneous estimation of physical clearance of the inhaled micro-organisms and a quantitation of iintrapulmonary bacteria1 inisitu inactivation, corrected for physical removal of the bacteria according to the following formula: Intrapulmonary Bacterial = 1 - Inactivation BACTERIA COUNT Time ISOTOPE COUNT Time BACTERIA COUNT Nebulizer X 100 O OPE COUNT Nebullizer

-7- Intrapulmonary bacterial inactivation rates can then be calculated, by the following relationship: Bt = Bo e-kt Bt = amount of bacteria at time t; Bo = amount of bacteria at time zero; k = bacterial inactivation rate constant; e = 2.71828 The data presented in Figure 5 reflect an index of intrapulmonary bacterial inactivation, as caliculated'relative to a bacterial-ilsotope ratio in the inoculating aerosol. When the bacterial colony counts, expressed as percent inactivation, are graphed separately from the change in isotope activity, it can be seen that the killing of the inhaled bacteria occurs within the lung. Since the isotope appears to be tightly bound to the bacterial DNA, this inactivation of bacteria is not due to physical removal of the organism, as the decline in isotope activity iis quite small relative to the rapid exponential decline in bacterial viability. The term "clearance" has been used to refer to the physical transport of the inhaled bacteria from the lung, as reflected by the decline in radioactivity, and the term "inactivation" used to describe the loss of bacteriaL viability, as reflected by the decline in the number of culturable organisms fromithe lung. This system represents an ideal in vivo bioassay for the evaluation of tobacco smoke or modification of tobacco smoke on the intrapulmonary inactivation of an inhaledibacterial challenge by the alveolar macrophage defense system.

8 14 INACTIVATION OF S oureus BY RAT LUNGS Aerosol Figure 5. Rapid decline in intrapulmonary bacteria:viability, with relatively little change inlisotope activity, indicating the bacteripl inactivation i~s due to in situ killing deep within the lunL rather than to physical removal~. METHODOLOGY OF IN VIVO' STZJDIES. All cigarettes employed in this study were 2R1 research cigarettes ctitained frorr the University of Kentucky (Lexington, Kentucky):. The liltered ver,ion of this cigarette, the 2R1'F, was utilized to evaluate tobacco smoke modification. The filter onithe 2R1F cigarette is designed primarily to remove the particulate or solid components ("tar") of the smoke rather than to specifica1ly alter the gas-phase of the smoke. Certain of these 2R1F cigarettes were treated with a liipid soluble anti- oaidant, N,N1-diphenyl-p-phenylienedilamine (DPPD), apparently with or without glutathione. Additional cigarettes were "sham" treated and were as,umed equivalent to the standard 2R1iF cigarette. The purpose of such filter treatment is to remove gas-phase oxi~dants in tobacco smoke that might be potentially harmful to pulmonary alveolar macrophage function. All cigarettes were prepared, coded and supplied to us by Dr. Nicholas. DiLuzio, Chairman and Professor, Department of Physiology, Tulane Medical

\ 1 3. OIVANT/F/CAT /ON OF /NTRAPUL IIGWARY ANT/BACTER/AL ACT/V/TY • Intropulmonory Bocterial Inactivation • Inactivation Rate Constants School (Appendix C). The cigarettes were received in two separate shipments. The first shipment contained "'M" and "D!' cigarettes, and were used for the majority of experiments. Additional cigarettes, needed for limited'studies on in vitro macrophage viability, were received in a:second shipment and were labeled "A" and "B"' cigarettes. None of the participants in the Harvard' Tobacco and Health Research Program were aware of the code meaning. The scheme for evaluationlof the acute effects of tobacco using this system is shown in Figure 6. Control, and experimental animals received an aerosol inoculation of radiolabeled S. aureus. Immediately after intra- pulmonary bacterial deposition, randomly chosen animals were sacrificed and processed to determine the "zero hour" bacterial inoculationilevel. The remaining animals were divided into unsmoked control~s and into animals receiving smoke delivered by a 30-port automated smoking machine (note Appendix A). On six separate occasions over a six-hour period animals received the freshismoke from 12 cigarettes or from unfiltered 2R1 research cigarettes. At six hours post completion of the bacterial inoculation, both control and experimental animals were sacrificed in order to quantify the percent viable bacteria remaining in the lung. Each experiment was repl~icated on two-to-three occasions, and the data analyzed by standard statistical methodologies employed inistudies of thils nature in our research program over the past several years. 2. SMOiYE /NHAL AT/GYV I CONTROL NON-SMOKEO, 21: ~ ~ Figure 6. Diagrammatic representation of the model used for the evaluation ~ of the acute effects of tobacco smoke inhalation on intrapulmonary 01 bacterial inactivation. X

- 10 - \ After inoculation with aerosolized bacteria, the animals were divided into the following four groups: Group I - Animals exposed to the smoke of filtered "M" 2RZF cigarettes Group II - Animals exposed to the smoke of filtered "D" 2RZF cigarettes Group III, - Animals exposed to the smoke of unfiltered 2R1NF cigarettes Group IV' - Controll group of unexposed animals. RESULTS OF IN VIVO STUDIES BtzctericidaZ' Effectiveness: The amount of bacteria killed six hours post- inoculation by the control group was set arbitrarily to a standar&value of one. Animals of Group I, which were exposed to "M" cigarettes, inactivated 97% of the control value during the six hours of smoke exposure. Animals in Group II, exposed to "D" cigarettes, inactivated 81% of the control value. Group III, representing animals exposed to unfiltered 2RINF cigarettes, inacti- vated 50% of the control value. A t-test for statistically significant differences yielded a p-value of 0.12 between Group I an&Group IV, and p<0.01 between Group I("M" cigarettes) an&Group II ('"D" cigarettes). These data are summarized in Table I. BacteriaZ Inactivation Time: In order to aid in the interpretation of bacterial effectiveness of the host defense system under these conditions of study, the data:in Table I (Bactericidal Effectiveness) may be alternatively consideredlin terms of the time required to inactivate a fixed proportion of bacteria. Setting the time required by control to inactivate the bacterial challenge to a standard value of one (hour), Group I("M" cigarettes) required 1.2 (hours), Group II ("D`cigarettes)~ required 1.47 (hours) and Group III (unfilteredicigarettes)I 2.1'7 (hours). These data are also further summarized in Table I. TABLE I COMPARATIVE EVALUATION OF BACTERIAL INACTIVATION Bactericidal Effectiveness Bacterial Inactivation Time (± Standard error of inean)~ ~ Bacteria Killed relative % (hours) relative to control to control Control, (No cigarettes) 100 ± .02 100 2R1F "M" cigarettes 97 ± .02 120 N11 2R1F "D" cigarettes 81 t.03 147 0 N arettes f rence ci 2RLNF 11 50t 217 ~ g re e . 0 1 4.)k

Carboxyhemoqlobin~(HbCO): The mean value of carboxyhemoglobin, a measure of the amount of carbonimonoxide found in the blood, after six smoke exposures was calculated from individtial blood analyses for each group of animals. The results presented in Table II are 11.9% for the "M" cigarette group, 13.0% for the "D" cigarette group and,15.6% for the unfiltered 2R1NF reference tobacco group, while the amount of HbCO in the control group exposed to ambient environmental carbon monoxide levels was essentially negligible. TABLE II COMPARATIVE ANIMAL DOSIMETRY Carboxyhemoglobin (%) (±' Standard error of mean) Particulate Deposition (ug/lung) (± Standard error of mean) Control (No cigarettes) Negligible None 2R1F "M" cigarettes 11.9% ± 0.9% 458 ± 60.7 2R1F "D" cigarettes 13.0% ± 0.7% 185 ± 13.5 2R1NF reference cigarettes 15.6% ± 0.6% 1238 ± 108 The difference between the mean values inithe "M" and "D" groups is not significant (p=.16), while both "M" and "D" groups do differ significantly from;those animals exposed to unfiltered 2R1NF cigarettes (p<.01). All data are summarized in~Table II. Deposition of PtrrticuZate: All cigarettes tested in the present study were laced beforehand with a chlorinated hydrocarbon, d'ecachlorobiphenyl' (DCBP)i, using the cigarette lacing machine described in Appendix B. The aerodynamic and chemical properties of vaporized DCBP make it suitable as a tracer of the particulate matter in fresh cigarette smoke. The amount of DCBP per milligram of particulate matter was obtained in all smoke exposures by passing the smoke through collecting filters and measuring the corresponding quantities of DCBP and particulate matter deposited on the filter. The total amount of DCBP in the lungs of each animal was quantified by chromatographic analysis. Finally, the deposition of particulate matter in the animal lungs was calculated by comparing the ratio of DCBP to particulate matter on the filter with the known amount of DCBP in the lung. In Table II are displayed the results of the deposition of particulate matter in the lungs for each group of animals. The mean value in animals exposed to "M" cigarettes was 458 ug of particulate deposition per

it exposed animal lung, for exposure to "D" cigarettes 185 ug, and'to the untreated and unfiltered cigarette 1238 ug. A1i1 differences are statis- ticalily significant to the p<.01 level. INTERFRE'TATION OF RL'.SULTS' OF IN VIVO STUDIES Exposure of experimental animals to both "M" and "D"'tobacco smoke produces some impairment of the pulmonary bactericidal ability relative to untreated animals. However, the additional time required by animalis treated with smoke from type "D" cigarettes (47% relative to the control group) ils more than twice the additional time requirediby the animals treated with smoke from type "M" cigarettes (20%), which would indicate that the exposure to the smoke of "D" cigarettes impairs macrophage functionisubstantially more than exposure to 'M1' cigarettes. In addition, however, the deposition of particulate matter in the lungs of animals following exposure to "M"'cigarettes is more than double the amount deposited following exposure to "D" cigarettes, which would indicate a gas-phase cytotoxin, other than carbon monoxide, present in the I'D" exposure to a greater degree. The investigation of a gas-phase cytotoxin deserves further evaluation.

