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\ ~. ALTERATIONS IN LUNG PARENCHYMA FOLLO!JING EXPERIMENTAL CHRONIC INHALATION OF TOBACCO SMOKE AMERICAN COLLEGE OF CHEST PHYSICIANS LAS VEGAS, NEVADA NOVEMBER 1, 1977 - 4:15 P,M, MGM GRAND HOTEL (GARY L, HUBER) THE FREQUENCY OF DEATH AND THE PREVALENCE OF MORBIDITY ATTRIBUTED TO~DISEASES OF CHRONIC OBSTRUCTIVE AlRFLOW'HAVE REACHED ALMOST EPIDEMIC IMPORTANCE IN THIS COUNTRY (1). A NUMBER OF EPIDEMIOLOGIC STUDIES HAVE 1MPLICATED TOBACCO CIGARETTE CONSUMPTION AS A POTENTIAL, BUT NOT EXCLUSIVE, PATHOGEN,IC FACTOR IN THE DEVELOPMENT OF TWO COMMONIOBSTRUCTIVf AIRFLOW' DISEASES, CHRONIC BRONCHITIS"AND EMPHYSEMA (2-8). NEGLIGIBLE QUANTITATIVE EVIDENCE, HOWEVER, HAS EMERGED FROM ANIMAL STUDIES TO LINK EXPERIMENTAL TOBACCO SMOKE INHALATION DIRECTLY TO PARENCHYMAL ALTERATIONS IN THE LUNG (9). As THURLBECK HAS SUMMARIZED, LACK OF RELIABLE DATA DEMONSTRATING TOBACCO- INDUCED EMPHYSEMA OR BRONCHITIS IN LABORATORY ANIMALS MAY WELL BE DUE TO THE DIFFICULTY OF PRODUCING A PROPER ANALOG TO~HUMAN SMOKING UNDER EXPERIMENTAL CONDITIONS (9). THE PURPOSE OF THIS COMMUNICATION IS TO REPORT THE DEVELOPMENT OF A HIGHLY QUANTITATIVE MORPHOLOGIC BIOASSAY FOR EVALUATING ALTERATIONS IN THE LUNG PARENCHYMA FOLLOWING CHR'ONIC EXPERIMENTAL EXPOSURE ~ TO TOBACCO SMOKE UNDER PHYSIOLOGIC CONDITION'S, COULD WE HAVE ~D THE FIRST SLIDE, PLEASE? ~

SLIDE 1 OUT OF A POOL OF 300 CHRONICALLY SMOKE-EXPOSED MALE RATS OF THE CD STRAIN, AND THEIR MATCHED CONTROLS, 1&SMOKE- EXPOSED ANIMALS AND 16 SHELF-CONTROLS WERE SELECTED ON A RANDOM BASIS. THE LUNGS IN EACH GROUP OF ANIMALS WERE INSUFFLATED WITH 2.3% GLUTARALDEHYDE IN 0.045 MOLAR CACODYLATE BUFFER AT PH 7,2 TO A CONSTANT INFLATION PRESSURE OF 25 CENTIMETERS OF WATER. ANIMAL BODY AND ORGAN WEIGHTS WERE RECORDED, LUNG VOLUMES FROM EACH ANIMAL, FIXED AT THE SAME INFLATING PRESSURE, WERE MEASURED BY A VOLUME DISPLACEMENT TECHNIQUE. FOLLOWING POST-FIXATION IN BUFFERED OSMIUM, MORPHOMETRIC EVALUATIONS, . . . . . USING THE PREVIOUSLY PUBLISHED TECHNIQUES OF WEIBEL AND OTHERS (10-30), WERE UNDERTAKEN TO DETERMINE THE ALVEOLAR PARENCHYMAL DENSITY AND THE ALVEOLAR SURFACE EPITH!ELIAL DENSITY, AS WELL AS THE RELATIVE PROPORTION OF ALVEO,LAR AIR SPACE IN EACH GROUP OF ANIMALS, A TOPOGRAPHIC ANALYSIS WAS ALSO UNDERTAKEN TO DETERMINE THE NUMERICAL DENSITY AND CHARACTER OF TYPE I AND TYPE II ALVEOLAR EPITHELIAL LINING CELLS. COULD WE HAVE THE NEXT SLIDE, PLEASE?

