Document 4QBwN9EOBJNqqbOarqXv7Vmpj

^ THE ROLE AND ACTION OF ENVIRONMENTAL AGENTS IN THE PATHOGENESIS OF LUNG CANCER 1. Air Pollutants L.Paul Kotin, m.d., and Hans Falk, ph.d. /-VurMHZNsiYE epidemiological studies L iiave demonstrated a world-wide increase s> the frequency of and absolute mortality area lung cancer.***3 These investigations little question that a portion of the averred increase is both real and, at least mil very recently, progressive. An analysis of the data in the various studies reveals a num ber of consistent findings that appear to have vsnifiont eti logical implications. These com- factors include: (1) a greater frequency f lung ameer in men than in women;11* ** ~ a greater liability to the development of ;**njr ameer in urban residents than in rural w^idents^*1***0*** (3) an increased incidence iwioated with a history of prolonged exces* <ne cigarette smoking;**T*1S* ** and (4) a temwwal pattern of increasing frequency cora- ; with a possible etiological influence B .* influenza pandemic of 1917-1920.**43 considerations indicate the need for Judies aimed at: (1) establishing the exact cuure of and the significance of the various *w<tiations (eg., spurious, cause and effect, re.;; and (2) elucidating the biological raech"rrrts reponsible for and compatible with Ac epidemiological characteristics of lung can*rr- In all instances in which an association between a disease state and environmental uaots has been demonstrated, the isolation r*on tlw departments at Pathology and BiochemisTI. Lairernty of Southern California School of McdL oar and the Pathology Department, Lot Angela CT?"5T Hapital. Lo* Angeles. CaliL Th investigation ha been supported br * grant 'A156> from the Field Investigations and Demon- Brandi of the National Cancer Institute, of w Nanonai Innitutea of Health. Public Health *ad by a grant (LC-3) bum tbe Amcrion c?f Society. Inc. ,,** photographic work was done by Mr. Lloyd ^uiwifcy of tbe Department of Photography, Uni- f Southern California School of Medicine; Los Win. Cahf. .r'~are'PFLlUf._i_r_1t6. 1a4. ndis 1re7pAroad.nudceBd awritthrep*perrimntiesdsiofnromof TMCTI- *nd Fig, 15 is reproduced wiih permit- W>tooof;ACiramPollution Connttrrooll District, County of Los ~Aved lor publication April 28, 1958. Table 1 QUANTITATIVE ANALYSIS OF ATMOS PHERIC SAMPLES COLLECTED IN LOS ANGELES* Sample 1. mg.t Sample 2, rag.f Compound Per 1.000. Per 1.000, 000 000 Tot. cu. ft. Tot. eu. ft. Pyrene 3,4-Benzpyrene 1,12-Benzperylene 0.28 0.14 1.84 0.92 1.45 1.00 0.90 2.33 0.72 0.32 0.84 0.35 Data are reprinted from Canter 9:905,1956. fSempled from Aug. 1, 1952, to Oct. 15, 1952 (42 days of actual sampling). ^Sampled from Oct. 21, 1952, to June 1, 1953 (59 day* of actual sampling). and identification of specific etiological agents and the determination of their mode of action have been primarily laboratory undertakings. In the panorama of lung cancer, all available evidence points to a dominant etiological role for exogenous environmental agents.*1 On the valid assumption that bronchogenic cancer develops subsequent to the action of multiple factors, a broad experimental pro gram was undertaken for the purpose of evaluating the relative etiological significance of several of these factors. Atmospheric pollu tion, cigarette smoking, and viral agents were selected as the primary environmental condi tions to be investigated since they were pre eminently compatible with the epidemiologi cal pattern of lung cancer. Two cardinal facts were recognized at the onset. First, there are unequivocal instances of lung cancer in which one or several of these environmental agents appear to have played no role (e.g.t nonsmokers, residents in rural areas or in agri cultural economies, eta) and second, when jl the factors under study have been present, the etiological contribution of each presumably has varied from case to case. Nevertheless, one may properly postulate the existence of a pathogenetic milieu compatible with the ac tivity of several or all of the suspected en vironmental factors operating, at differenz- o 'r-\ Ll; inrjj c; C: cr C". Lu O gB 0020_ 422~7 -- " w So cannotbe authenticated 'PPG INDUSTRIES. INC. 2315 Cancer January'February 1959 CUfMUM* t!tM OTMZ rrxritHC aF:4 aa*w**r)^wwr 3-'* ar*tepr*rA*r. fa/ umfJ nuorm*/rH**/e \s.;4 n/arur ----*--- /O/ruram/rr/jr --.... atteuAi//vans*- I4n sjU. 3 X*. 4X*-- / taoi//r or omn/rnc srtoo -- 96 nou/*s or ///avmu smog- Fic. 1. Recovery of aromatic polycylic hydrocarbons after exposure to washed air and synthetic smog. intensities, in the development of lung cancer. Representative experimental findings in chem istry'. physio, and biology that are related to atmospheric pollution will be presented. Then the data will be utilized in formulating a concept of the pathogenesis of lung cancer that is consistent with the epidemiological characteristics of the disease. The role of cig arette smoking and viral infection will be presented in a subsequent article and will be integrated with the concepts being currently presented. The mechanism whereby these en vironmental agents working singly or in com ic bination may produce a neoplastic response M $ill be discussed in the final article of this & cf-5- Method & Soot, an experimentally and clinically recO ^ ognized carcinogen, is a major component of ^ Q Uj smoke emitted into the atmosphere incidental co *5 if3 to the use of solid, liquid, and gaseous ^ O' P "* fuels.* ** A product of the incomplete com0 fr- bustinn of most organic materials when they $&3g are heated or burned in the absence of an o, adequate supply of oxygen, soot's carcino" genic properties are presumably due to the presence in it of certain aromatic polycyclic hydrocarbons.40*41 Alth ugh quantitative data are lacking, the chemistry and method i formation of soot established its continuous presence in the atmosphere f r centuries. T>- ascribe an etiological role to soot and iu aromatic polycyclic hydrocarbons present iu the air, therefore, requires either the identi fication of significant new sources for thee compounds or the recent introduction ini the environment of potentiating or cofactors. An additional reason for considering the at mosphere in the pathogenesis of lung cancer may be the presence in the air of a new dass of carcinogenic agents capable f acting iu association with or independently of the tra ditional aromatic polycyclic hydrocarbon carcinogens. We, therefore, established as ini tial goals the quantitation of aromatic poly cyclic hydrocarbons in the atmosphere ami the simultaneous determinati n of