IN VITRO STUDIES

- 13 - IN VITRO STUDIES' ' The intrapulmonary antibacterial defense of the intact animal in vivo is a complex, multifactorial system that involves the pulmonary alveolar macrophage, alveolar lining materiaL,and several immunological factors that have as yet not been fully defined for the lung. In an effort to develop a reliable bioassay in1which effects of an environmental influence, such as tobacco smoke, could be precisely defined relative to its inter- action with lung defenses, several investigators have studied the in vitro function of pulmonary alveolar macrophages. Macrophages can be rea'd!ily harvested from the lungs of man and experimental animals by bronchopulmonary lavage. These cells can be maintained for variable periods of time in tissue culture systems, wherein their bactericidal function,cell viability, mobility, phagocytic capacity and other key functions can be quantitatively defined, both in the basal state and following in vitro exposures to tobacco smoke or individual components of smoke. Such in vitro macrophage bioassay systems have been used to test the potential cytotoxicity of various smoking products. In such a system, shown in Figure 7, coagulase negative Staphylococcus albus, maintained on agar slants imovernight cultures, are prepared for phagocytosi~s studies. The bacteria are incubated with freshly obtained alveolar macrophages from experimental animals, agitated in a water bath and the parameters of in vitro phagocytosis monitored. Cytologic survival is d'ocumented by direct cell counts with bright field light microscopy, viability ascertained with supravital staining and phagocytosis characterized by appropriately staining bacteria within macrophages. Bactericidal activity of the macrophages is determined by quantifying the presence of viable bacteria in pour-plate dilutions of an aliquot of the macrophage- bacterial suspension. Imthis manner, we have been able to determine some of the functional characteristics, particularly phagocytic and bactericidal activity of the alveolar macrophage in vitro following incubation with a variety of smoking products. This system is not the optimum bioassay, unfortunately„to best measure phagocytosis per se or intramacrophage bacterial killing Per se. It is the optimumisystem, however, to evaluate the direct in vitro effects of whole smoke or smoke components on isolated macrophages in that smoke cannot be feasibly added:to the optimum systems designed for evaluation of particle uptake or intracellular killing.

\ Y~aEI1 Bocteria 0 l.rJ, _ E~\ti~ ~~ == cELL Grsa wrTlr ( Pour Ptate DilutYons 0/STPbLED WAT£17 Figure 7. Schematic diagram for testing the direct in vitro effects of tobacco smoke on alveolar macrophage function. In previous studies published from our laboratory, smoke from several different smoking materials was introduced into a tissue culture flask along with freshly harvested alveolar macrophages and the staphylococcal bacterial challenge. At time zero an&at various intervals after initiation of the incubation, aliquots of the mixture were removed from the flask and cultured for bacterial viability. In each study, an additional control was employed that evaluated the biologic effect of the smoking product on in vitro bacterial replication, in the absence of phagocytic cells. This is a control that had been lacking in all previous work in this area to date. There was a significant impairment of bacterial replication in vitro induced by exposure to all smoking products. Similar degrees sf depressed in vitro replication were noted with exposure to whole smoke or to gas-phase components of either product. Thus, in all bioassays performed with mononuclear phagocytic cells, this in vitro impairment of bacterial replicati>n was corrected for in the results on the macro- phage bioassay system analyses.

\ Shown in Figure 8 are the results of exposure to 8 ml of fresh whol~e smoke from~the smoking products on the inactivation of bacteria imthis in vitro system~. Control macrophages inactivated all' but 23.6 ± 2.0% of the bacterial, challenge. Tobacco smoke impaired alveolar macrophage bactericidal function inia dose-dependent manner, with relatively complete impairment following addition of 8 ml of smoke to the test system. There was no discernible difference i~nduced by various smoking materials. DOSE RESPONSE EFFECT OF SMOKING MATERIALS ON MACROPHAGE BACTERICIDAL FUNCTION ~ V ` sp1oor ,Morijpona ,-Cytrel z i , V ~ I I _ ~~3- KrMirYr * TODOCDa Q CigOt Q so~- e ~ Figure 8. The dose-dependent nature of impaired alveolar macrophage bactericidal activity following exposure to various smoking products. To further evaluate these results, the smoke from each product was )assed throughlan absolute fillter to remove all of the so called: ''particulate matter," as shown in Figure 9. In that the filter that we used is absolute, only the gas-phase of the smoke entered the test system flask. Shovrnlin Figure 11 is the effect of this gas-phase component on alveolar macrophage function. Although there is a slight difference when the macrophages are exposed to the gas-phase component only, this difference is not significant and it thus would appear that the alveolar macrophage cytotoxin demonstrable in such an in vitro system

- 16 - is contained in the gas-phase of the smoke. The slight differences noted might be attributed to a very small amount of absorption~of some of the gas-phase components by particulate phase on the filter as the material passes through it. In our next set of experiments, we passed the gas-phase components through a water dispersion trap, as shown in Figure 10. This process removes all water soluble components from the smoke. As can be seen in further evaluation of Figure 11, this resulted in complete removal of the macrophage cytotoxin, implying that the macrophage cytotoxin was highly water soluble. Millipore Filter, Holder I i FILTERED GAS PHASE ~--•OF i CIGARETTE SMOKE Lighted Cigarette Wire Screen All Glass Fiber Absolute Filter Disc Figure 9. Experimental design for removing the particulate aerosol and prodiicing filtered gas-phase smoke components. N ~ N 0 ~ ~ GO +"1 ~

~. GAS PHASE AND WATER FILTRATION SYSTEM FOR CIGARETTE SMOKE Lighted Ciqoretfe All Glass Fiber Absolute Filter WATER-FILFERED GAS PPIASE ~ OF SMOKE Distilled Water Fritted Gas Dispersion Filter Figure 10. Experimental design for producing water-filtered gas-phase smoke.

INDIVIDUAL EFFECTS OF SMOKING MATERIAL COMPONENTS ON ALVEOLAR MACROPHAGE FUNCTION' ~ 40- - oKentucMyToDaccoQ ' •Cytrel SmoMinq Mcterioli h 2Q- oCipar~ +Marijuano M 0 1 1 2 HOURS 1 3 Figure 11. The effect of whole smoke, gas-phase, and water filtered gas-phase smoke on the bactericidal ability of alveolar macrophages. The alveolar macrophage derives part of its cellular energy for phagocytosis from glycolysis. It is presently believed that there is a concurrent burst of hexosemonophosphate shunt activity occurring with pliagocytosis, presumably generating the energy needed for intracellular killing. Cytochrome-linked respiration and oxidative phosphorylation in alveolar macrophages do not appear to be altered by gas-phase components or aqueous extracts of gas-phase tobacco smoke. Glycolysis and glyceraldehyde-3-phosphate dehydrogenase activity on the other hand, seem to be directly impaired by gas-phase components of tobacco smoke, and it has been demonstrated that a close parallel between the dose-dependent depression of phagocytosis by alveolar macrophages and the depression of glycerald'ehyde-3-phosphate dehydrogenase activity with tobacco smoke occurs. Iodoacetate, a sulfhydryl reagent, is a metabolic inhibitor of alveolar macrophage metabolism that directly impairs phagocytosis.

- 19 - ' In addition, cystein and glutathione, sulfhydryl protectors, inhibit the cytotoxicity of tobacco smoke on~alveolar macrophages. We would propose, as a pathophysiologic mechani~sm of cell injury, that the close similarities in the effects of smoke cytotoxicity implicate a highly water-soluble component of the gas-phase of alli products and that this cytotoxin may modify the sulfhydryl groups of glyceraldehyde-3-phosphate dehydrogenase activity, with a resultant impairment in phagocytosis, as evaluated in~this particular system. One cannot extrapolate totally these in vitro studies on murine alveolar macrophages to the lungs of man or to the pathogenesis of any specific lung disease in man. One should emphasize, however, the role of the pulmonary alveolar macrophage in the normal physiology of the lung. This cell is the first line of the pulmonary defense network for all but the smallest fractionlof the interna1 surface of the lung and has as its major function the uptake, transport, and elimination of all potentially pathogenic agents that are inhaled. As the key cell for the phagocytosis and detoxification of inhaled poisons, the alveolar macrophage plays a central role in the pathogenesis of a wide variety of pulmonary disorders. It is the cornerstone of defense against both environmental and endogenous pathogenic influences. METHODOLOGY AND RESULTS OF IN VITRO STUDIES A Cigarette 5)noke and tYie ViabiZity of AZyeoZar Maerophages: One "A" filtered, "B" filteredlor 2R1F cigarette was puffed once every 15 seconds and:the cumulative smoke generated from 12-15 puffs bubbled through separate 10 ml aliquots of Hank's balanced salts soluti~on in a 16 X 125 mm tube. Each puff had a volume of 35 milli~liters and was passed into the solution through a 14 gauge cannula. The pH of all the smoke solutions was acidic and had to be readjusted with sodium bicarbonate to a level of 7.0 to 7.2. Macrophages were harvested from laboratory rats by bronchopulmonary lavage with heparinized saline solution. The cells were centrifuged, resuspended in Hank's balanced salt solution and cultured in pollyprolylene tubes. The cells numbered approximately 5 X 105/m1. Tubes containingg two milliliters of cell suspension were tumbled end for end four times per minute and samples were taken for viability determination assays at 0, 30, 60, and 120 minutes after the addition of one milliliters of smoke solution to each of the culture tubes. Cell suspensions were diluted into an equal volume of Trypan blue solutioniand loaded into hemocytometer chambers. The percentage of cells excluding the stain were considered to be viable, with the results from this method'shown in Table IIII.