SLIDE 2 SHOWN IN THIS SLIDE IS A PHOTOGRAPH OF THE 30 PORT AUTOMATED SMOKING MACHINE USED IN ALL STUDlES, THE RATS WERE EXPOSED IN GROUPS OF 50 TO TOBACCO SMOKE. NON-EXPOSED, MATCHED SHELF CONTROLS WERE MAINTAINED UNDER THE SAME ENVIRONMENTAL CONDITIONS. APPROPRIATELY CONDITIONED CIGARETTES WERE SMOKED CONSECUTIVELY WITH A REVERSED~PRESSURE, SQUARE-WAVE PUFF PROFILE OF TWO SECONDS DURATION. FRESHLY GENERATED SMOKE WAS STABILIZED BY IMMEDIATE DILUTION WITH NINE PARTS OF ROOM AIR AND DELIVERED TO EXPERIMENTAL ANIMALS IN AN INTERMITTENT STREAM OF 20 SECONDS OF SMOKE ALTERNATING WITH 40 SECONDS OF FRESH AIR. THE DILUTED FRESH SMOKE WAS CHANNELED THROUGH A SEALED PASSAGE INTO WHICH PROJECTED THE SNOUTS OF LIGHTLY RESTRAINED ANIMALS, WITH A DELIVERY OF TIME OF LESS THAN FOUR SECONDS BETWEEN SMOKE GENERATION AND INHALATION. COULD WE HAVE THE NEXT SLIDE, PLEASE?

!` 'SLIDE 3 SHOWN IN THIS SLIDE IS A SCHEMATIC SUMMARY OF STUDIES ON TOBACCO~SMOKE DOSIMETRY, KENTUCKY ZR1 REFERENCE RESEARCH CIGARETTES WERE USED IN ALL SMOKE EXPOSURES. EACH CIGARETTE GENERATED TEN 35 MILLIMETER PUFFS UNDER CONDITIONS RECOMMENDED FOR EXPERIMENTAL ANIMAL INHALATION BY THE HUNTER COMMITTEE. THE ZRI CIGARETTE HAS A VERY HIGH TAR CONTENT, WITH 26.8 MILLIGRAMS OF TOTAL PARTICULATE MATTER GENERATED .PER CIGARETTE UNDER OUR SMOKING CONDITIONS. IN SELECTED EXPERIMENTS, CIGARETTES WERE LACED WITH DECACHLOROBIPHENYL, OR DCBP, A NON-RADIOACTIVE CHLORINATED HYDROCARBON TOBACCO SMOKE TRACER. THIS RESULTED IN AN AVERAGE OF 12,4 MICROGRAMS OF DCBP PER MILLIGRAM OF TOTAL PARTICULATE MATTER IN'THE FRESH SMOKE GENERATED. TOBACCO SMOKE DOSIMETRY WAS EVALUATED IN INDIVIDUAL AN'IMALS BY DETERMINING THE AMOUNT OF SMOKE RETENTION. WITH EACH EXPOSURE TO TOBACCO, THERE WAS, UNDER OUR EXPERIMENTAL CONDITIONS, APPROXIMATELY 516 MICROGRAMS OF TOTAL PARTICULATE MATTER PER GRAM OF LUNG RETAINED BY THE ANIMALS. CARBOXYHEMOGLOBIN, AS AN INDICATOR OF GAS PHASE EXPOSURE, WAS ON THE AVERAGE 16.6% IN THE SMOKE EXPOSED ANIMALS, ALL ANIMALS WERE EXPOSED THREE TIMES PER DAY FOR 180 CONSECUTIVE DAYS. ON THE BASIS OF THIS INFORMATION AND RELATIVE BODY WEIGHTS, AS WELL AS ADDITIONAL DATA, WE WOULD ESTIMATE THAT THE DOSE DELIVERED1WAS EQUIVALENT TO APPROXIMATELY ,. ONE AND ONE-HALF PACKS OF UNFILTERED CIGARETTES PER DAY IN MAN. COULD WE HAVE THE NEXT SLIDE, PLEASE?

