Document Ra2EZD3oG4Q638doDnkezM4Xv

PLAINTIFF'S EXHIBIT K. - t 'y G ' ^ Kdalionship Ixilwuen ivvposurr to Asbestos, < 'ollt<rc*fi Formation, Ferruginous liotlirs. and (.aninoma EARLS. MOWERS. Ph D. S< hitul tif I'ltbltt Ifrultli. f nnt i\il\ if ( /m -fifC'/ik. 11*\ .-UiUi-lr). ( ahlttnutt WMI24 Kipodirt lo wkctti, U avtorulrd Kill rfcruatr fifcruMag prarwn, formation ol Icmicwout bodirv and aa inrreaatd ri%k ol nrciniau. Il n puxtiblr lo relate (be ttfiotn bWminil rrvponfcrs to aUirWo, tipown ia lirn of faaUaaMatal eflnu 00 Ibe redox %tatt of cettx or Ibtun. CotUcti vyathexfc fav ocfialtf by the level of "-krtugiutiirK' acid. nod Ibe rale of rullaRea finuliM k directly related lo Ibe rale, of fibrottv, iactudiaa elavtia foraudioa. The iarreaxe la oxidative reactioa* aavoruded abb rollaaea forawtioe i> ibotaebt to iacieace Ibe awwat of feme iroa buaad lo cellular prutcla. revulllaa ia furmattoo of a viUcate-lrootlll) bood oa aa aibevtoa fiber. Tbc iroa-coalalaiag protcia hoaad lo Ibe mbevlu* fiber b> tbc vtruag vilicate toot? bead b a femtxiaouv body. Tbe formpodding decrcmc ia 1 be ferroox-terric redox couple iabibitx electron transport. Ihue vtahdiriag eiupcroxides aad free radical, (bought lo be involved ia tbe iaitiatioa of carrinumav. hh sf.veral biological and TTISSUE RESPONSES on exposure to asbestos have been regarded as being unre lated one to another or. specifically, as in dication that the formation of ferruginous bodies was not related lo chronic fibrosing processes nor lo an increased risk of car cinoma. To the contrary, when the various responses to asbestos exposure are viewed in terms of molecular mechanisms, it be come. increasingly apparent that ail biologi cal responses lo asbestos are intimately re lated. The relationship results primarily from modification in the energetic pathways of the macrophage cells that first encounter the fiber and is ultimately expressed by a perturbation and removal of ferric-contain ing proteins. To reestablish redox equilib rium. the macrophage cells increase oxygen consumption, and the associated increase in oxidative metabolism leads to export of a fibrogenie factor with concomitant increase in collagen, claslin. and reticulin in surround ing cells and tissues. Development of carcinoma is poorly un derstood at cellular and tissue levels, but it is possible to note that disturbances in cofactor requirements for oxidorcductascs and perturbation of tissue energetics most likely increase the probability for expression of a carcinogen. There is also evidence that cal cium metabolism involved in energy trans port by mitochondria as well as in other biochemical pathways is disrupted, leading to calicification of pleural membranes pos sibly due to dihturbunco at the hormonal level of action. With the premise that all biological responses to asbestos exposure are related, it is of interest to examine var ious implications of the premise from a molecular biology viewpoint. Bkmergtfics It is possible to view defense mechanisms of cells and tissues in terms of the genera tion and flow of energy-rich compounds. Since biological systems derive energy by oxidation of organic substrates.1 a funda mental response to challenge is an increase in energy production leading to increased consumption of oxygen. The observation thjt bronchial epithelial slices increase ovv- 7;a . / . iJl'tUiMjH I' llllllC Sill, . j ihis gi.ii... : coiisumptioi ..I teirou. I .... I o Cl 'til . > Vase ills Is ' I . .1(11 to J l SsJ |.. bleb' ami lllssd lo .SIM . j i, i ovidi/sd h r, moved I'm hi finis' mo mis inkier alkaline . tii.ni solution .. ilk i mody nanus.. It IS |VlSSlhls pioducliou asso ovveen eoiisun, alt an ms re o-.. isilijps belts i i. nipcrature nlo i"sleased rates . . ..ii s sport eiis i . i niotuxic etiei . iges. Ivmphos s to aiJ in rophages ai h.iblv relate. : ice the tliobi- emein ol j. kvl be USSOCIu ors, repress