- 20 - \ TABLE III VIABILITY ('%) OF SUSPENDED ALVEOLAR MACROPHAGES IN'THE PRESENCE OF SMOKE SOLUTION Incubation Time 0 min. 30 min. 60 min. 120 min. Control Macrophages 97.5% 88.5% 88.6% 86.4% Macrophages plus "A" Smoke 96.7% 94.7% 77.0% 5.2% Macrophages plus "B" Smoke 95.3% 88.4% 55.7% 7.1% Macrophages plus "2R1F" Smoke 95.0% 89.8% 42.7% 5.5% ViabiZity in Monolayer Cell CuZtures: Alveolar macrophages were harvested by lavage and suspended in Hank's balanced salts solution at a concentration of 1 X 106 cells/ml. One milliliter aliquots were placed into Leighton tubes containing 10.5 X 35 mm glass cover slips and incubated for 30 minutes to allow cell attachments. The culture fluid was aspired and one milliliter of either stock HBSS or of "A," "B"'or "2R1F" smoke-treated solutions, prepared as noted above, was added to the tubes. Cover slips were removed from the Leighton tubes at 60, 90 and 120'minutes after the addition of smoke solution, and the cells incubated with Trypamblue to determine the celi1 viability, with the results from~this method given in Table IV. The time- zero value of cell viability was determined on cells in suspension and all subsequent values obtained from the cultured monolayers. ,

TABLE IV THE EFFECTS OF SMOKE SOLUTION ON THE VIABILITY (,o) OF CULTURED MONOLAYERS OF ALVEOLAR MACROPHAGES Incubation Time 0 min. 60 min. 90 min. 1201min. Contro li Macrophages 96.0% 64.0% 63.5% 64.0% Macrophages plus "A" Smoke 96.Oa 64.0% 41.2% 23.5% Macrophages plus "'B" Smoke 96.0% 63. 4% 58. 8 0 33. 0 0 Macrophages plus "2R1F" Smoke 96.0% 4'5.5% 41.6% 20.8%. Ciaarette Smoke cmd BactericiduZ Fzacction of AiZveoZar Macrophaaes: Freshly harvested al~veolar macrophages from laboratory rats that had not been exposed in vivo to smoke were harvested by saline bronchopulmonary lavage. Macrophages and the staphylococcal challenge were mixed with eight milliliters of the fourth puff of fresh,whole smoke from the 2R1 an&the 2R1iF research cigarettes and with similar smoke from "M!' treated!and'"D" treated 2R1F filtered cigarettes. The percentage of bacterial growth or inhibition is shown in Table V. The percentages of bacteria cleared from the bioassay system, expressed as colony forming units from pour-pLatee dilutions after three-hours of incubation, relative to zero-hour control, values and corrected for the effect of the smoking products on bacterial growth, are aresented in Table VI. TABLE V THE EFFECT OF FRESH WHOLE SMOKE ON BACTERIAL VIABILITY Bacteria remaining (%) Bacteria alone 197.9% Bacteria + 2R1 Smoke 131.0% Bacteria + 2RIF Smoke 121.3% Bacteria + "M" Smoke 118 0% . ~ ~ Bacteria + "D" Smoke 98.8% N

-22- 'FABLE VI THE'EFFECT OF FRESH WHOLE SMOKE ON MACROPHAGE BACTERICIDAL FUNCTION Bacteria remaining (o) Control Macrophages 26.5% Macrophages + 2RL Smoke 111.4% Macrophages + 2R1F Smoke 106.3% Macrophages + "M" Smoke 109.1% Macrophages + "Ds' Smoke 131.2% IiVTERPRET:ITION OF RESl1LTS OF IN VITRO' STI,IDIES The smoke solutions fromiall cigarettes tested was toxic to macrophages, both in suspension and cultured onimonolayers, with the number of macrophages dramatically declining below that of the control cultures after the initial 601minutes of incubation. When incubated in solution, wherein relatively high doses of smoke were administered, the system was virtually totally lethal to the macrophages by the end of two hours. There were no discernible differences among the cigarettes tested by this method. By the alternative method of evaluation, in which monolayers of macrophages were cultured after the treatment with lesser concentrations of smoke, there also was significant decreased macrophage viability after the first 60 minutes of incubation. At 120 minutes, the viable macrophages treated with "A" smoke or "2R1F" smoke was approximately 33% of the control group. The "B" smoke was also toxic to the macrophages in this system, but less so than the "A" smoke. The smoke from all cigarettes was toxic to bacteria in our tissue culture bioassay system. The smoke from "D"-treated cigarettes was the most toxic, and there were no discernible differences between the effects of "M"-treated cigarettes and untreated 2R1F research units. Although there is stati~stical significance in~the differences between the other individual values, the biologicaL significance may have little meaning. Basically, staphylococci do not replicate at a normal rate in the presence of smoke, and "M"' or "D" treatment do not have a major effect on.this phenomenon.

- 23 - Control macrophages phagocytized and inactivated all but 26:.50 of the staphylococci over the period of evaluation of bactericidal effective- ness. Phagocytosis and inactivation were completely inhibited by the smoke from all cigarettes, with the severity of inhibition following exposure to smoke from "D-treated" cigarettes being over 20 percent greater than the effect of smoke from "M-treated1' units. Although the particulate load of the 2R1 research cigarette is significantly reduced by the standard 2R1F filter, it would appear that the gas-phase macrophage cytotoxins are not comparably reduced~. It woulid further appear the "M1' and "D" treatment had no significant biological effect on altering these gas phase cytotoxins, when the results, as presented in Table VI, were corrected for the effect of the smoke on the bacteria alone. It must be emphasized that although these in vitro tilssue culture systems are effective bioassay systems for comparative product evaluations, they are not particularly representative of the biologic effects of tobacco smoke on macrophage function in the intact animal in vivo. Currently ongoing work in our laboratory (Appendix D) indilcates that the concentrations of tobacco smoke gas-phase components in these in vitro viability and functional bioassay systems is probably in the magnitude of 105~or so higher than occurs in the airways or alveoli of man. In other words, these in vitro procedures, as welil as those of others, basically "overkill" relative to the actual concentrations of smoke product delivery. Direct extrapolation of the effects of these products, based on such in vitro~results, to related biologic function in the intact host, then, should not be made.

\ PARTICLE SIZE ANALYSES

- 24 - I% PARTICLE SIZE ANALYSES The properties of the tobacco smoke aerosol can be altered by various filtration procedures. In that such potential alterations might affect the site of smoke particle deposition within the lung, or might chemically alter the smoke itself, it is of crucial importance to evaluate this variabl~e in all biologic studies of experimental, tobacco smoke filtration. METHODOLOGY OF PARTICLE SIZE ANALYSIS The 2R1F filter cigarettes were laced with the DCBP tracer and were smoked to a 23 mm butt length in a modified Homberger smoking machine. This machine has been modified to produce a steady flow during each two- second puff of mainstream smoke diluted 100:1 with cl'eaniair. This high dilution is necessary tolavoid instability in our aerosol centrifuge. For each run, two cigarettes were smoked 12'puffs each. The diLuted smoke was sampled'directly at the outlet of the smoking machine with a cylindrical aerosol centrifuge. The centrifuge, operating at 3000 rpm, continuously fractionates a stream of tobacco smoke according to aerodynamic diameter along a removable 40 cm stainless steel foil. After sampling, the foil was removed and cut into 11, sections, which were extracted and analyzed'for tracer by chromatography. Each foil section corresponded to a known range of aerodynamic diameters. A fil~ter, which aliso was analyzed for tracer, was positioned at the outlet end of the deposition channel to capture all particles too small to be deposited on the foil. The aerodynamic size calibration along the foil ha&been previously determined, using polystyrene latex spheres of known aerodynamic size. No correction for coagulation or evaporation was made, however, and the time and mixing conditions were the same for all runs. RESULTS OF PARTICLE SIZE ANALYSIS' All size distributions have a reasonable fit to a lognormal diistribution. Results, showniin Table VII, are the mass median diameter (h1MD)i and geometric standard deviation (GSD) for the best fitting lognormal distribution.