SLIDE 4 SHOWN HERE IS A SUMMARY OF OUR ANIMAL SELECTION AND TISSUE ANALYSIS PROCESS,. THE SIXTEEN EXPERIMENTAL AND CONTROL ANIMALS WERE SELECTED ON A RANDOM,BASIS FROM MUCH LARGER REPRESENTATIVE GROUPS. THE OTHER AN'IMALS INVOLVED IN THESE STUDIES WERE USED FOR INVESTIGATIONS ON ALVEOLAR MACROPHAGE FUNCTION, STRUCTURE AND BIOCHEMISTRY, AND FOR STUDIES ON AIRWAY MORPHOLOGY AND GLAND SECRETION, OF THE SIXTEEN INSUFFLATED LUNGS IN EACH GROUP, TEN LUNGS WERE SELECTED ON A RANDOM BASIS FOR FURTHER STUDY. THE FIXED LUNGS WERE DICED INTO CUBES APPROXIMATELY 3 X 3 MILLIMETERS IN SIZE. FIVE BLOCKS FROM EACH LUNG WERE SELECTED FOR SECTIONING, AGAIN ON A RANDOM BASIS. USING A WILD AUTOMATED STAGE SAMPLING LIGHT MICROSCOPE, FITTED WITH A PROJECTION HEAD AND A WEIBEL COHERENT MULTIPURPOSE LATTICE, AN AVERAGE OF FIVE FIELDS AT A MAGNIFICATION OF 725 POWER FROM EACH SECTION WAS SELECTED1BY SYSTEMATIC SAMPLING FOR MORPHOMETRIC MEASUREMENTS. POINT AND INTERSECTION COUNTS WERE MADE ON 250 LIGHT MICROSCOPIC FIELDS OF LUNG FROM SMOKE EXPOSED ANIMALS AND ON 2501FIELDS OF LUNGS FROM CONTROLS TO DETERMINE THE VOLUME DENSITY OF THE ALVEOLAR AIR SPACE COMPONENT IN THE LUNG PARENCHYMA, THE VOLUME DENSITY OF THE TISSUE COMPONENT IN THE LUNG PAR'ENCHYMA AND THE SURFACE DENSITY OF ALVEOLAR. EPITHELIUM. COULD WE HAVE THE NEXT SLIDE, PLEASE?

SLIDE 5 SHOWN IN THIS SLIDE ARE THE ABSOLUTE BODY WEIGHTS AND ORGAN MEASUREMENTS. CONTROL ANIMALS WEIGHED 565 GRAMS AND EXPERIMENTAL ANIMALS WEIGHED 335 GRAMS. THE BODY LENGTH IN CONTROLS WAS 23.3 CENTIMETERS, AND 21.2 CENTIMETERS IN SMOKE EXPOSED ANIMALS. THE LUNG VOLUMES, AT AN INFLATION PRESSURE OF 25 CENTIMETERS OF WATER, WERE ESSENTIALLY IDENTICAL IN THE TWO GROUPS, WITH 11.7 MILLILITERS VOLUME IN CONTROL LUNGS AND 11.9 MILLILITERS VOLUME IN SMOKE-EXPOSED LUNGS. THE WEIGHT OF THE HEART, LIVER, KIDNEYS, SPLEEN AND TESTES WERE ALL SIGNIFICANTLY SMALLER IN THE SMOKE EXPOSED ANIMALS. THE ADRENAL GLANDS WERE LARGER, HOWEVfR, IN THE SMOKE- TREATED ANIMALS. COULD WE HAVE THE NEXT SLIDE, PLEASE? SLIDE 6 SHOWN IN THIS SLIDE ARE THE RELATIVE LUNG VOLUMES, CORRECTED FOR DIFFERENCES IN BODY SIZE. LUNG VOLUMES, WHEN EXPRESSED IN PROPORTION TO BODY WEIGHT, WERE 71% LARGER IN THE SMOKE-EXPOSED ANIMALS, WHEN FIXED~UNDER A CONSTANT INFLATION PRESSURE. LUNG VOLUMES WERE ALSO LARGER, WHEN EXPRESSED IN PROPORTION TO BODY LENGTH, WITH~THE DIFFERENCES IN THE TWO METHODS PRESUMABLY DUE TO A SIGNIFICANT LEANER BODY MASS, BUT ONLY A SMALL REDUCTION IN BODY LENGTH, IN SMOKE-TREATED ANIMALS. COULDiWE HAVE THE NEXT SLIDE, PLEASE?