i' '.liar comma . rs i.i rclatk a is parties., jsss'd later rugiiMMis Hvh \ terruemou- i. STOOD5053 7:s ST009585I ,, consumption in iIk' presence of asbestos. .O.iIIiiic silica. and polygonal glass xup ii> ilux general observation.7 Ultimately, c consumption of oxygen leads lo oxtda.'ii ul ferrous in lerric ions, and lor the ;tk.ess lo continue ii becomes necessary lo kciease the level of ferric ion in order lo ciurn lo a redox equilibrium as described :<x Krebs and V'eeeh.1 Ferric ion can be ; educed to .cirous ion which would create . >ilicr oxidi/cd substrates, or ferric ion could tv removed by precipitation or coniplcxing hi some manner. Ferric ions are unstable under alkaline conditions, and their removal tium solution and or their reduction are iliermody namically favored.1 It is possible that an increase m energy production associated with an increase' in oxygen eonsumpiion has survival value. XV all an meiease in cucigy. the tissues ,ue ivrliaps better prepared lor delense. the icinpcrature may be increased, leading to increased rales of enzyme activity, and cells can export energetic compounds forming a chcmotaxic energy gradient to attract macro phages. lymphocytes, and other wandering cells to aid in the defense. Aggregation of macrophages around an asbestos liber is probably related lo energetic phenomena, lienee the mobility and apparently random movement of phagocytic cells in general ould be associated wtih chcmotaxic energy 'actors, representing the principle means ol ellular communication. Biocncrgclics ot amors i.i re'ation to induction of mesothe lioma is particularly significant and will he discussed later. ferruginous Body Fomwlion \ ferruginous body refers to an iron- koiil.mimg protein body with a lihrous core. I lie lioilies are thought to be lormed by macrophage cells attempting to phagocyti/e a foreign liber. I here are various concepts regarding formation of ferruginous bodies, and the term "ferruginous'' was coined by Ciross m I Wih.1 This general term has re sulted in part from uncertainty regarding the nature ol the fiber core and observations that asbestos fibers are not unique in induc ing lerruginous bodies. Ferruginous body formation has been induced by asbestos, fi brous glass, various vegetable fibers, and other silicate-containing minerals. Ferruginous bodies occur in a variety of shapes and sizes. There can be evenly dis tributed deposits, a senes of clumphkc de posits. or large deposits on the ends assum ing a barbell shape. Sizes vary, but the fiber core approximates llic lengths ami Jiameleis ol asbestos and other fibers found in human lungs. Formation of the lerruginous body is thought to occur by deposition of ferritin and/or hemosiderin in the ferric oxidation state on an electronegative surface. On phagocytosis of foreign substances, macrophage cells encapsulate the substance in a mucopolysaccharide coat.1'7 The coal appears to have a high affinity for ironcontaining substances, hut (his does not necessarily explain formation of ferruginous bodies by asbestos and similar substances. Examination of elements arranged by Paul ing's electronegativity scale in Table lK shows that silicates tend to form a nucleophilic conjugate base while magnesium oxides tend to form magnesium and hyroxidc ions. It is possible that an electronegative mucopoly saccharide coat acting in concert with nu cleophilic silicates represents the nieehan- r.xHi i- i Hlceironetl.iliv il> of C cnlr.il Mollis ` 1 11 1 i-OH l.ll N..