-25- ~ TABLE VII SUMMARY OF RESULTS 2R1F "D" (filter)' 2R1F "M" (filter) 2R1NF (non-filtered) Mass Median Diameter 0.42 um 0.40 um 0.50 um Geometric Standard; Deviation 1.79 M L.75 wm 1.40 um Based on the results shown in Table VII there is little difference between the "M" and "D" cigarette smoke, but both are different from the non-filter 2R1 cigarette smoke. These comparisons are better shown in Figure 12, a plot of particle size versus cumulative percent. Cumulative data from lognormal distribution produced a straight line on this log probability plot. Again we see a close similarity between the "M" and "D"' cigarette, an&both differ from the 2R1 results. The discrepancy between the lowest points for the "Wand "D"' may be a real effect, however, it is more likely that this is the result of an artifact, such as leakage around the filter or analytical error.

- 26 - 1 5 10 30 50 70 90 95 99 % LESS THAN INDICATED SIZE Figure 12. Comparison of smoke particle sizes in~untreate&2R1 reference cigarettes and treated'"M" and "D" cigarettes. INTERPRETATION OF PARTICLE'SIZE ANALYSIS Based on these preliminary measurements, subject to the limitations described above, it can be concluded that there is no significant difference in p article size distribution between the "M" and "D" versions of the 2R1F (igarettes that were tested,. Both treated ("M^' and "D") cigarettes differed rrom the unfil'tered variant, which may be attributable to the effect of the filtration alone.

CONCLUSIONS AND RECOWENDATIONS

- 27 - , CONCLUSIONS AND RECCJNVr;NDATIONS On the basis of the experiments summarized herein, it would appear that the "D-treated`cigarettes are more toxic in both acute in vivo and in vitro bioassay systems of alveolar macrophage bactericidal function. It would also appear that, in terms of macrophage viability, that smoke from "A-treated" ci~garettes is slightly more toxic in one, but not both, in vitro bioassay systems. Our studies, including the preparation of this report, were completed without knowledge as to the coding of the experimental cigarettes. It appears that "M,treated" andi'"A-treated" ccigarettes do not differ in their biologic effects, under the conditions of the studies included herein, from the standard 2R1F untreated research cigarette. If thee filters of the "M" and "A" cigarettes were impregnated with N,N1'-diphenyl- p-phenylenediamine, we would recommend no further study as there appears to be no major biological benefit from these units. Our studies imply that the "D-treated" cigarette is more toxic to the biologic functions evaluated in our studies. Tf the "D" cigarette is the one treated'with N,Nl-diphenyl-p-phenylenediamine, iit would appear that such treatment is potentially hazardous to:the host an&we would recommend no further study, except in the interest of identifying the potential toxin. We would;recommend no further studies on the characterization of the aerosol properties of the smoke from these cigarettes. Any proposed evaluationss on experim_ental, human consumption _would--g- stron 1 contrain3icate&until t e a ove noted observat ons are urt er rlarifi ic animall in alation studies.

2010048930 r

, SELECTED REFERENCES 1. Cohn, Z.A.: The Fate of Bacteria Within Phagocytic Cells: 1. The Degradation of Isotopically Labelle&Bacteria by Polymorphonuclear Leucocytes and Macrophages. J. Exp. Med. 117:43, 1963. 2. Cohn, Z.A.: The Fate of Bacteria Within Phagocytic Cells: II. The Modification of Intracellular Degradation. J. Exp. Med. 117:43, 1963. 3. Green, G.M.: Pulmonary Clearance of Infectious Agents. Annu. Rev. Med. 19:315, 1968. 4. Green, G.M?: Cigarette Smoke: Protection of Alveolar Macrophages by Glutathi,one and'Cysteim. Science 62:810, 1968. 5. Green, G.M.: Protection of Alveolar Macrophages from the Cytotoxilc Activity of Cigarette Smoke by Glutathione and Cysteine. J. Clin.. Invest. 47:42a, 1968. 6. Green, G.M.: The J. Burns Emberson Lecture-In Defense of the Lung. Amer. Rev. Respir. Dis. 102:691. 1970. Green,, G.M.: Defenses of the Lung. Clin. Notes Resp. Dis. 11:3, 1972. Green, G.M.: Alveolobronchiolar Transport Mechanisms. Arch. Int. Med. 131:109, 1973. Green, G.M.: In Defense of the Lung. Amer. Lung Assoc. Bull'. April, 10. Green, G.M. and Carolin, D.: The Depressant Effect of Cigarette Smoke on the In Vitro Antibacterial, Activity of the Alveolar Macrophages. New Eng1. J. Med. 276:421, 1967. 11. Green, G.M. and Goldstein, E.: A Method for Quantitating Intrapulmonary Bacterial Inactivation in Individual Animals. J. Lab. Clin. Med. 68:669, 1966. 12. Green, G.M. and Kass, E.H.: The Role of the Alveolar Macrophage in the Clearance of Bacteria from the Lung. J. Exp. Me&. 119:167, 1964. 13. Green, G.M. and Kass, E.H.: Factors Influencing the Clearance of Bacteria by the Murine Lung. J. Clin. Invest. 43:360, 11965. 14. Green, G.M., Powell, G.M. and Morris, T.G.: Specific Chemical Enzyme ~ Mediation of Tobacco Smoke Toxicity for Pulmonary Alveolar Macrophages 0 (PAM). J. Clin. Invest. 50:40a, 1971. ~ - o 0 ~ ~ co w N

15. Greenwood, M.F. and Holland, P.: Mammal~ian Respiratory Tract Surface: A Scanning ElectroniMicroscopic Study. Lab. Invest. 27:296, 1972. 16. Harris, H.W., Meneely, G.R., Renzetti, A.D. Jr., Steele, J.D. and Wyatt, J.P.: Definition and Classification of Chronic Bronchitis, Asthma and Pulmonary Emphysema. Amer. Rev. Respir. Dils. 85:762, 1962. 17. Hinds, W.C.: Silze Characteristics of Cigarette Smoke. Amer. Ind. Hyg. Assoc. J:., in press, 1977. 18. Holland, P.: Personal Communication. (We gratefully acknowledge Dr. Holland in providing us with these electronimicrographs.) 19. Kass, E.H., Green, G.M. and Goldstein, E.: Mechanisms of Antibacterial Action in the Respiratory System. Bact. Rev. 30:488, 1966. 20. Kendall, L. Jr., Laguarda, R., O'Donnell, C., Sornberger, C. and Huber, G.: Patterns of Breathing in Humans During Tobacco Cigarette Consumption. C1iin. Res. 23:598, 1975. 21. Laurenzi, G., Berman, L., First, M. and Kass, E.H.: A Quantitative Study of the Deposition and Clearance of Bacteria in the Murine Lung. J. CUin. Invest. 43:759, 1964. 22. Lewis, C.I., McGeady, J!.C., Wagner, J.R., Schultz, F.J. and Spears, A.W.: Dichlorbenzophenone as a Nonradioactive Tracer for Cigarette Smoke - Gas Chromatographic Analysis of Tracer. Amer. Rev. Respir. Dis. 106:480, 1972. 23. Lewis, C.I., McGeady, J.C., Tong, H.S., Schultz, F.J. and Spears, A.W.: Cigarette Smoke Tracers - Gas Chromatographic Analysis of Decachloro- biphenyli. Amer. Rev. Respir. Dis. 108:367, 1973. 241. Schultz, F.J. and Wagner, J.R.: A Thirty-Port Smoking Machine for Continuous Smoke Generation. Beitrage zue Tabakforschung 8:53, 1975.

• APPENDIX A SM0KE INHALATlON APPARATUS

\ -Ali- APPENDIX A SMOKE INHALATTON APPARATUS Although several laboratories have published informationion the response of the lung following the experimental exposure of animals to tobacco smoke, most of these results are controversial and subject to considerable criticism. The primary reason that experimental smoke inhalation studies have been so unsuccessful, inithe past i!s that there have been no smoking machines that could create a stable aerosol of tobacco smoke, comparable to that created by human smokers, which could be delivered to the lungs of experimental animalls. It is our evaluation that all existing commercial machines now used fail in this crucial requirement. To solve this probl~em, we have built a total of three Lorillard smoking machines for our laboratory. This machine, initially developed by the tobacco industry, ils shown in Figure A-1. Figure A-1. The multiportal smoking machine, capable of generating a stable cigarette smoke aerosol for delivery to the lungs of experimental animals.

- A2 - Figure A-2. A view of the loading cassette of the smoking machine. There are 30'portals for cigarettes, with 15 on each of the two rotating wheels. Cigarettes are automaticalily loaded, and ejected after a predetermined number of puffs, usually 10. A1'1 cigarettes are conditioned with standardized humidity and temperature prior to use, an&burned, to a consistent butt length.

-A3- \ Figure A-3. Shown in this photograph is a view of the cigarette being loaded into a portal, by necessity under slight pressure. Tobacco cigarettes that are tightly packed immediately reform their original shape, as shown in the lower of the two cigarettes seen above.