their sources. Special interest was sh wn in axn- pounds recently introduced into our environ ment. The exhaust products of the internal combustion engine were chosen as the firu materials for quantitative studies. Widespread home and industrial use of petroleum pri- ucts, especially in gasoline and diesel engine*, is the hallmark of our industrial society. The entry of these products into our environment is temporally compatible with a possible-eu'"* logical role. _* :TK!" NOT COiVi V I Environment & Lung Cancer. I. Kotin i- Falk 149 Findings Table 3 "' ' CHEMICAL V broad spectrum of aromatic polycyclic AROMATIC HYDROCARBONS ESTIMATED IN I-MINUTE SAMPLES OF DIESEL EXHAUST, USING VARIOUS LOADS AND ENGINE REVOLUTION SPEEDS -i.iroorbons was found in the atmosphere in the exhaust products from both gaso- ;engines and diesel engines.1** ** " The -.vent carcinogen 3,4-benzpyrene was identimi and quantitated in all of them. It is our i<lid that 3,4-benzpyrene is best regarded as tar one of several possible carcinogenic pollu tants in the atmosphere. Rather than assum- it to be- the only or even necessarily the :tti significant carcinogen present, we let its prrvmcSserve as both a chemical index of aro matic polycyclic hydrocarbon contamination m the air and a biological index of cardnof-rtic potency. As antiripated, in an essen tial* liquid or gaseous fuel-consuming com munity, such as Los Angeles, 3,4-benzpyrene *av identified in concentrations lower than reported4 in ceruin other urban com- Load* Py- rent At 1,000 r.p.m. 0 137 i 267 \ 536 f- 1,800 1 2,500 At 1,200 r.p.m. 0 208 i 257 l 448 f 888 1 1,912 At 1,400 r.p.m. 0 188 i 177 4 220 } 734 1 822 pG./minutc of: Com pound X Ben*- py rene Benx- Anpery- thanlene threne 22 146 22 0. 76 465 42 43. 175 772 124 223. 640 1,320 610 472. 639 876 1,265 469. 0 9 79 4.3 0 47 40 24. 278 437 171 197. 488 432 930 320. 614 1,706 976 944. 0 80 0 20. 56 78 0 16. 76 1,372 368 69. 337 982 1,071 577. 346 1,687 944 666. unities (Table 1). A listing of the vehicular All with compression release. evhaust products, as shown in Tables 2 and 3, ^'abiishes thC presence of significant amounts *: hrnzpyrenc and allied hydrocarbons. the first possibility proved unsuccessful. An Comparison of the analytical findings from investigadon of the stability and survival of *r*'Vular exhausts with those from atmos- aromadc polycyclic hydrocarbons under vary ; studies revealed a reversal of the ratio ing atmospheric conditions was then under .nzprrene to pyrene. Benzpyrene was con- taken. Our data readily disdosed that the re *:rntly emitted from vehicular exhausts in versal in the benzpyrene to pyrene ratio was a t--.i[]er quantities than was pyrene. Atmos- reflection of differences in hydrocarbon sta Arric analyses showed benzpyrene to be pres- bility.11 As can be seen in Fig. 1, pyrene in larger amounts than pyrene. Two al- proved to be the most labile of this group of 'rmative explanadons for this finding sug- hydrocarbons while 3,4-benzpyrene, even un ^*(td themselves. First, a significant emission der the most adverse atmospheric conditions, <irce of benzpyrene existed of which we were proved to be notably more stable. araware, or second, marked variations in the ability of the various aromatic polycydic kmmearbons existed. A search- for proof of PHYSICAL Having established the presence of 3,4- Tablk 2 AROMATIC HYDROCARBONS ESTIMATED inj-iwntjte SAMPLES OF GASOLINE EXHAUST. USING VARIOUS ENGINE REVOLUTION SPEEDS benzpyrene and allied hydrocarbons in com mon and consistent sources of air pollution and having demonstrated their survival in the atmosphere, we then became concerned with the relation of these hydrocarbons to the soot At 0 load, nff./nrinuta of: partides on which they are adsorbed. Physical factors play a significant role in the deposition ScKrd, Py- TL rent Cm* prnnd Bea*pyrene Ben*. . Aapery- thanlehe threne of particulate matter in the respiratory tract. The biological potential of ardnogen-Iaden 500 I.fOO :.no I.ftO :..vo 5.fro 223 439 507 374 340 121 289 325 266 142 127 25 120 235 153 soot is, therefore, significantly related- to the 61 177 102 size of the soot partides present in the atmos 33 40 25 60 73 70 36 27 31 phere, which in turn determines their epithe lial deposition capability. The fundamental 13 83 14 biological importance of parade size resides 48 J 10 39 13 this i2ow?infte terAFtewiwP=*- PPG INDUSTRIES, INC. DID NOT COMt FROM ITS FILES AND CANNOT BE AUTHENTICA1ED BY PPG INDUSTRIES, INC. CO 1 'J 'jjafap-jsa*w;>JBaa Ha- aidafyi* 150 Cancer January-February 1959 VL u |y 1 -- | 1 N0TE7------rM7Qy L i i PCTCMT70A/ M /U9 ~~s Ml P*#riCiC Jitr --; n NCi CUivit iv.w ,-v'QfiL|s > | ^ JO J Fie. 2. Rate of retention of particulate matter in lung in relation to par- tide tire. (After Dautrebande et al.`) to ll I) to , II III VI .it JO . La 20 Panels Sot ajg 4J) i; rl i1 .1 i1 i| ' > 1 t f i : , 1 1 "A don of inhaled soot parades (larger in size than 2 p) are arrested in the mucous mem branes of the nose, accessory nasal sinuses, pharynx, and oral cavity. At the other ex treme, a lesser but still significant segment of exceedingly small soot partides (less than 125 Angstrom units in size) remain suspended in ddal air, except for that small amount of ultra, ultra microscopic partides that are precipitated by brownian movement. The retendon of paniculate matter in the lung in reladon to parade size is shown in Fig. 2. Dautrebande et aL* correlated the locadon of paniculate deposition with spedfic sites in the tracheobronchial tree (Fig. 5). We have been able to confirm the pattern of pulmonary in halation deposition and retention through the use of soots of spedfic partide size. The elution of carcinogens by appropriate organic solvents or plasma proteins is also critically related to the size of the partide on which the carcinogens are adsorbed. Aromatic polycyclic hydrocarbons cannot be very read ily eluted from soots of very small partide size. Further, partides with an average di ameter of less than 400 A will actually remove aromatic polycylic hydrocarbons from their immediate environment because of their high surface adsorption. Partides beyond the 400-A average size range will release adsorbed aro matic polycylic hydrocarbons in the presence of appropriate solvents. As partide size in creases, these compounds are released more rapidly and in larger amounts. p Tina DOCUMENT ^0t STfROM Studies of the atmosphere and of the ex haust products from vehicular engines show j predominance of. soot partides within thr 0.1- to l.