-OII ii a Be-OH IX Me-OH i: I (Hill I'.llltlllL' III H-OH Mi MOM 1x IV < -OH y.x Si-OH 1X V N-OH VI) l*-OH M VI O-OH vx S-OH :x VII ton 4 1) ('l-OII VO 726 Isms In w Inch asbestos .llld ntlkl libel's .iiir.nl Kim ions Nimbici pmsihiiuy n iti.it lIk coaling ol tnrcign IvhIks by .in 11.niprotein ali.tchcd in ,1 m;ku|Hil\s.kdi.ii uk' IIIJI could Iv .1 luiklailklll.il dcli'llsc IIICi.llattism relalcd to lhe immune response. ami asbestos and other silicate-containing miner als eould ael in disiupt the immuik-risimnsc or foreign lvx.lv reaction, producing a pro long'd and continuing stress. It is of interest to note tiiat Icriugimnis hods formation represents a mechanism lor rentosal ol ferric ions recardless nt the na ture or mechanism lor formation ol the ferruginous deposit. I lie increase in oxida tive pa ill wavs most likely results in export of fihrogenic factors including lysozymes. Kreh's cycle intermediates, or polssaccha rides. II encapsulation of a liber m a mueopoly saccharide coat is involved, then a depletion of mucopolysaccharides in the mac rophage cell membrane might account for its increased permeability with leaking ol cellular material. It is possible to conclude that perturbation of the redox equilibrium of the phagoevtic cell by removal of ferric ion to lorm ferruginous bodies sets the stage lor successive events. Collagen Synthesis Ihe synthesis and increasing tissue con centration of ihe various forms of collagen have been associated with chronic fibrosing ir.r.aulioc of Av>vtov lnO*iN*vl Gl.iin* .o. Profits Krti ** J 4 iI o "JvrtujIuUric ' Mid . . ^ OKV-il 6cun ... * Aa.no t. ' I. Figure I. Imliklion ul e< 11.1 I si Milieus .Hill by j'lvsii s inli.il.ilitin \ .m pioCesscs. a livpnllklk.il desciiplloil ul ilk pini.ss Is sill,llll III I IHIII . I Ills' til'll,Ms li'lllllilg 11 *1111 aslk si, ISIS IS dill us. Ill II.Ill- p.i11s,>11 lii divide ilmliil.ii obinsis .iss,K.ialed wilh ev|xisure to mIk.i. I lie dillusc librosis may Iv related in the tact llial the length ol libers precludes complete pliagocvlosis. Webster.'' in similes with South Aliic.m vervel monkeys exposed to two dillereitl lypes ot airborne asbestos dusts, tound llial the lead ions ol the asbestos ItIvi's vviib the body were ol three distinct tyjvs: ( I I an organized desquamative avcolitis; (2) an obliterative bronchiolitis; ami (.1) an interstitial fibrosis not directly asso ciated with the presence of asbestos needles or Ix'dies. Ihe observation of an interstitial lihrosis not directly associated with the fiber supports l leppleston's hy|>oihesis in regard to the formation and cvjxitt of a librogeme factor by macrophage cells. Hcppleslon1" contends that Ihe factor is nonlipid in nature, possibly a protein or derivative thereof, or perhaps a galaet.m may he involved. Hcpplesion's hypothesis also suggests that the fihrogenic factor acts by stimulating hy droxy proline production by fibroblasts. How ever. as shown in Figure I. hydroxyprolme is formed by hydroxyla'ion of prohne resi dues in protoeollagen. and apparently is not tound as a free amino acid in the cell." Thus the librogeme factor or factors must be intimately related to the ratc-conirollmg step in collagen synthcsis-liydroxylalion o. prolme residues in protoeollagen mediated by protoeollagen hydroxylase. Protoeollagen is composed of relatively few types of amino acids, primarily proline and glycine. This results in an extremely hydrophobic molecule, and the protoeollagen is thought to Iv stiongly bound to an KNA templet. Hydroxy lation of certain proline residues is carried out hy reaction witii mo lecular oxygen and a reducing agent, cata lyzed by protoeollagen hydroxylase. The activity of this enzyme is regulated by the level of o-ketoglutane acid in the cell. Hydrovyl.aion ot the protoeollagen forms a .i .,'11. i'o I I ..'la I,. II 'lulk i. >: t k111. ti Hulk. II: iump Ik .s" k I ' llll, . I led hv h , ,1 it l.k- k sultlli 1,'ii.i.ilinil Jll i .,1c X i >| cl iviii and r ( amiHiina I Ik' gi'lli I . Jlr UIKtHlif' 't v \ v 1 1 Hlli .1 tjx. iml' 'n death Cain.s nt ill, hex ii dcliniti cu .' deal nl Gsi' ui.' a llosl -XI i.il. Ilk ill. lit In til' ol Hide ui: to an<n Sh^ lo '-rs m., rn. al m i El) aay he i m ..nts, m >i Vir to '"dudmzmg V ' v 11 h a V and cl arc c a suic : tr Olllct V niu an ,iiinni ary