SLIDE 7 • SHOWN IN THIS SLIDE ARE THE RESULTS OF MORPHOMETRIC MEASUREMENTS OF THE LUNG. BLOOD VESSELS AND AIRWAYS WERE EXCLUDED FROM ALL ANALYSES, AND THE DATA PRESENTED REPRESENT THE ALTERATIONS IN THE LUNG TISSUE DISTAL TO THE TERMINAL AIRWAYS. THERE WAS A 21.7% REDUCTION IN THE VOLUME DENSITY OF THE TISSUE COMPONENT OF LUNG PARENCHYMA IN THE SMOKE-EXPOSED ANIMALS WITH A COMPARABLE INCREASE IN THE VOLUME DENSITY OF THE ALVEOLAR AIR SPACE COMPONENT OF THE LUNG PARENCHYMA. COULD WE HAVE THE NEXT SLIDE, PLEASE? SIL DE 8 SHOWN IN THIS SLIDE ARE THE MEASUREMENTS OF PULMONARY ALVEOLAR SURFACE DENSITY, THE ALVEOLAR SURFACE DENSITY IS EXPRESSED IN'SQUARE MICROMETERS OF ALVEOLAR SURFACE AREA PER CUBIC MICROMETER OF LUNG ANALYZED, THE REDUCTIONS IN ALVEOLAR PARENCHYMA PRESENTED IN THE PREVIOUS SLIDE WERE ACCOMPANIED BY A IZ% DECREASE OR REDUCTION IN THE ALVEOLAR SURFACE OF THE SMOKE-EXPOSED ANIMALS, INTERESTINGLY, WHEN SURFACE AREA WAS EXPRESSfD~RELATIVE TO THE VOLUME OF PARENCHYMAL TISSUE, THERE WAS AN llo MEAN INCREASE IN SURFACE AREA PER CUBIC MICROMETER OF PARENCHYMAL TISSUE IN THE SMOKE-EXPOSED ANIMALS. WE MAY POSTULATE THAT THIS MAY BE BROUGHT ABOUT BY A MORE IRREGULAR EPITHELIAL SURFACE LINING THE AIR SPACES OR SOME OTHER CHANGE IN SHAPE IN THE SMOKE-EXPOSED ANIMALS. COULD WE HAVE THE NEXT SLIDE, PLEASE?

SLIDE 9 SHOWN ON THIS SLIDE ARE THE RESULTS IN THE INDIVIDUAL LUNGS FOLLOWING EXPERIMENTAL EXPOSURE TO TOBACCO SMOKE, WITH THE VALUES OF THE VOLUME DENSITIES OF THE TISSUE COMPONENT OF THE LUNG PARENCHYMA PLOTTED ON THE SCATTERGRAM FOR CONTROL AND SMOKE-EXPOSED ANIMALS. IT IS NOT POSSIBLE TO DETERMINE FROM THE NUMBER OF ANIMALS EVALUATED IN THIS STUDY WHETHER OR NOT EACH SMOKE-EXPOSED ANIMAL WAS EQUALLY SUSCEPTIBLE TO THE EFFECTS OF TOBACCO INHALATION. STUDIES WITH CONSIDERABLY LARGER POPULATIONS AND AUTOMATED IMAGE ANALYSIS WILL BE REQUIRED TO DETERMINE THE RELATIVE RISK OF INDIVIDUAL SUSCEPTIBILITY UNDER THESE EXPERIMENTAL CONDITIONS. COULD WE HAVE. THE NEXT SLIDE, PLEASE? $ ID TOPOGRAPHICAL ANALYSES OF EPITHELIUM LINING THE ALVEOLAR SURFACES IN BOTH GROUPS OF EXPERIMENTAL AN'IMALS INDICATED THAT THERE WAS A SIGNIFICANT DECREASE IN THE NUMERICAL DENSITY OF TYPE I PNEUMOCYTES IN THE SMOKE-EXPOSED:AN'IMALS, WITH AN AVERAGE REDUCTION FROM 35 To 23 TIMES 106 TYPE I CELLS PER CUBIC CENTIMETER OF LUNG. ON THE AVERAGE, THERE WAS A COMPARABLE REDUCTION IN TYPE II PNEUMOCYTES, WITH A SLIGHT TREND TOWARDS A HIGER TYPE II TO TYPE I EPITHELIAL CELL RATIO IN INDIVIDUAL LUNGS WITH HIGH SURFACE DENSITIES. ELECTRON MICROSCOPIC ANALYSES OF THE TYPE ~I PNEUMOCYTES REVEALED A . REDUCTION IN THE VOLUME PROPORTION OF LAMELLATED BODIES IN SMOKE- TREATED ANIMALS, RELATIVE TO CONTROLS, CONSIDERABLE ADDITIONAL WORK W I LL HAVE TO BE UNDERTAKEN, US I NG A VAR I ETY OF L I GHT M03'7J~7 AND ULTRASTRUCTURAL QUANTITATIVE TECHNIQUES, TO DETERMINE THE EXPLANATIONS AT THE CELLULAR LEVEL FOR THE VARIATIONS IN LUNG PARENCHYMA NOTED BETWEEN,THE SMOKE-EXPOSED AND CONTROL ANIMALS. COULD WE HAVE THE NEXT SLIDE, PLEASE?