-p diameter range. As will be demon strated, this range is consistent with the bi-- logical action of various partides on the ba4* of both partide retention in the lung. a. shown in Fig. 3, and elution f carcinogenic hydrocarbons, as shown in Table 4. BIOLOGICAL Respiratory Epithelial Studies. Experi mental. After inhalation, a period of contact with the respiratory tract epithelium is necosary before the carcinogen can be liberated from its soot partide and enter the cell Ordi narily the necessary prolonged interval (or this carcinogenic stimulus to be in contact with the respiratory epithelium is predtided by the physiologic defenses of the tracheo bronchial tree. The principal and mo*i potent factors responsible for the prevention of prolonged residence of partides on the lining cells of the tracheobronchial tree are. the mucous stream covering the epithelial cells and the activity of riliated cells that h* their action constantly move the mucus cephalad. Particles settling out on this stream are as a result, immediately set in motion. The normal rate of movement of these settled pa* tides is sufficiently rapid to mate elution and local carcinogenic stimulus in the tracin'* bronchial tree unlikely to occur, Tire initio PPG INDUSTRIES 'NC DID aUTHNTICATED IT'S FILES AND CANNOT BE : INDUSTRIES, INC. | BB 00 20!M j- - 2918 Environment & Lino. Canclr. I. Kotin w- I-'ulk i i 1-2 1 10 rj- A \ .4 -t .z < * _LL a, n/ -B-pm U2 j: a a */, V- - i ; . I i I- Ai us V l/ 5 TT -r T * m IPachc* .or Spoa/cn/ol w 49 Oaoncm* ./i PASSAGE 4.02 r/M f4rcj Fin. 3. Relatiorf at par. tide sire to deposition tile and time of panage within tracheobronchial tree. icriod required for elution or removal of the were exposed to controlled test environments ardnogen from the soot and the added time for varying periods. After the animals were cquired for entry into the cell are fulfilled removed, the tradieobrondiial trees were ex when the ciliary-mucus defense barrier is teriorized and opened so that dliary activity #' ed or lost. Carcinogenic panicles pen- and mucous secretion could be observed in j more deeply into the pulmonary the intact animal The second method of >arc.tdiyma to the level of the alveoli are ins*tudying the atmosphere's effect consisted of -i J :ested by the macrophages in the lung. Phago- using isolated animal tracheobronchial epithe J ytoiis is effective in removing these particles. lial strips for in vitro studies. Carlson par- hich then may ultimately be disposed of by tides of a size, configuration, and chemical heir entry into the systemic lymphatics or by structure identical to those recovered from eing advanced to the level of the mucous the atmosphere were used for die determina cream. tion of dliary rate. A steromicroscope with 0 > Under certain conditions, however, the host scaled ocular lenses was adapted for this pur 1 ,, J >* T, i iefenses can be interfered with, and abnor* nally long residence of particles ensues. Nu merous irritants of a nonspecific type are presnt in polluted atmosphere that, when pose. In addition, studies were carried out using the esophageal tract of frogs. During study by the first method, one initial response of the exposed epithelium was ..; --I.,* ^ *" O- readied in sufficient concentration, are cap- apparent exdtation as manifested by an in UJ O' ble first of slowing mucous .flow and ulti creased rate in the movement of particles. In mately of stopping ciliary activity. These response to a few speafic irritants, however, OO ompounds include both organic and inor- this primary effect could not be observed. anic adds, the oxidation reaction products With exposure to these, there followed a >f hydrocarbons, the aldehydes, the oxides of period of decreased activity that, if allowed to itrogen, and the oxides of sulfur. They may persist, often resulted in total cessation of >e present in the air as gases and aerosols. As partide movement. Prolonged or intense ex rule, the effect of these compounds is tran- posure was followed by limited and delayed ient, and when their concentration drops, recovery. Total ciliary paralysis could be ___ lormal activity returns. maintained for a considerable length of time,------ """""oO 20 The modifying effect of atmospheric irri- after which partial recovery was still possible. *\ __ ant substances (ozonized gasoline, aldehydes, Structurally, it was possible to demonstrate a nic adds, etc.) on the normal functions of considerable degree of parallelism between | 'teobronchial tree was studied in two alterations in physiological functions and cell - .rst, animals in inhalation chambersular abno^n^t^sQRgp-esentarive respon--^ THIS. DOCUMENT come 4919 , "oUSTR'ES, INC. BY PPS an as ess* sa O tu OOayr, HuC.c 0 gs'5 ax Kg? -9 m o c/> Q k S 5 C) 9 - zftSg WSqW 5?^ gg5* w 2t -J (JJ w *r c S: K Q. t m 152 Cakc Janvary-February 1959 VoL ij Table 4 RECOVERY OF POLYCYCLIC AROMATIC HYDROCARBONS FROM 300 m OF SOOT AND PLASMA AFTER INCUBATION % recovered Alter incubation period oi:m Compound, ./100 mjr. soot Soot It hr. 16 hr. 96 hr. Pla*. Tot. Soot Pla*. Tot. Soot Pla*. Tot. 192 hr. Soot Pla*. Tol s*. line s^T Pyrene 8.4 Fluoranthene 1.0 Compound X 28 1.2-Betupyrene 2.9 3,^Benzpyrene 1.7 1,12-Benzpery* lene 9.2 Anthanthrene 2.4 Coranene 10.0 9 81 T 100 0 100 T 52 18. 82 31 66 25 54 40 34 90 34 61 95 PP 100 T 93 ... 17 41 58 too 41 59 . 100 97 67 35 100 79 54 33 87 76 66 26 92 9 50 0P 0P 0 21 6 -23 5 15 T 17 12 8 59 6 P 0 21 T 29 J1L 20 11 * * - 13 20 15 61 P 39 21 .13 13 13 10 67 100 . 100 39 100 * * 100 36 100 24 100 26 100 25 too The symbol* in this table are T for trace and P for present. to irritant exposure are shown in Figs. 4 to 6. to determine the biological and chemical face Mucous secretion was also affected by ex* of aromatic polycyclic hydrocarbons as they posure of the respiratory tract to-tmtanet. occur on retained sool We wished to learn: Physiologically, alterations were first charac (1) if carcinogenic hydrocarbons remain un terized by an increase in secretion. This* re altered subsequent to their deposition on the sponse could be demonstrated in microscopic respiratory epithelium or if they change quali sections from the respiratory epithelium. tatively or quantitatively; (2) if any changes An increase in the number of and activity that might be demonstrated are associated of goblet ceils was the first histological change with the activity of exogen us substances (re noted.' This progressed until there was almost active atmospheric pollutants) or endogenous complete replacement of the normal epithe factors (metabolic degradation): and (3) if a lium by an