i Iks, i(| GO --t o CD GO cn CO cn cn nt iitl huhtMrml // V/r'J/1 ` .ImiH tatimt Jnttt mil ..vr-soluble coiLgcu that ftcea the UNA for synthesis of more protocollagen. ,.;J .is king us the stimulus cf o-ketc.Jutaric .aid is present, the cycle continues. The >:;'Je-sirjRded soluble collagen forms a ,uore stable tripie-stranded collagen by ai'-NS-linldng at random in solution. The triple-stranded collagen continues to form more complex collagen fibers and fibrotic nundles." It is possible that lysozymes, krclx's cycle intermediates, or saccharides exported by macrophages stimulate higher levels of a-ketogiutaric acid in fibroblast cells resulting in increased rates of collagen formation. The collagen formation is also an index of synthesis for other clastic fibers, eiastin and reiicuiin. that make up fibrin. Carcinoma The general view of carcinoma is one of an uncontrolled growth of cells and tissues exerting a debilitating effect eventually lead ing to death of the affected organism. The causes of this debilitating disease have not fven definitely identified, but there is a great deal of circumstantial evidence impli cating a host of environmental factors. In general, the induction of carcinoma is thought to involve interference with the flow "f genetic information, particularly the in formation that controls ceil growth. Asbes tos fibers may be direct carcinogens, imply ing that no secondary agent is required; or hey may he secondary carcinogens, promot ing agents, or carriers of carcinogenic subtanccs. Viruses, radiomimctic chemicals, 'oxjc products of combustion, certain metals, md ionizing radiation have all been asso ciated with an increased risk of developing -anccr,1- and it is possible that all these agents are capable of producing cancer. Exposure to asbestos is associated with two distinct types of cancer: bronchiogcnic -arcinoma and mesothelioma. In bronchio-enie carcinoma, asbestos most likely acts as secondary carcinogen or promoting agent, ml in mesothcliama, the evidence strongly izi suggests that asbestos may act as a direct carcinogen. To understand the mechanisms by which asbestos could exert a promoting or sec ondary effect, it is necessary to identify pos sible relationships between the biological processes associated with exposure to as bestos and the mechanisms for action of potential carcinogens. One possible mech anism is induction of changes in membrane permeability due to release of lysozymal enzymes to surrounding tissues. By modify ing membrane permeability, potentially car cinogenic substances such as viruses, metals, toxic products of combustion, and carcino genic chemicals would have easier access to critical sites in cells and tissues. This would essentially he an adverse effect on the cells' first line of defense--selective permeability of the cytoplasmic and/or nuclear mem branes. A promoting effect could arise in a different fashion as a result of alterations in the major biochemical reactions in the cell, particularly in the oxidorcductasc sys tem--the main detoxification system of most cells and tissues." The synthesis of collagen and related substances, eiastin and rcticuiin, requires cofactors from this system, and there is possible inhibition of oxidoreductases by competition for cofactors required for metabolism and elimination of potential carcinogens. Such cofactor competition could explain the relationship between exposum to asbestos, smoking, and the high in cidence of bronchiogcnic carcinoma that has been reported among asbestos workers who smoke.13 An example of inhibition of the metabo lism of a potential carcinogen is inhibition of electron transport in the hydroxylation of 3,4-bcnzopyrcnc. The hydroxylation of of 3,4-bcnzopyrcnc involves the consump tion of one mole of molecular oxygen and two moles of hydrogen from NADPH. The hydroxylation reaction involves two distinct steps; formation of an