SLIDE 11 IN SUMMARY, WE HAVE PRESENTED THE RESULTS OF A HIGHLY QUANTITATIVE MORPHOLOGIC BIOASSAY FOR EVALUATING ALTERATIONS IN THE LUNG PARENCHYMA FOLLOWING CHRONIC EXPERIMENTAL EXPOSURE TO TOBACCO SMOKE UNDER PHYSIOLOGIC CONDITIONS. THE ANALOGS OF THIS MODEL TO HUMAN DISEASE MUST BE CONSIDERED. FOR EXAMPLE, EMPHYSEMA IS A CONDITION'OF THE LUNG CHARACTERIZED BY ABNORMAL, PERMANENT ENLARGEMENT OF THE AIR SPACES DISTAL TO THE TERMINAL BRONCHIOLES ACCOMPANIED BY DESTRUCTION OF THEIR WALLS (9), OUR EXPERIMENTAL ANIMALS HAD, IN LUNGS FIXED AT CONSTANT INSUFFLATION PRESSURES, A SIGNIFICANT ENLARGEMENT IN THE VOLUME DENSITY OF THE AIR SPACES OF TOBACCO-EXPOSED LUNGS DISTAL TO THE TERMINAL BRONCHIOLES, WITH A 21 PERCENT REDUCTION OR DESTRUCTION OF THE ALVEOLAR WALLS AND A 12 PERCENT DECREASE IN THE ALVEOLAR SURFACE AREA. THESE STUDIES IN EXPERIMENTAL RODENTS CANNOT BE EXTRAPOLATED DIRECTLY TO THE INTERACTION OF TOBACCO CIGARETTE SMOKE IN THE LUNGS OF HUMANS, IT MAY BE, HOWEVER, THAT THE ALTERATIONS OBSERVED IN THE LUNGS OF OUR TOBACCO-TREATED ANIMALS, WHIlCH WERE EXPOSED FOR ONLY SIX MONTHS, REPRESENT SOME OF THE EARLIEST RESPONSES AND ALTERATIONS OF THE PULMONARY PARENCHYMA TO TOBACCO SMOKE, AND AS SUCH THIS MODEL MAY PROVIDE A QUANTITATIVE BIOASSAY FOR COMPARATIVE PRODUCT ANALYSIS. ADDITIONAL RESEARCH WILL BE NEEDED TO DETERMINE WHETHER OR NOT THESE ALTERATIONS ARE ANALAGOUS TO EMPHYSEMA, AND IN WHAT MANNER THEY ARE RELATED TO EXPERIMENTAL TOBACCO SMOKE INHALATION, CONSIDERABLE ADDITIONAL STUDIES, ESPECIALLY THOSE 03'748G88 EVALUATING THE EFFECT ON THE LUNG PARENCHYMA OF THE ALTERATIONS IN MACROPHAGE STRUCTURE AND METABOLISMiTHAT WE HAVE PREVIOUSLY DESCRIBED, WILL BE REQUIRED TO ANSWER THE NOW CRUCIALLY APPARENT QUESTIONS THAT THESE OBSERVATIONS RAISE, THANK YOU.

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