overgrowth of these mucus-produc relationship exists between the amount of ing cells. Subsequently, pools or lakes of. soot and carcinogenic hydrocarbons recovered mucus were formed in the epithelium, and from human lungs and the presence of "pre- finally, the mucous lakes emptied into the neoplastic" morphological changes or oven lumen of the tracheobronchial tree, with com lung cancer. plete epithelial desquamation down to but 3,4-BenzpyTene, a potent carcinogenic aro not including the basal cells. The liiitopatho- matic polycyclic hydrocarbon, was chosen for logical sequence is shown in Figs. 7 to 11. This study as the prototype of a compound belong phenomenon is of considerable interest in ing to this group of hydrocarbons. Lungs for that it represents a step in the pathogenetic analytical study were obtained with the short sequence in which compounds that are not in est possible postmortem delay from the au themselves carcinogenic theoretically facilitate topsy sen-ice of the Los Angeles County Hos the biological activity of compounds that are pital.10 presumably endowed with the property of in Of all the aromatic polycyclic hydrocarbon* ducing cancer in the respiratory tract. presumed to be present, pyrene alone was Human Lung Studies. Two mechanisms capable of quantitation. Our inability to de may explain the almost universal finding of tea the presence of the other aromatic polycy soot-laden lungs in urban residents at autopsy clic hydrocarbons, most n tablv 3.4-benrpy- --first, the action of pulmonary phagocytes in rene. necessitated an investigation of whether the ingestion of the particulates and second, the absence reflected artifactitious destruction the method previously elucidated whereby of 3,-1-benzpyrene and related compounds..in- soot may be abnormally retained in the respir cidentai to our experimental methods or atory tract. An investigation was undertaken whether it represented their true in vivo fate. NOTE: T?'"' ^ tj.cstT oiDr BB 00 20427" 39Z0 mot - ...... ..... I E.W1RONMLNT & Ll'NG Cancer. I. Kutin e- Falk SMoa 3M04 /* M9M cone. SMoa j" met CONC. 133 i i Ftc. 4. Effect of smog in various concentrations on frog's esophagus. Graphic proentiuon of effect of irritant (tmo*) on movement of particles deposited on dliaied epi thelium. Note greater in hibitory effect and de layed recovery in area of impingement. The lines indicate: ....... - reading before exposure; x------x impinged area; and ---- unimpinged ares. Two methods were selected for the resolu* :i**n of this problem. First, up to 20-ug. ^uoumi of 3,4-benzpyrene were added to rep^Hnutxve samples of lung tissue at the start luring the various sequential stages of faction procedures in a series of experi ments. The presence of the added 3,4-benzt uene was detectable at levels of 10 pg. for - :u>ie lung samples. Second, carotenoids nor-ally present in lung tissue were identified throughout the enure procedure. In view of the extreme susceptibility of this group of compounds to oxidation, their persistence strongly suggests that benzpyrene, a less labile compound, would have survived the analyti cal method. The disappearance of certain aromatic polycyclic hydrocarbons, including 3,4-benzpyrene, must, therefore, be regarded as being due to factors other than our experi mental method and techniques. SSssr: 22 o 2J * *[*BB 0020428^ ^ Pbotomiaugrapb from taction of bronchial epithelium piepeted immediately after Gestation of ciliary Minify and flow of mucous stream. (X230.) * Photomicrograph showing deposition and retention of toot on bronchial epithelium after neutralisation ousry action and mucous flow by irritant inhalation. <x30.) THIS DOCUMENT WAS NOT A RECORD OF PPG INDUSTRIES, INC. DID NOT COME FROM IT'S FILES AND CANNOT BE AUTHENTICATED .2921 mti 154 Cancx* January-February 1959 VoL i; .Fro. 7. Initial rroponse of bronchial epithaiium to irritant, a* evidenced by increased goblet ceil activity. (xl23.) Fro, 8 Later stage showing marked increase in mucotta cell iccretioa. with mucoua droplets in lumen. (x230.) Fro. 9. Fooling of mucus in bronchial epithelium, with compression of nuclei, beginning obliteration of cell membranes, and thick static mucous stream overlying epithelium. (x250.) Fw. 10. Beginning separation of superficial bronchial epithelium, with persistence of basal later of ceils. (x250.) The physical characteristics, including the particle sue of the. soots recovered from hu man lungs, were studied under the electron microscope. It should be noted that the size range primarily included panicles from which 3,4-benzpyrene could be readily eluted. The disappearance of aromatic polycyclic hydro carbons from representative human lungs is shown in Table 5. The significance of these data will be discussed. Histopathological examination of sections of respiratory epithelium removed from the lungs frequently revealed hyperplastic 'and metaplastic changes. These were most fio- quently seen at the arborizations and other impingement sites in the tracheobronchial tree (Figs. 12 and 13). This is consistent with the exaggerated experimental effect of the im pingement of irritants on respirat ry epithe lium when contrasted with sites of passive passage. Attempts to establish a correlation between the amount of soot recovered front the human lungs and die presence of meta plastic or neoplastic changes were unsuccess ful. Carcinogenic Studies. Painting and Injec tion. A necessary preliminary step in the as sessment of the carcinogenic agents incidental MHT mror ror-* 1 DB 0020429 | t 2923 Environment & Lung Cancer. I. Kotin i- Falk 155 -osplieric contaminants is their biologi.ing. Extracts of exliaust products of .-upline engines, diesel engines, and the atmos phere were bioassaved for tumorigenic propinies. Samples were painted on the skin or injected subcutaneously in C57BL and strain \ mice. Representative tumor yields for the various materials studied are shown in Table 6. The solvents used in the carcinogenic tudies and the aerosol fraction of the atmos phere collected in Shepherd traps were used i.>r control experiments. The studies of Shep herd et at13 and of Haagen-Smit14 have shown the aerosol phase to consist primarily of the nxidation products of aliphatic hydrocarbons. U'e have confirmed these findings and have further demonstrated that the aerosol phase i< free of carcinogenic aromatic polycylic hydrocarbons.2* An unexpected finding was the production of tumors after painting and injection with the control fraction, presumably a noncardnogenic aromatic polycyclic hydrocarbon-free fraction of the aerosol phase of the atmos phere. This finding was duplicated in a second cries of experiments.2* The tumor yield with the use of these aerosol materials is shown in le 6. An additional immediate unex- # Ne finding was the production of a number of turnon in the mice painted injected with atmospheric and vehicular i xhaust extracts than could be accounted for !