-A4- \ In our machine, the cigarettes are loaded into a cassette, as shown in Figure A-2 and automatically supplied to the cigarette holders as shown imFigure A-3. Once loaded, the puff chamber closes on the rubber cams and a standard amount of tobacco smoke is generated, created as a stable aerosol which remains stable until delivery to the lungs of the experimental animals. Shown in Figure A-4 is the puff-chamber approaching the loaded and burning cigarette, and shown in Figure A-5 is the puff-chamber tightly closed around the rubber cam and the cigarette, delivering i~ts puff that creates the stable exposure aerosol. The smoke generated is then passed through a dispersion system, shown in Figure A-6 and a series of tubes. These are special conductors that do not allow the aerosol, to precipitate on their waLls in any significant manner and permit delivery of the generated smoke to the lungs of the animals in less than 2.0 seconds. ?h O Figure A-4. The puff-chamber approaching the loaded and burning cigarette. 0 O ~ ~ ~ W I

- AS - \ Figure A-5. The puff-chamber tightly closed around the rubber carwand cigarette.

-A6- \ Figure A-6. An oblique view of the smoking machine with the puff-chamber dispersion conducting system. 11

T4 APPENDIX B' CIGA.RETTE TRACERS

1 - B1 - APPENDIX B' CIGARETTE TRACERS The most important measurement for determining the particulate dose of tobacco smoke the animal receives is the average concentration it is exposed to ddring a run, the dose being the product of minute volume, duration, retention and average concentration. The average concentrati~on of exposure is measured directly by taking filter samples at the exposure site and correlated in selected studies with particulate deposition in the lung.. Lacing Ciqarettes: It ils essential to equate exposure levels (',smoke delivered to the whole animals) with dosimetry (actual amount of smoke inhaled). To achieve this, we have a:method of lacing all, cigarettes with a non-rad'ioacti~ve chlorinated hydrocarbon, decachlorobiphenyl (DCBP). The lacing machine shown in Figure B-1 is used to evenly deposit tracer individually into single cigarettes. Figure B-2 shows anienlargement of the lacing machine holder in which the cigarettes are manually placed. When the machine is operational, the holder rotates the cigarettes slowly, allowing for even deposition of tracer as the lacing probe is withdrawn. Figure B-1. Cigarette tracer lacing machine.

- B2 - Figure B-2. Enlargement of the lacing machine cigarette holder.

- B3 - Shown in Figure B-3 is the carousel used to hold the exposed animals, with the delivery or conducting tubes at the top and the exhaust system at the bottom. The flow of the aerosol is rapid and does not allow agglomeration or condensation of the fresh smoke aerosol nuclei in any significant degree. As can be seen, fifty animals can be exposed simul- taneously in this system. The animals are heUin small containers, shown in greater detail in Figure B-4 and Figure B-S. These containers employ an interlocking system for efficient and rapid loading and placement in the chamber. Once in place, the animals are relatively comfortable and inhale the smoke product as it passes through the exposure system. Figure B-3. The smoke-exposure carousel. Fifty animals can be exposed at one time. Each carousel is aounted on wheels for mobility and easy transport of animals from their housing to the exposure room and the smoking machines.

- B4 - • Figure B-4. The animal-holding chambers are transparent and can be easily removed from and placed in the exposure carousel. Animals are comfortably held in place by the sponge-rubber plug in the rear of the chamber.

- B5 - \ Figure B-S. An animaU in place during exposure to smoke. This system has the versatility to expose any sized animal, from the smallest rodent to an animal, the size of a horse or larger, to the same stable aerosol of fresh smoke. By using this system, we deliver "laced" smoke to the animals. Total "tar" in the delivery stream is quantified by gravimetric methods and' cross-calibrated with tracer content. The total amount of tracer in the lungs of each animal is quantified by chromatographic analysis. Then, by back calculating to the tar-lacer ratio in the smoke delivered, the exact amount of "tar" or total particulate matter of whole smoke delivered to the lungs of animals can be accurately determined. By using these techniques, and by also simultaneously measuring CO in the gas phase of the whole smoke delivered'in the exposure system and HbCO in each animal, we have an accurate assessemnt of both gas phase and total particulate delivery of tobacco smoke to each individual animal.

APPENDIX C CIGARETTE FILTER'MODIFICATIC.9

- C1 - 1 "................... "' 2 TOBACCOSMOF:E FILTERS FIG. 1, FIG. 2 and FIG. 3 show views in elevation, partlyin The present invention rclatcs to tobacco smoke filters section, of cigarettes having difTerent arrangcments of filters which are surtahle fur emplormenf in esgarettes, pipes, and different types of chemscal filter compositrons. cigarette holders or cigar holders In FIG. I the numtra1110 represents a cigarette consisting of The prescnt filtcr, classrfird as a chemical fdter, has 5 a paper Iube I1 packed, except for tip np poruon, with tobac- profuund' advantages over other frlters known to the an by co 16. One end'of the papertube I I(or a separate butt tube ) being.ble to nwddy the toi,c andlor lethalleflects of csgarette eonststs of a section 12 camposed of parttcles of DPPD with or smoke estracts on alveolar macrophagcs• the primary host without glut>fthsone between filter sectrons 13 and 14 com- defcnsccell'r.(Ihelung posed of cellulose acetate or other known tobacco smoke The filters currently ivarlable are essentrally emplored'to 10 filter material. The section 12 may also contatn,DPPD mised remnve particulate and sol d componentrof smoke as well of withiacttve charcoal creating a simultaneous mechanical and to ddutc the gasoous component% of smoke with air The gase- chemical filter having dual advantages ous phase of the smoke readrlv passes throueh such filters In FIG. 2 the numerals 10, 11 and 16 are the same as in FIG resulting in irritation to the lung -rssue and. as will be nwed' I and the section 12 is composed of a ccllulbsic filter materral later, profound lethalny, to ssolatcd~aivcolar macrophages, a 115 impregnated with DPPD, with a plain cellulosic filter 13 authc cell whose primary function is to maintain a proper lung en- tip of the cigarette. I rircnmcntby detoxificauon and destroying a rartety of agents. In FIG. 3 the numerals 10„11 and 16 are the same asan FIG. The gases derived from burning tobacco smoke contatn 1stnd the numeral 11 represents eellulosic filter material or highly reactive, low molecular wught; volaule components, of other absorbent filter material impregnated with, DPPD with which acetaldehy,de is charaetenstic, as well as free radicals 201 or without glutatAione. The numeral 15 represents a recess and largc quantities of gaseous rons induced by the high tem- portion of the ttp of the cigarette. peratures, which c:n,ap,proach I,000' C resulting in pyro:rza- Rearrangements of the various combinations shown in the tion and duti;lauon. drawings may, of course, be made, and it is not neeessary that In addrtion, free rad:cals which have been demonstrated to there be any filter other than the composttron conststtng ofor be eytotoric have also been demonstrated to occur in the 25 corrmpnsing DPPD. gaseous phase of smoke The chcmtcal filtering material incor- By having a plain (nonchemical) filter at the tip of the porated in the present invention has the abrlity, in the classical cigarette (FIGS 1 and it no contact of the smoke wtth the role of anuosrdants as tree radical stoppers, to lower the chemical agent occurs Likewise„the uscof a recess portion ofi eytotosic substances oGcigarctte smok'e, possrbly by reducing the tip (flG 3) will prevent contact of the smoker wIth the the attack of free radicals or other such react,.c agents on put- 30 chemical agent. monary al•colar cells and therefore matntaining cell!vtnbduy. Since frec rad cah have been implicated in the genesis of neoplasia, a b'eneficial cffect of the antroxidant filler may, be implied In aGd tion, the chemicali frlter has the ,.b h(y to ptevent smoke-induced dccrerscs in srculatsng,plasma lipid 35 anuosrdants. It irthe object of the present invention to provide a filter which, I have discovered, reduces the disclosed tosteity of, cilarette smoke on alvcalarmacropha;es. I have discovered that~ the above object can be accom- 40 plished by having a tobacco smoke ficer such as a cylindrical eonuinerfilled .. ith a smoke permeable composition compris- ing the lipid soluble anuoa dant \•Iw'-drphenyllp-phen- yltnediaminelDFPDI. preferably mtsed with the water solu- ble antiosidant glutathione The DPPD or the DPPD mixed 45 with glutathiune may be used alone or mrxcd wrth other In- gredscnls such as w• th aclivatcd charcoal or rmpregnat%d into a filter carrier material such as celluloseacetate, paper, wood pulp, eotton. Gbrous polyulefins, cellulose esters in f brous or plastic form, regenerated cellulose, sdrca gel, alumtna„tobac- 50 co and'thc hke, Likewise the DPPD may be rosxed with other chemical filters such as the amrdc Gtter of U.S. Pat. No. 3.426,765 . The amounuof DPPD required to beeRective in reducing the tosrc effect of tobacco smoke on alveolar Ilung) 55 maorophages is from 0.025 to-0 500 grams in a filter for use in eijarettes and prpes, irrespective of the type and size of the cigarette or pipe in present day use. Preferably the amount rillibe from 0 0501ro 0:00 granss. The perccntattc of DPPD W rth cartier materral is preferably 60 from 5 - 70 pcrcent by weight based on the weight of carrier material, althoagh fngher anrounts mav„of coursc, be orescnr sir.cc the invcntrun is uperatnc with 100 per ccnt DPPD. The proportion oftlutathronc to DPPD may ary widely up IL•• 65 but preferabl y the her g f 10-1 r hr g t i ht o o a w o u e g ti , ra tathione. in ordento:r.e rncrcasedwffecuveness to the DPPD, should be present in a ratio oGatdkast 1-10 K'Acn active ca•`on is present the werght ration likewise may vary upau : ratio uf a:trva:ed cart:un to DPPD of10-1 or higher, preferah'• 0 5-I to 5-I. Other chemical filtrrrng msteri.l may be used in varying amounts, but Preferab,y the UI'?D shoulJ he presant Inmalur amount eomparedho uthrr nutcrul The In.entrun is rllustrattd t~y, reference to the aeeumpany. • sng dra. nll in - hteB, l he DPPD and DPPD with glutathione additi+es specifi• eally employed to reduce the tosrc effects of cigarettes on al- veolar (lung) macrophages well also restnct certain biochemr cal changes whicA occur following exposure of animalt and humens to cigarette smoke. ihese.inuos dant~filtcrn,+dl conr tribute to the reduction of chronic pulmonary duease, whreh can be credited to impaired macrophage functton. The DPPD antioxidant permits to superimposition of a technique ts hich might be described as"solecti.e chem eallfil- tration•' onto the current commonly usedproceas ofmechant• cal filtration. Hys•tng in a gencrallray indicated the nature and purpose of my invention, the followtng are examples and data by w hrch specrf~c illustrations of the practice of the incent on and the adcantages to be obtarned'are demonstrated and,are not to be construedas Ismsting the same., EXAMPLE I Toaic Efieets of aqueous Extracts of Cigarette Smoke on, Isolated Alveolar \tacrophagcs. Alvcolar macrophaees were isolated from rat lungs b~ the technique uflung •aasMng and equal aliquuts .~f .~btaineacells were added to three flasks containmgthe fullowtng Flask A. 3ml of Krebs-Rrnger Phosphate kledtum t KRP1. Flask B. 2.9mI of K RP plus 0.l ml of KRP smoke solution Flask C. 2.5 mI of KRP plus 0.5 mllofKRP smoke solution. Flask D. 2.0 ml of KRP plus 1.0 ml of kRP smoke solution. The KRP smoke solution was prepared by bubbling thraugh 10 r+sl of KRP solution the smoke from one unfdtered cigarette. This solution was used within 10 minutes of as preparation. The isolhted alveolar macrophages ( II x 10' ) were added to each flask and the flasks ere incubated for 30 mtnutes at 37* C. Viability was oetermtr.ece at 30 mtr,utes after the tncu. bation in the fint eapearmcnt„and 301and 60'msnutes in the second eaperrment. Eaperilnent I Er;erirem IL, dell Cell Smote Sur„td, iunnal. -ercrnt Flask Solauoa : 'n P.irn 1.0'%tre bu %Ira A 0 10 Ta 2 H.1, B t1,1 nJ 74 3 ea 2 C 0J ral 41 9 3tIJ 70 75