exoperoxide-enzymesubstrate complex, and reduction of the complex by NADPH. If the cofaetor for ST 0095056 Vfnctnhrr, l(H4 reduction is not available, then a stable species are leached from the fiber. I he ef- 3-4-bcnzopyrcne e.xopcroxide is available Icet ol magnesium ions is to change mito for addition to DNA or other maeromole- chondria from an orthodox fO) energy-rich eules. perhaps increasing the probability of form to an aggregated (At uncoupled energy- a cancer." poor form, or an O-to-A transition, while 'A Asbestos has also been characterized as calcium ions with hormones such as thyrox a potential carrier for other substances that ine produce the opposite transition, A to O.1'1 may result in production of a cancer. Cral- In effect, calcium ions allow mitochondria ley1, has postulated that certain metal con to take in and store energy, while magnesium taminants on asbestos fibers may be respon ions cause the energy to be dissipated. The sible for the associated carcinogenesis, and leaching of magnesium ions from asbestos he has shown that many of the metals as fibers or fibrils in mcsothelial tissues could sociated with the fibers produce tumors. favor development of tumor cells. It is pos Polyaromatic hydrocarbons have also been sible !hot tumor cells arc able to survive the found on asbestos fibers in trace amounts, challenge of magnesium ions leaching from and it is possible that these substances are asbestos fibers better than normal cells. In introduced into tissues, exerting a tumori- time, the norma! cells arc weakened or de genic effect. Metals or polyaromatie hydro stroyed owing to energy deprivation, and carbons arc possibly introduced in process the tumor cells, being more energetic, re ing. as natural contaminants in asbestos, or cover and begin to develop and spread rapid by absorption from the environment. ly in the absence of norma! cells to hold Mesothelioma is a tumor of the mcsothelial them in check. Calcification of the pleural tissues. Development of this tumor is poorly is most likely an alternate manifestation of understood, but it seems to be highly asso the interplay of calcium and magnesium ciated with the presence of asbestos. It is ions. The deposition of calcium is controlled possible that the mechanism for induction by a delicate hormonal balance that could of mesothelioma is related to the effect of be upset by introduction of other divalent asbestos on bioenergetics of the mcsothelial cations.19 cells. The increase in glycolysis associated with tumor cells was described by Warburg'1' Summary in 1930; he attributed it to an increase in In Figure 2, the possible relationship be operation of the respiratory chain. The idea tween exposure to asbestos, ferruginous body of an increase in glycolysis being responsible formation, collagen synthesis, and carcinoma for lumorigcncsis was subsequently dis is outlined. Calcification of the pleural mem- counted,1' but RackcrIH has revived the idea in a modified form in his hypothesis involv ing glycolysis and pH changes, pointing out that the increase in glycolysis is not neces "vtiaw i .irvos 3wvt> l I **' I Astute* 9 to sarily associated with the respiratory chain. Interference with energy flow in cells and tissues can have severe consequences for the whole organism, and divalent cations, cal >\c ..'v" t s# l n< ktivvWtsultww .