*v the presence of 5,4-benzpyrene alone. The implications of these findings will be dismissed Uter. Inhalation. The production of skin tumon in mice with both the aromatic polycydic hydrocarbon fraction and the aerosol non- Fie. 11. Pertinent basal layer o( bronchial epithelium subsequent to desquamation at superficial epitltelium. Note tubeplthelial inflammatory cell infiltration. (x250.) aromatic polycydic hydrocarbon fraction re covered from the atmosphere made it neces sary to choose the proper environment for exposure of animals in our inhalation studies. The non-oromatic polycydic hydrocarbon fraction was selected for several reasons. First, essentially it represents a recently introduced atmospheric pollutant, the cardnogenidty of which has apparently been demonstrated for the first time. Second, in contrast to rural areas, these pollutants are present in greatest concentration in urban areas in which epi demiological studies have shown a consist ently higher lung cancer mortality rate. Third, chemically related compounds have been , r; '- ? f ' -- A- III v ', ; LSJ |-- i-- oo Taau5 DISAPPEARANCE OF AROMATIC POLYCYCLIC HYDROCARBONS FROM SOOT IN HUMAN LUNGS* Amount of: Lung cu. Pl *. Vr. Wt luflff, gtn. Soot, mf. Aah, n>C- Pyrene Found, . Expected, X- 3 90 71 4 4 , 3 <* n 70 74 71 81 62 70 66 950 1,333 1.570 1.570 710 1,260 1,120 910 1,420 720 360 660 360 390 190 540 830 5,010 480 200 770 170 1,070 150 510 310 3,460 0.9 6-27 1.9 3-13 1.9 6-24 3.6 3-13 1.3 3-14 2.3 2-7 4.4 5-20 5.1 7-31 1.3 ` 43-185 am reprinted from Canctr 11:486, 1958. WHIS DOCUMENT WAS NOT A RECORD OF PPG INDUSTRIES, INC. DID KOf COME FROM - IT'S FILES AND CANNOf BE AUTHENTICATED NOTE: NOT i JPG INDl Beatpy icjic A benxperylene Found, 4- **. ,, .. Trace Expected, X- 34-172 17-86 31-138 17-86 18-93 9-45 25-130 38-200 233-1,200 JIO J i" i~- -jaKajsrr i Z323 '" r- , J 1 > ; -n ' - - -- I ! .'. E tRCi/lCancer JanuaryAlflJoGQW l**U `Z Fic. 12. Focal area of metaplasia presumable at site of impingement. Note dilated epithelium on either ml-of metaplauic area. (xl25.) Fie. 13. Higher power of area adjacent to squamous metaplasia adjacent to site of dilated bronchial epithelium (X250.) fcSQ QW cc o 8S 8g o- 3tr f- 2 < O ,, uj . 29mo W5S S ^ 2 c/> >Zu 5 y d 3 sp:?^ ^ t- Q 3 oo ^ -O<Jndwllj - i8 5 g7t/) " demonstrated to be carcinogenic in injection dation products, were similar with both studies by workers in Great Britain***** and methods. Mass spectrometer** and* infrared by Fieser et al.1* in their injection studies spectroscopic studies** were used to demon on the carcinogenicity of oxidation products strate the essential similarity between the of cholesterol. Data from British studies in naturally occurring pollutants and those re dicated that these compounds are capable of produced in exposure chambers (Figs. 14 and intracellular activity.*** ** Fourth, general 15). agreement exists that the latent development period of lung cancer is approximately 2 dec ades, and these aliphatic phase materials were Tana 6 introduced into the atmospheric environment sufficiently long ago to have allowed for any PRODUCTION OF SKIN TUMORS IN MICE BY PAINTING 3 TIMES A WEEK* carcinogenic influence present to have become manifest at the time of die recorded epidemio Appearance of tumors in gp.f Tumor-bearing mice logical increases. Finally, from a technical viewpoint the synthetic reproduction of these materials in inhalation chambers is more feasible than is the use of aromatic polycyclic hydrocarbons adsorbed on soot. Analytical studies by others and by our selves hare shown the aliphatic phase of hydrocarbons in the atmosphere to consist primarily of the oxidation products of straight chain, branched, and cyclic olefinic hydrocarbons. Gasoline provided a convenient and realistic source of these hydrocarbons, and it was reacted with oxides of nitrogen using either sunlight or artificial ultraviolet Moust train No. mica Appear. 1st. tlf Sample mar, material dayi No. mkt surv. Nat % No. with mult. turn. Painting vt'ih aromatic hydrocarbons C37BL Partic. 76 phase atmoe. 463 31 13 W C57BL Partic, 4230 86 41 108 phase (22) 42 468 13 A fSrtic. 440022 2200 1177 8833 8* 23 phase dies. cxh. (11) Painting vitk oxidation products of aliphatic hydrocarbon C37BL Aerosol 42] 35 7 20 30 phase (2) atmo*. ` light as sources of photochemical energy for. Data are reprinted from Cancer 9 : 906-907, 1936 the production of ozone. For the major por t Eleven per cent of the mice that devcloped.tumoc* tion of die study, however, gasoline was di had malignant lymphoma*. JNun,>r in parentheses indicate the number <>' rectly reacted with ozone. XI1*? resultant oxi- mice with malignant tumor*. NOT COME r; I BB 0020431 | 2S24 ii Environment 8: Li'xc Cancer. I. Katin Falk 157 ,..iuin A mice, |x>cssing a high spontane ous pulmonary tumor incidence along wi[h presumably a low threshold of pulmonary re activity to tuniorigenic agents, were exposed to an atmosphere of ozonized gasoline for a tumor-dose study.31 In contrast, C57BL mice, a *tnin possessing a low spontaneous pulmonary tumor rate with presumably a high resistance threshold to the induction of pulmonary neo plasms. were used for the primary induction .if tumors.3 Two control environments were ti*ed for contrasting the strains of mice under -tudy. One consisted of an atmosphere washed hr passage thr ugh successive banks of metlianicai and activated carbon filters. The other environment was an open room in which the atmosphere was unaltered. The hu man experience of exposure to daily variation in pollutant concentration was thus dupli- iatL ' As noted earlier, a modified tumor-doseexposure study was undertaken with the -train A mice. The significance of experimen tal induction of pulmonary tumors in strain A mice in terms of extrapolations of the data to humans is but one facet of the much larger problem concerned widi the applica tion to man of data from carcinogenic studies in animals. While discussion of this problem is beyond the scope of this presentation, cer tain `facts relative to spontaneously occurring and induced pulmonary turnon are pertinent The true neoplastic character of these turnon has been established by the observation of metastascs both in regional lymph nodes and in distant organs and by successful transplantation.1T- *T Pathogenetic and histological dif ferences notwithstanding, the commonness of spontaneous development of lung tumon es pecially in mice and man adds to rather than militates against the extrapolation of data from the former to the latter. Finally, it has been shown that the response of pulmonary tumor formation to meticulously controlled doses of carcinogens given intravenously is so sensitive that lung nodule formation has been advanced as a method for bioassay of susfiected carcinogenic agents.3* J*- *- Mam spectrometer studies thawing similarity between naturally occurring and synthetically reproduced .Hr"*. *7* **. Th* Crs* spectrogram is that of a concentrate from 130 liters of air sampled at El Rancho del located on the doen near Palmdale, Calif., ahout 60 miles from Los Angeles. The second spectro'hat of a Los Angeles smog sample, taken on Oct. 19. 