- C2 - PATENTE 14 '16 ~ ./Io t:-~„ - ,-~r ~ -- - - _ - . -- - - ~_ 2

-C3- 3 4 D 1.001 160 16 1•7 lhest studlca. ar wcll as thnse to (oliow„cleanly,denote the quenua rstracts uf clgautle smoke eaert a lethal ef(ect on ur,latcd rat alvenlar aaacroptiagcs which is dose dependent. While no red'uetlon in,snbtluy wss obstrved when Oil ml of smoke solution~was added! a 42 and 71 per cent'decrease in viability was observed at 30 and'60 mtnutes, respeett.ely upon the addition of U.5 ml of smoke solution. 1i'hen 1.0 ml of smoke solut on was added• or a.olume equivalent to 0 I' of the original smoke extraeTobta nod (romione ctgarette„an al, most total loss of viability was oesenved Thus.,exposuro ofia1F veolar macrophages to gaseous, water soluble eiemenu of eigarette smoke is assoctated wlth significant cellular monafi- tT: EXAMPLE 2 Further Studies on the thaToxie Effect7ofCiearette Smoke. Additior:al studcs were conducted on the effects of vaning eoncentrat-ons of c garette smoke on al.colal macrophage via6lllty. kat alveolar macrophages, 7 5 z 10s macrophagcs per flask, uerc incubated in the presence or absence of aque- ous estra;ls of ctgzrette smoke prepared as Example II from one unfiltcrcd ctgarette.. Ftad Added Eetract 0M'n. A - )6 4 8 0 tl 74 1 C 03 709 D 1 0 tf • A - 77.v • 0.11 C 0 5 D 1.0 Viabllity„ Fercent 30,A(m. 6OMtn.. 8i FIa.+! Flltrr ntraet 3 A ._. ............... ... ... .. - M ..................... . ...... t C.,.,....... AnaosfJ.nt,.,. ., . .......... Innil~rnrnilral..-. + L, . _---.,., (IUl; ulofe .ceA4_. + A .... .• ........................ - n ........................•..... ~ C .......... Anttor.d.nt'....... + D..-........ lh.rt,chrnleal. -.. t E-..-...,.-.. CsWulox arelate„ + 10 Cr11rInH111ty, I.rrrntO 1in1n, WI rynw 1.4/ udrr. tr~ l 7i a rtl' Ir.a u] :LU 1~ u e~.t I+ ts 7 r h 140.1 m 4' 4 1.7 11  M.! 41 3 4 1.s u; tr These studies elearly denote the toxic lethall effects of cigarette smoke tstract oo ccll':vteb~illty as essentially 1G0'per cent mortal:ty of' al~eolar macrophages is observed after 2 15 hours of incubation~(;B'GROI,'P) During the inltlal hour of m- eubation, the mean 62 pen cent decrease in viability, which characterized the cellsancubated in aqueous estrocts;,was not observed whcn the antioxidant chemical filter conslst ng of equal parts of DPPD and glutathione was emploved. Slgnifi- Z0, cant protecu.e effects are observed at the 120 mtnute period when the antroxldant flter, is employed Likewise. essentially 100 per cont'mortaltty, was seen in the cells incubated with aqueous extracts of cigsrette smoke derived from fil(ered eigarettcs, chemical control filtered ctgarettes i.e.,,the non- 25 antioxidant type filter, ar,d the nocfi-tered'agarette. Thus, the beneRcial effect ofD'P'Janttoxtdant on negating the toxic effect of cig,arette smoke on lung, macrophagcs is manifested. These macroph'ag,es• an important host defense cell of the body, function in phagec}tosls and destruct on of 30 bacteria, viruses altered or aged eedls'• as well as the rcmovall and digestion of toxic macromolecular compounds which are present in their environment. EXAMPLE 4 The influence of Antioxtdant Impregnated Filters on AI- veo)ar tit+crophage l',tabJity. The effect of DPPD aotloxtdant with glutat'llone tm- pregnatcd'on cellulose acetate filters (FIGS 2 and 3) in maln- tatning alveolar macrophage viability was also tested'. Our tests indicated that approxtn:ately I ml ofi Krebs-Ringer phosphate solul on ts taken up by a cellulose acetate filter„ whlch were sectuonedi to enhance surface area. A KYebs- Ringer phosphate medium was prepared, whtch had a concen- tration of 50 mg of gluuthtone and 50 mg of DPPDlper mil- liliter. The sectioned filters were immersed in the aqueous suspension for one minute, removed and dned under ~acuum over night. The approximate total uptake of antiosidants by each Glterwas 100 mg Filters prepared in such a1ash:on w ere then inserted back Into tho end of tlie cigarette from which the filters .ere origina!lv removed and aqueous estracts of cigarette smoke prepared from the resulting cigarettes as prertously, drscrlbedl Incubatlon of the aqueous smoke cx- tracu were isolated macropages were conducted at 37' C(or 60iand I!20 minutes. Thc results are as follows: 35 40 These studies confirm the dose dependent toxic effect of aqueous cxtracts of cigarette smoke oni isolkted lung QS macrophagcs. EXAMPLE 3 Atoddicauon of Toxie Effects of Cigareue Smoke by An- 50 l ic id F ter. t s ant l In vlawrof the dcfinuile toxic effects of ctgarette smoke ex- tract on isolated alveolar macrophafes. attempts were made to modify the toxlc effects of smoke extract on alveolar macrophages An anuox dant filter was designed and developed (FIG. 1); consisting ofi 100 mg of antioxldant filter 55 (S0 mg of glutathrone and 50 mg of V;N"-diphenyl4p-phen- .Ienedumtne (DPPD). Control filters were employed using non-antiosrdant,chemlcalst such as sodium chloride (50 mg)„ and sodium bicarbonate (SO mg); which have comparable physical appearances of the antlossdants. Thase filters art 60 designated as ••Ihert Chemical Filters• having no:antioctdant aelivity '• Aqueous estrscu were ptepared:using the followsog type cigarettes. A. Nonfil(ered Cigarcetes. B. Antiosidant Fdtered Cigarettes. C. Control Inert or `on-anuostdant Containing Chemical Filtered Crgarettcs- D.Rcgular Commercial Cellulose Ar-tate Filtered Time tn minutrs Group Fltter Smoke estnot. 0 60 I.C A•- ---•----- --_.......- t;o.o ;0.1 r* a n.-..•-..._... _ -• + u : o a, C•---_-„ ~mtoiil.r. irnnrctn.ird:..+ .....~._ D..-..-.. Cellnorr.;eurr•....-.-..-. + ........ a~ t r These studies, in essential agreemeni pres ious observa- tiona, elearly d'emonstrate the toxlclty of cigarette smoke (jroup. 8)) and the mabtlitvv of currently employed f.lten (group D) to modify the lettiallactionoficagaret¢e smoke on al- rtolar macrophages. In profound contrast, the imeregr•ataon of DPPD and glutatRlone anuoscdants rr•to a cellulose acetate fsltcr results in complete inha,tton of the !cthal efT:ets of cigarette smoke extract (group C)'. The protective effect of the L1PPD giut.th;one fi,ter was due principally to the DPPD• snce glutsthione dtd not cicrt ar•y protective aca.rtv on r,•zrntatnong alscolar macrophage v:ablhty in the presence of aqueous smokc extracts. These fsndmas are presentod in the following table. As can be ob- served. the tncorporation of glutathlnne with DPPD resulted ES Cigarctus- The aqueous smoke estracls were prcpared and added in, 70 the amount of I ml to incubation f)asks contarntng 1'.2 x IO' al.ei macrophaje cells isolated from ratlungs. ln:uhattons trere carrlcd out at 37' C for 0. 00 snd' ! 20 mtnutcs And'cell viability detcrmtned at each pcrlod by means of the trypu. 715 Slueescluslon test. The follow,ng data •ere obutnedi,.