1950. at -13* C.: the third is that of a Los Angeles le taken on Nov. 7, 1950, at --S3* C. and the fourth is that of "witches' brew" synthetic smug, taken this document WAS NOT A h;ECO?D OF I BB 00 20432 2925 158 Cancxx January-February 1959 VoL i* Tails 7 PRODUCTION' OF LUNG TUMORS IN* STRAIN A MICE- Tumor-bearing mice XVk. of Set of study mice Nostud ied t Tot no. % Oteuised lasolutt wasked air chamber 16 9 15 2 13 30 9 15 4 27 24 9 15 4 27 23 9 45 11 24 32 9 45 8 18 36 9 & 75 19 25 40 9 89 19 21 52 9 d* 39 16 11 % with mult, turnon ** . 4.4 4.4 4. 1.1 10. Total 333 33 PiaCEXTACC 24 Aliphatic hydrocarbon-polluted air chamber 16 9 14 1 7 20 9 14 5 36 24 9 15 6 40 23 9 44 21 48 32 9 45 20 45 36 9d* 75 40 53 40 . 9d* 90 57 63 52 9 if 39 31 80 * * 13.3 11.3 11.1 17.3 31.1 48.6 No. with sing, turnon 2 4 4 9 6 16 18 12 71 1 5. 4 16 15 27 29 12 No. with mult, turnon 2 tu 3 tu 4 tu rnon rnon rnon ..4 ** . 1 2 3 l 3 10 ... 4 4. 4.4 1 ..4 *4. ... 1 1~ 2 ..4 4 * ... 4.. ,.. ..4 ---- . 4< 1 3 5 12 21 9 44. . . ft 1 1 * 4W 1 5 8 *. ..4 4*. If ..4 2 2 Total tumors No. %i 2 13 4 2T 4 27 14 31 10 22 22 29 20 23 21 54 97 29 17 5 36 9 60 32 73 25 56 54 72 94 104 62 159 Total 336 181 PZtCEXTAGK 54 109 31 16 5 282 84 Data an reprinted from Cancer 9;911 and 913, 195$. tMic* ware removed at 4 week intervals for ttudy, in the numbers indicated. At the end of the fifty-second week, all remaiaing mice were removed for study. {This percentage represents the total percentage of tumors in the total number of surviving mica. }Thia mouse had 7 tumors. The accumulated knowledge serves to re emphasize certain pie-experimental considera tions. First, the target tissue selected for study was the respiratory tract Epidemiological data relating to cancer of this system served as the basis for this investigation. Second, the method mice. The changes observed in the mice ex posed to the daily variati ns in air .pollution as they naturally occur were essentially simi lar to those seen in the test chamber mice. The dau relating to these experiments have been previously reported.** of exposure chosen was that of inhalation. Third, the spontaneous pulmonary tumor for mation observed in the strain A mice under * TnwtMrmJcx *-| -w. study provided a sensitive target for the rapid procurement of data. ____ The results of exposing strain A mice to a washed air control atmosphere and polluted air test atmosphere are shown in Table 7. re a 14 The C57BL mice were exposed in inhala tion chambers for a maximum of 92 weeks at % TltMIMinSMCt which time surviving mice were killed. The incidence of lung tumors and certain data relating to them are shown in Table *8. Two associated findings of marked interest were observed in the C57BL mice. The first was a consistent and often intense hyperplasia and metaplasia in the test chamber mice as com pared to the controls. The second finding of interest was a significant decrease in the in cidence of extTapulmonary tumors in the test Fic. 13. Similarity between naturally occurring an. synthetically reproduced ether-soluble aerosols. * Typiol infrared spectrogram of synthetic aerosols. C Typical infrared spectrogram of natural aerosols. NOT ' ' NOT UV-'MiL, l ; i. J M U 1 .Z.0 |_BB 0020433 j 2926 SsSffiSilg \.' 1 L I.Environment St unc Ca.nczjl Kotin & Falk 159 T-Jj Jg^r & I'm. IS. Intnbroncbial papillary tumor in C57BL <ne subsequent to inhalation at synthetic smog. IT.) ,, readily apparent that the ozonized 'Ouiie atmosphere had a significant tumori-nic effect on the respiratory epithelium of :'.c exposed mice. The tumors produced were 'cniieal with those referred to as classical heologenic carcinomas, although our find* z<` indicated that these tumors were probably rminal bronchiolar rather than alveolar in "ithn. Representative tumors are shown in itrs* 16 and 17A and B. Several observations bring to these tumors are worthy of note. ' A significant number of tumors were pres et in assodation with intensive surrounding neumonitis. (2) In areas noncontiguous with imor areas and frequently in non-tumor* 'volved lobes, hyperplastic and so-called 'etaplastic changes could be seen in the ronchial and bronchiolar epithelium. (3) reater pleomorplusm and invasiveness were cmonstrable in the induced tumors than ere seen in the tumors among the controls. Comment The identification of carcinogenic agents in dilutant sources and in the atmosphere does >t inevitably connote an adverse biological effect. Nevertheless, despite significant omis sions in our data and- despite the limitations imposed by the absence of or deficiencies in certain analytical and biological techniques, our chemical, physical, and biological data unite to form a constellation that strongly im plicates the atmosphere as one dominant factor in the pathogenesis of lung cancer. The data are accorded additional significance by virtue of their edngruity with the epidemiological pattern of lung cancer. The following considerations are presented in sequence to support our concepts. Carcinogenic agents have been identified in several ubiquitous sources of atmospheric pol lution. Materials identified include 3,4-benzpy rene, aliphatic hydrocarbon oxidation prod ucts, and specific inorganic materials such as chromium and nickel compounds. Ever in creasing pollution of the atmosphere with carcinogenic agents has been a concomitance of the industrialization and urbanization of society. One of the most ubiquitous of the carcinogenic agents belonging to the aromatic polyclic hydrocarbon group of chemicals is 3,4-benzpyrene. It is emitted into the air from many sources including gasoline and diesel engine exhausts, soot secondary to the incom plete combustion of organic matter, soot in cidental to rubber rire Wear, tear, and deg* radation, and, in isolated areas, soot from specific industrial effluents in the manufacture of coal tar and its derivatives. Experimental studies have shown that the aromatic polycyclic hydrocarbons vary in their stability in the atmosphere, with 3,4-benzpy rene belonging to the group with maximum relative stability. The destruction race of aro matic hydrocarbons in the atmosphere indicates that their survival in the atmosphere is consistent with their being inhaled by exposed populations. Even in a strong oxidizing at mosphere, such as occurs in Los Angeles, the rate of destruction of 3,4-benzpyrene is lower Tails 8 LUNC TUMOR INCIDENCE IN C37BL MICE* Motm cp. Appear. 