5 the smoke exposed pulntunary - C4 af - 6 •d in 10 n)illtliters of KRP solution arid its r tn enhancemenl tn~survtval of maerophagc% This suggests a syncrgisuc action between, the phos- horescene acttvtty determtned prior to, and after, the lipn).olufile and water snluble anttnudant on maintaining pul• addr an of, 1' ml of ml peroxide Thc hydrogen peroxide . as munary niacruphage vrabtltty The UPPD fdter was found to added to I,nt of solution prepared frcm unfiltcred c;garcttc Ae cqually cffecuvc d11ie DPPD was suspcnded'etther tn v.ater 5 smoke, antioxidant ftltercd. commerclal etiarcoal filtcred, and r„ tmpregnate the Gller or dtssolved in etliyl'alcohol prtor to a comtirnauon of charcoal plus Fiutath,nnrand DPPU'anoox,• fillet tmpregnatron d.nt mnture and counted one minute later n.v rneams of a liquid sctnttllatton system The adournn of hvdrngon peroxide INFLU'F:NCE OF DPPD AND'GLI:TATHIONE. ALONE OR resulls tn tmmedtate emtsaton of c!ectromagneue radtarton, nr ( ON)OiNTLY tIN ALVEOLAR MACROPFIAGE 10 light, from as ycu an untdcntified compound present, in the VIABILIT Y1N THE PRESENCE OF AQVEOI.'S SMOKE smoke e3tract. EXTRACT These studies, and those conducted w,th toluene trappsng CetLNiebr6ty, 4 Smoke Incubtnon:Trme FUter Extract 0.Mrn. eO~Mm.. . 0 •S 77 + II Cetlulo.e Acetate + DPQD- + 1M 33 DPPD•• + )4 DPPD . Glutatarunt"' • 31'. 111.11tMont ~ 9 •- DrrD t7W.rt Vy.r.a r...r.r....pa.ue..ro ~n.pr.l.u. nlrtr •a - DrP.D 1 I00n// J~rwt+et ~n tl/rt llcenal la~rn pr.in.ra.fller u. -. D rPD r a0. a i.ea rt.r.r..en.e.t )o.m / I.• u u.~ ro iw.~retn.r. nuv. EXAMPLE S Evaluation ofthe Abditr of Antiostdants. Added Direcaly to the TrapVing Solution. to Modify Cellular Toxicity of Citarette Smoke Since antioudants sveae protecttve when incorporated into ftlttrs,stuJtes were undertakcn to dctermtne d:the adtauon of antiondints d rectl} into the smoke trapptng solution would modify the lethal effects of cigarette smoke extracts. To this end„glutathtone anJ/on DPPD wrre +ddetl tn the amount of 100!mg/10 ml of the Kiebs-Rtnger phosphate trapping solu- lion and I ml of thc resulting smoke extract obtained from un- filtered cigarettes was added to cach fl~sk ><hicli eontatned ap- solutions, indicate that unknown phosphorescent substances IS are present in cigarette zmoke extracts ><hrch produce photons. The excited molecular spectes• as sec untdcnttfted'. prod'uct light quanta which can~be detected by a phototube of a hquid~ scintillation counur,g system The cnergy, of the excited molecular species is appreciably bclo~ that'of Trntt- 20 um, i.e., less than 0.018 K1EV It can be noted in the table below that the Krebs-Ringer. Phosphate buffeo had background: actr.ttv t I0-.Ocounts per minute). Simtlarly in aqueous soluttons„ like Krebs•Rmgcr Phosphate medium, the snto'rr soluuons prepared from the 25 various fiiters wcre com;arable Howeser., s.hcn hadrogen peroxide was added to aarlou3 Y:RP~smoke solt,ttons- an ap- proximate twentyfold increase occurred in the photon produc- ing activity of,the sample Thts contrasted markedli+ to but a fourfold increase when DPPD,plus,elutathtone -as emplo,ed 30 in the filter tn prepare the KRP smoke solutton Commercial eharcoal-eellulose acetate ftitcr ~as effec!ive in reducing tne increase in photoacttsat+on wnen compared to Ihe unliitered cagarette; howcvcr: theaztt.t!•..`as%tee that nbscr.td , hen the chemical antioxidant filter .,ascmploved Emp,lbatmg the act,seted canbon plus anttottdantl felter„ pre;ared as' pre.t- ousl'y descrt'edc complbee suppression of ltght energ~ tnom the aqueous soluttonir as obtamed. 35 .tqw-on+ emnl. ao Cour.r+ v~: t trttr~~ttr x,,, FI;u1Filter .:ctranr unnut :t` t:~ proximately 1.2 x 10' alveolar macropt~ages. A.. ... Nntre.-- .. 11 . _...., \nnr........... ............. = 45 C _....... U.I`1'U~ni~~I CIuUtMnn::•--'- -- +rnnl.• It..•t tirvntro.:ruti.f .r~~wralJh~~t .~ert.~+t. t.:.t. rtnr+ I t' A It. .... . . it ttrrlttut.ctri tltr, ._ + l:al.nrnrnt.. Ttutr i,wlnatc.. (n W. O.ara..b -. 1l : n;ltt' ... ..•. t.a r:,7 U_._....CI~.u,onL._ . . . . . - E...._..CGarnn:uV,t~:llrP..U:n~~~t~~ - 50 n.. ....._.._... ... . . .. _...- :a..1 a:,n ......, ... . .... ....... ..... + ........ tt.t'......, v.... ... f)t`t`le....... ,.. + ... ..-. ~..r..... tt.... . . ututawtn~~ ._ + t+u .... u._...... lrrrlr rw+elrn urr~n+•,._ + ........ s.r ...... 55 A B In agrecment' with our previous obserratton• aqueous cx- through 20 ml!of toluene contatntng ?.5~dtphenylolat,:;e and tracts of eigarette smoke were toxic to alrtolar macrophages p•bis-(2-5-phenyfoxazolyl/'-bcnzene to enhance detection of (Flask B and E vs. Flask A and D. respectr.cly). In conuast:to light energy emitted by the sample unuer study w hen samples manifested~cytotoxictty. the addition .,f the ..ater soluble an- 60 were prepared and counted one mtnute follb~ tng completion tioxidant. llutathtone, and the lipid soluble antioxtdant DPPD of prcparattont the photoacttstty .:f thc to!uene ram?le `% as tn. lu the smoke trapping solutrun markedly reduced the lethality creased approximately one tliousanufolil u hcn smoke from~an Df cigarette smoke (,Ffrsks C and H) Gtutattitone byttsetf dtd unfiltered ctgarette was used tsamplc B ss. sample A 1 Thts nut appear tu exert %rgntfic+nt protecarse eRect 6S was stgnrftcantly decreascd'6g pen :ent w hen tRe D'PPD-glu These stuJtes eliminate the pusstbtltty of a non-speetfie tathtontcantrotidant Gltcr -as emplu%•d.. mechanism of protection uf anuo.rd'onts„and'tndicatc that the addtttonof DPPD. or DPPD and glutathtone to the KRP medr- rm ir, vitro. wdl modify the touc effects cf ngarette smoke on atreolat macrophages. 70 Sampie EXAMPLE h~ AaltwuJant. Jnd Phntn anr.atrun S'tudtcs. 1n a furlhcr eflivt tu evaluatt the rule nf DPPD antwxidant tn~reducsng alve+dar macruphage injury following exposure to csgaretlc smoke ertrac,t and tu evaluate the concept of free 11 radical generatton by ctgarette smoke• cigarette smoke wu . .1) ta :~ li SL -t IVI Footnote to above table One ml ofiaqueous extract was placed in scintillation stul s.ithout phosphorous and counted \o photoactrstts was manifestcd~ tnuu)ly in all samples 'v1'hen H.Oy A as addeil.. photoacttvation occurred to ~arving degrees m the aquecvs smoke eitracv solution and' was dependent cpon tilter cm• ploycd. .ldditional studies wcre conducted where smoke w as passed rIlrrirttnnnv: Acirutv in Filter Sn'ott <our.UrMmutt. C CellYutow Ac-urt D DP?D-Glutatnnsnc a0 I?,77}