1st. tu mor, wit. Na Tumor-bear, mice mitt urv. No. % Washed sir dumber Smog chamber 56 71 376 6 ... - *-**""* 155 15 9.6 " 474, 1958. r JjJ - * " ' 11 this document was not a Dp/-n PPG INDUSTRIES iNrn.nL REC0RD OP as 2327 _ a 160 Cancer Janunry-l'ebruary 1959 VoL 1 Fig. 17. A. Papillary pulmonary tumor in C37T5L mouse adjacent to site of intensive pneumonitis. (xlU.) 1 High power of marked area of Fig. 17A, allowing stream of neoplastic tissue into surrounding pneumonic lung. Note arrow. (x730.) than that of many other hydrocarbons. In others areas, in which the oxidizing atmos pheric property is less significant, the stability of this carcinogen is greater. In. addition to the traditional carcinogenic agents belonging to the group of aromatic polycyclic hydrocarbon compounds, we have characterized two heretofore unidentified polycyclic hydrocarbons in the atmosphere (Fig. 18). The structure of diese compounds warrants their bioassay for carcinogenicity.. Should they indeed be carcinogenic, these compounds will help explain the discrepancy observed by ourselves and by other investi Sa @s gators between tumor yield and benzpyrene content of samples from various environmen tal sources. [A third aromatic polycyclic hydro tun * 0= ,eC of- oK carbon that has been recently identified is 3,4benzfiuoranthene. Subcutaneous injection of 5-mg. doses of this compound into C57BL mice has resulted in a tumor yield of more *3# CO 2 5 d -9. 9 * than 30% in experiments currently in prog ress.] The aliphatic materials are primarily in troduced into the atmosphere as a result of pollution by raw gasoline vapors. These vapors under certain meteorological conditions react in the presenre of sunlight and oxides of ni trogen to form a broad spectrum of hydrocar bon oxidation products. These agents have Q Sr1 co ^- S~ become significant atmospheric pollutants X-Pa 4 simultaneous with intensive industrialization. N0-H7.-- ................................................. ... Their temporal presence coinrides with th epidemiological increase in lung cancer. The are capable of direct cellular entry, and thu the necessity for concern with the mechanism relating to the elution of aromatic polycycli hydrocarbons from soot is obviated. As lum cancer, in common with all neoplastic diseases must surely have many agents involved in it inception, the simultaneous or sequential ac tion of both the traditional carcinogenic arc matic polycyclic hydrocarbons and the ali phatic oxidation products in the pathogenesi of lung ancer is a reasonable possibility Chromium and nickel though present only it infinitesimal amounts must also be considered by virtue of the occupati nal and/or expen mental data that incriminate them as card nogenic agents.1 A critical factor in the elicitation of th< carcinogenic response is the concentration a which the etiological agents are present. It i our belief that as of the m ment we must asume that any exposure is hazard us.1* .is th carcinogenic dose is the product of the cor centration of the environmental enrrinogeni agent and the exposure time, current dat strongly suggest that in general we are bein exposed to low dosages of carcinogens. IVTiil little is really known concerning carrinogeni dosages in man, the exposure to a "low dose over a long period of time is compatible wid the induction of <he neoplastic state. Abuc NOT U.. _ ' < / II BB 00 20435 | So. I Environmen r & Lt'Nc Cancer. I. Kotin ir Falk penmenc;il evidence exists to indicate initial response of the respiratory epithelium liu. dm is so. Further, both epidemiological to dig deposition of foreign particulate irri ..nd clinical data indicate that the develop tants. (2) Persistence of the insuldng agents ment of oncer usually requires a prolonged rapidly leads to a neutralization of the pro mierval. In the instance of human lung cm- tective factors, widi resultant slowing of par- ,cr, a period of from 10 to 20 years is posui* tide movement; diis, in turn, leads to pro Ijied. This seems entirely reasonable when gressive accumulation of partides at selected one considers that experimentally approxi sites in the respiratory tract. (3) A demon mately 25% to 50% of the life span of an strable difference in die effect on the epi animal is usually required for the induction thelium can be observed, depending upon ..f cancer. Spontaneous tumors in experimen- whether the respired material directly im nl animals usually occur in the second half pinges on or passively flows over tradieo- ..f the life span. This is similar to the experi bronchial epidielium-. In the instance of the ence in man. former, the effect is more intense and of The physical state of the carcinogenic hy greater duration. (4) Reversibility of the ad drocarbons present in the atmosphere is one verse effect of atmospheric environmental I adsorption on soot particles in a size range agents persists for an unantidpatedly long pe iinpatible with the deposition of and reten- riod, although restoration to base line levels :'<ju of a portion of the breathed carcinogen- of activity seldom occurs. (5) Denudation of :den particles. The anatomical site of deposi- die superfirial epithelium permits die im -inn is a specific reflection of the size of mediate apposition of inhaled particulate car >articies. The relative infrequency of primary cinogenic matter to the persisting layer of -mcheal carcinoma is perhaps, among other basal cells, which presumably may give rise to :icton, die result of the rarity of partide depo- hyperplastic, metaplastic, and ultimately neo Irion on the tracheal epithelium. Those par- plastic change. ides destined, on the basis of their size, to Soot recovered from human lungs has been vide on tracheal epithelium are die ones that shown to be free of the carcinogen 3.-f-benzpy readily pass beyond the nose, accessory rene. Pyrene, although demonstrable, is pres iuses, and pharynx. Further, the ad- ent in signifleandy less than anticipated p.e bond between particles settling on amounts. A gradient of disappearance exists, 'ic bronchial and bronchiolar epithelium and varying from partial in the case of pyrene to lie cardnogenic hydrocarbons is, such that total in the case of 3,4-benzpyrene. The differ iiuion of die bitter readily occurs in the ence between the total disappearance of the :e<ence of tissue and plasma proteins.12 cardnogeu, 3,4-henzpyrene, and the partial Respiratory epithelial changes facilitating disappearance of the noncardnogen, pyrene, " deposition of and retention of particulates may be a reflection of the cardnogenidty of mr in response to a broad spectrum of irri- the former and innocuousness of die latter. ats present in the atmosphere. The as- vmbled data from studies suggest the follow(1) An apparent purposefully exaggerated Summary and Conclusions aivitv of ciliated cells and goblet cells is the The cardnogenic implications of atmos pheric pollutants reside in at least two indi spensable factors relating to the pathogenesis of lung cancer. The first is, of course, the en vironmental presence of and the host entry of agents proved experimentally to be tumori- genic and epidemiologically to be associated with an increased liability to the development of the disease. The second factor is the atmos pheric existence of host-modifying factors that, by virtue of their effect on the abated epithe lium of the tracheobronchial tree, make pos xr-<B.a,o)-nnnui(Tazia sixz-oo -rujouumtD*! af". -I?- "*'wo *roml,lc polycyclic hydrocarbon* re"*ly menlifted in atmosphere and now under bioas' for -dnogcnidly. sible the abnormal deposition and retendon of paniculate matter in the lungs. In the in stance of cardnogenic aromatic polycyclic hy drocarbons, die elution of benzpyrene from THIS DOCUMENT WAS NOT A RECORD OF PPG INDUSTRIES, INC. DID NOT COME F^OM IT'S FILES AND CAN.NOf BE AUTHENTICATED BY PPG INDUSTRIES, INC. -aeK Oo BB 00 20436^ 2929 aw* m 162 Cancer January-Febmary 1959 Vol. i* soot by intracellular proteins is thereby facili tated. A significant local concentration of de sorbed aromatic polycyclic hydrocarbons, in cluding 3,4-benzpyrene, results. Atmospheric irritants may, in addition, periodically and intermittently cause denudation of the super ficial epithelium, so that the basal cell layer is directly exposed to the carcinogenic stimulus. This periodic epithelial desquamation and regeneration in the presence of an abnormal growth stimulus is regarded as providing a favorable environment for subsequent abnor mal growth patterns. Carcinogenic aliphatic oxidation products present in the atmosphere in aerosal form are experimentally capable of direct cellular en try, so that the respiratory epithelial cells are subjected to simultaneous or sequential ex posure to tumorigenic agents. An endogenous environment favorable to the biological ac tivity of the carcinogenic hydrocarbons results. Bronchogenic carcinoma represents one of the current critical problems in the field of pulmonary disease. Laboratory investigation can contribute much information to the ulti mate solution of this problem. In the physical science area, finite analytical data are possi ble. In the biological realm, strong supporting data can be secured, despite the fact that ex- perimentai investigations are necessarily lim ited to nonhuman animal species. It is neces sary, of course, to remember certain deficien cies inherent in the biological studies. Choice of species, selection of appropriate animal strain, duration of exposure, concentration ot test material, and routes of administration are all variables that modify the extrapolation or experimental data from other animals to man. Despite these shortcomings, past experience has shown a high index of meaningfulness of animal experiments for the human species. The broad spectrum of agents carcinogenic for visceral organs in experimental animals and apparently for those in man should make one proceed with caution in attributing the absolute dominance of any one agent over an other. It is perhaps safest to regard the develop ment of lung cancer as the end stage of 2 series ot sequential changes that require the presence of a carcinogenic agent, environmen tal host-modifying factors, and the innate susceptibility of the host. All of the links ir the chain are not of equal magnitude. Rather than searching exclusively for a "cause" o: lung cancer, it might well be more rewarding to investigate the series of changes that consti tute its pathogenesis. REFERENCES 1. BAETTn. A. Pulmonary carcinoma in chro matn workers; I, review of literature and report of Arch. Indust. Hyg. 2: 487-504, 1950. 2. Campbell, J. 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F, and Guam, A. G-: Lun; oncer mortaiitv. geographic distribution in United States for 1948-1949. Pub. Health Rep. 69: 1033-KH2 1954. 17. Klein, M.: Transplantability and growth ' urethan-indured pulmonary adenomas of mouse. Car. cer Res. 17: 635-638, 1957. 18. Koetovec, R.: Mortality from-lung cancer ir. Netherlands during and after-last war. Brit. J. Cancr 8:34-39.1934. 19. Ktrnx. P.: Esperimentallr weak carcinogen[Guest edit.] Cnnrcr /let. 18: 1-3. 19'R. 20. Konx, P.. and Falk, H. L.: Production of u. mors in C57HL" wirr with atmosphere-extracted al. NOTE: NOT COM\ rf r:' - - * *'*5 :J J BB 0020437^2 2930 f\ LiisjasjKiiiii.-r' aUJ|iyi-wi RWSjjJl, iiii^ V. 1 Environment $: Ling Cancer. I. Kotin -- Falk 163 * 'drncarbom. [ \bjtr.l Prof. Am. A. Cancer 1935. ..in. P.. and Falk, H, Lu II. Experimental . diiL.ion of pulmonary turnon in strain-A mice after fir etposure to atmosphere of ozonized gasoline, mn-r 9: 910-917, 193G. 2* Kotin. P.; Falk. H. I_; Madfk. P.. and Thomas. 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Stocks, P.: Statistics of cancer of lung. J. Fee, Radiologistt 6: 160-173, 1935. 40. Stocks, P., and Cammell, J, Mu Lung cancer death rates among nonsmokers and pipe and cigarette smokers; an evaluation in relation to air pollution by benzpyrene and other substances. Brit. At. J. 2: 923929, 1955. 41. Waller. R_ Eu Benzpyrene content of town air. Brit. J. Cancer 6: 8-21, 1952. 42. WDerzanm. M. Cu Wajon, L M,, and Mc Namara, F. Pu Tlie Pathology bf Influenza. New Haven, Conn. Yale University Press. 1920; p. 48. THIS DOCUMENT WAS NOT A RECORD OF PPG INDUSTRIES, INC. DID NOT COME FROM IT'S FILES AND CANNOT BE AUTHENTICATED BY PPG INDUSTRIES, INC.