EXAMPLE 7 Influence of DPPD Antlosldant F+Iters on Lipid'Soluble An- tiostdant Activtty, of Plasma and Other Tissues of Smokc Ea- poscd Rats Studies .ere also conducted to determtne if DPPD antlosi- Jatnl filters could modify certain sperlfic blochemrcallevcnts rwctarcJ viIlh ugarette smoke exposure. Plasma and trssue IiprJ sulublt anlurrrdant .ctraty ras duermtned in normal; untrcatcJ cnnuul rata, and in rats cspascd to smokc (rum five unfiltered clgarcrtcs In,addltlon, groups of rats were also cs• posed lu •mnke from five regular filter Icellhlose acelate) cigarettes and to smoke derived from five anuoadant fdten ( FIG. 1 ) cigarettes Values belbw arc listed as means - stan. dard error, of inean, and are derived from, groups of, rats number from 6 to 13 Values of DPPD lipid anhosld'anu are ecpressed as mtcroequi.alents of anuosldant permtlhliter of plasma orlpcr Iram oGussue. DPPD An:lotld.nr Activity, GrDNP Pbtma uEqJml Lunj uEq!g Llver uEq/t 002 It 1411 i .11.V. Control Kltal_ -Ilill'A n.A Cr{.rttnc Srnose 0.0110 0 126 0.343 rUtiGU<red-I e0002 _01O11 =0A31 ('qut11e tm.,1'e 0009e 0 102 0 253 t1l.fditrvJ-~I _UoUT _0026' z00II7 r iancllt 1mla rrUt1V2 0 1a0 03F0 rltva.ln.(dltr _UOULII _0.0t97. :00l1 I ta.ralt ~m..1e 4 AnmeHAnt hdltrl 0 01 ao~ IU0U2 0173 a0 160 0337 e0:023 lioai! •rtt,filtcred clgarettcs had plasma antrosrdant lt;vcls that were •pprorlmately 100 per cent hlgh'en than that obscrved whcn legular filtered or nor.filtered cigarettes -ere employed Thus„the Jepl'cuon of plasmahptd solLble anllondant acuwny which follows cigarette smoke e:posure is stgnlf.cantlr in. hibitcd when DPPD antlotldants are incorporated dlrectly, Into the cigarette fiiter. ihus- by my Inventlon, I ha.e devised a novellfdtcr comprls- ing the lipid soluble anuoudant DPPD wMchcan hc rcadlly 5 adapted to reduce the totlc:ty,of cigarette smoke This cheml- eal antioudant addittve can readily be incorporated by Im- pregnauon lnto conventucnal lype f iters nr as inserts lnto con- vtntlOnal filten. The resulting cilgarette, yhtch has been smok'ed'by'the tnvestlgatorand othen• is a mlltl', pleasant smoke without appreciable loss of7la~oror aroma. 10 15 I claim: 1. A tobacco smoke filter comprising a cylindrical contarner having a cross sectlonal dimension conforming to that of a tob'acco smoke passage and defining a space having an inlet and outlet, said space being flltd with a smoke permeable eomposltion, comprising ti',ti'-dtphenyllp-phenylenedlarnlne in an emount from about 0'D25 to 0.500 grams to reduce the toslc efitct of tobacco smoke passing through the container on alveolar macrophages, the amount used r,ot being toztc to the smoker. 2 A tobacco smoke f ltcr according to claim I In ~ hlc(i the N;N'-dtpAenyl-p-phenylencdlamme is nnsed - Ith'iglutathlonc 3 A,tobaceo smoke filtecaecording to claim I , hcreln the N',N'-diphtnyl.p-phen~lenedlamine Is Impregnated Intu , ;el- 20 25 30 lulnslc smoke liltertng ntaterlal; 1'. A tobacco snwke filter according tu aamt I uhcrcln the N,N'•diphenyl-p-phenylenedlamine is Incorparatcd rlth .tc- tivated charcoal. S. A tobaccoamoke filter ,tccording to .lalm :+herctn the lhese studlcs denote that esposure of rats to alternating en- 35 mixture is impregnated into a eelluiose acctate filtering .irnnmenu nfcrgarette smoke and air. by mcans of a partial matorul. vacuum to drav. in alurnaung amounts of air and smoke into 6 A tobacco smoke filter adn?ted to remove undeslrabte an,enclosed chamber in whleh the rats W ere housed, mJuets a components from tobacco smoke, sa~d filter hastng a cro.,s• profo nd (all'/n plasma lipid soluble annoudant actwltv. The sectional dimension conforming to that of a tobacco smoke decrctse is observed alth as Ilttlb as one cigarette A regular 40 passage and comprising a carner material and an amount of eommerclallj avallabk cellulose acetate fi!ter does not modify from S per cent,to '0per cent by W ught based on the -clg,tit Ihe 72 per cent decrease in plasma hpld antiotldanes How- of said' carrier of \',\"-dlphcncl-p-phcnj}cncdlamise to ever. the decrease observed r• Aen the DPPD lipid soluble an, reduce the tozlc effect of tobacco smoke passing through said tiosidant filter -as employcd was bur 44 per cent In other Pusage, the amounlused notbctng,toancto the smoker .ordi, the animals exposed to smoke obtained itom DPPD an- <5 • • t • • 50 55 60 63 70 15

APPENDIX D IN' VITRO BI0.4SSAY SYSTEMS

-D1- DOES TOBACCO SMOKE IMPAIR ALVEOLAR MACROPHAGE FUNCTION?: G.L. Huber and J. Shea. Harvard Medical School, Boston, Massachusetts. The question of the health effects of cigarette consumption, shrouded in controversy and'partial truths since its inception, deserves definitive clarification. In an attempt to establishAirect causal relationships between tobacco and lung diseases, the in vitro effect of smoke on alveolar macrophages' (AM) bactericidal function has been extensively studied by many investigators. Publications on AM function from human smokers and from experimental animals, however, have been paradoxically contradictory, with resul~ts ranging from totally impaired AM function to no impairment or even enhance&function reported. To investigate this problem, studies were performe&comparing in vitro exposure of AM to tobacco smoke versus in vitro function after in vivo exposure. Rat AM were harvested from nonsmoke&animalis by pulmonary lavage and i-i vitro bactericidal inactivation of S. albus quantified. Control AM inactivated 74.8% of the bacteria after 3 hourv of incubation. Exposure in vitro to 2, 4, 6 and 8 ml of whole smoke impaired bacterial inactivation to 50.4, 37.4, 22.6 and 17.7%, respectively, with differential filtration revealing that the AMicytotoxin was a highly water-soluble component of the gas-phase of the smoke. However, AM harvested from animals acutely exposed inivivo to progressively increasing doses of tobacco smoke had no impairment on in vitro bactericidal activity, even when the in vivo tobacco exposures were at a near lethal level. Analysis of tobacco gas-phase components indicated that because of their very high water solubility, the AM cytotoxins reach an unrealistically high concentration when added to in vitro systems. Conversely, data generated from lung analog analyses indicated that when tobacco~smoke is inhaled the wet airways of man selectively remove these cytotoxiins and reduce the concentrations of these gas-phase components at the alveolus to levels not detectably toxic to AM. Thus, the adding of whole tobacco smoke or smoke components to AM in in vitro bactericidal systems does not provide a realistic bioassay. The seliective absorption of water soluble cytotoxins in these systems to concentrations five logs or higher than in the lungs of man may provide an explanation of these paradoxic results reported to date.

- D2 - CLEARANCE OF TOBACCO SMOKE GAS-PHASE ALVEOLAR MACROPHAGE CYTOTOXINS IN A MODEL AIRWAY SYSTEM. J. Shea, R. Weker, 0. Grubner, M. First, G. Sornberger, D. Drath and G. Huber. Harvard MedicaL School and Harvard School of Public Health, Boston, Massachusetts. Several researchers have demonstrated that exposure to water soluble gas-phase components of cigarette smoke in viltro impairs the bactericidal function of alveolar macrophages (AM). Some investigators have implied that the effects of these gas-phase cytotoxins on cultured cells may be similar to the effects of inhaled whole smoke on the Win the lung. Since inhaled smoke must traverse the airway before reaching alveolar cells, we have quantified the removal of gas-phase components passed over a moist surface comparable in area to human airways. Our model airway system consists of a gas-phase smoke generator and tubes lined with paper moistened with balanced salts solution. Control AM cultures inactivated 77.2% of a S. albus challenge in 3 hours, whereas exposure to a fractional puff of gas-phase tobacco smoke completely paralyzed AM phagocytosis (0% inactivation). AM exposed to the gas-phase of tobacco smoke passed through sequential quarterly lengths of the model airway inactivated 42.7%, 66.4% and 73.4% of the bacterial challenge, indicating progressive removal of AM cytotoxins by the wet surfaces. Concurrent chromatographic analyses of individual components of gas-phase tobacco smoke demonstrated progressive removal of several potential cytotoxins, including acrolein and acetaldehyde, along the moist airways. The results of these experiments suggest that the AM toxicity of gas-phase tobacco smoke, demonstrable only in in vitro systems, might be substantially reduced in~the airways prior to reaching the alveolar spaces and might explain the normaL inivitro bactericidal function of AM from human smokers and smoke-treated animals. J

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