* cium and magnesium, have dramatic effects -on storage and transport in mitochrondia.1'1 It is instructive to note that the magnesium |s;,.COntent of certain types of asbestos isolated exposed tissues is reported-11 to deSffcwase with time, suggesting that magnesium IIcure 2. Possible relationship between expo sure to jstscstos, ferruginous hotly formation, solt.igep synthesis, .mil carcinoma. tf tin It:,: ;cs and sidered : solutio: .wanly nscs In r nhalatior av L'oiar . .stuuerts [,,, f.hrogeL'..i.ician. mediate, bit: duction of . ami reticu!::' f.iie-control!!'hydroxy lation protixrollagen a reducing cf factor is nr:'.: linucx and :h; ary earcinogen cmogenic subs membrane^ am gens `o cells. colaciurx an-.J carcinogenic loi continue.! . reestablish :b: by removal of ftiruginous bit. ih.it ferruginous v'i significance ! ii'a.al hejith I!: : t -.acd. 1" Terences eitninger, ' Publishers. S; wtik /\ C. -ew . I Certain ln pla*e ( ip.i. I While Mi.; siebs I- \ 'elation ST 0095057 JsagggjfcS hnnuan huittstruil Hy^icnv Awotialmit Jam uni hrancs and induction of mesothelioma arc considered to be manifestations of leaching and solution of magnesium ions, but net necessarily related to other biological re sponses. In the biological and tissue response to inhalation of asbestos, fibers that reach the aveolar spaces arc phagccytizcd. As a result of changes in membrane permeability caused by direct irritation of the membrane by the fiber, there is a release of cellular constituents that contain fibrogcnic factor. The fibrogcnic factor is possibly a protein, guluclan, lysozyme, or Kreb's cycle inter mediate, but in any case it stimulates pro duction of collagen and assxxriatcd elastin and reticulin by increasing glycolysis. The rate-controlling step in collagen synthesis is hydroxylation of certain proline residues in prottrcollagcn, using molecular oxygen and a reducing cofactor. As long as the fibrogcnic factor is produced, collagen synthesis con tinues and the asbestos may act as a second ary carcinogen by acting as a carrier for car cinogenic substances, by damaging cell membranes and increasing entry of carcino gens to cells, or by competing for reducing cofactors and inhibiting the metabolism of carcinogenic substances. The driving force for continued consumption of oxygen is to reestablish the redox equilibrium disturbed by removal of ferric-containing proteins in ferruginous body formation. It is possible that ferruginous bodies arc perhaps of great er significance in environmental and occupa tional health than has previously been main tained. References 1. I.ehninger, A. L.: Biochemistry, p. 3. Worth Publishers. New York (19701. 2. .S.:me/ykrewiez. K.. and H. Wozniak: Effect <'f Certain Industrial Dust* on the Oxygen Uptake Capacity of the Pulmonary Tissue* of While Mice. Med. Proxy 17.TK5 (1966). v rsrehs. H. A., and R. I.. Vetch: Equilibrium Relations between Pyridine Nucleotides and 729 Their Roles in the Regulation of Metabolic Processes. Adroit. Enzyme Keg. 7 .197 (1969). 4. Yalsimirxkii. K. B.. and V. P. Vasil'ev: Insta bility Constants of Complex Compounds, p. 70. D. Van Noslrand Co., Princeton. New Jersey (I960). J. Gross. P.. R. T. P. de Trcville. I.. J. Crallcy. and J. M. G. Davis. Arch. Pathol <55.519 (I 96K). 6. Davis. J. M. G.: Further Observations on the L'ltrastructurc ;utd Chemistry of the Forma tion of Asbestos Bodies. Exp. Mol. Pathol. / d.146 (1970). 7. Governa. Vf,, and C. Rosanda: A Histochcmi- cal Study of the Asheslso Body Coating. llrit. J. huL Med. 29.154 (1972). X. Pauling. I..: The Nature of the Chemical Bond. 3rd cd., Cornell University Press. Ithaca, New York (I960). 9.Websicr, F. The Paihogcncsis of Asbes tos. Ini. Conf. Pneumoconioses, Republic of South Africa. Johannesburg, p. 62 (1969). 10. Meppleston, A. G.: Fibrogcnic Action of Silica. Brit. Med. Bull. 25.282 (1969). 11. White, A.. P. Flandlcr. and K. V.. Smith: Prin ciples of Biochemistry, 4th ed.. p. K7I, Mc- Graw-Flill. New York (1968). 12. F'ishbein. C., W. G. Ftamm. and FI. L. Falk: Chemical Mutagens, Academic Press, New York (1970). II. Selikoff, J. J.. F.. C. Hammond, and J. Chung: "Asbeslos Hxposure, Smoking, and Neoplasia. J. Amer. Med. Ass. 204 106 (1968). 14. Flowers, F.. S.: Aromatic Hydroxylase Inhibi tors as Environmental Factory in the Produc tion of Cancers. Doctoral Dissertation, Uni versity of California. Berkeley (1971). 15. Cralley, L. J.: Flectromotive Phenomenon in Metal and Mineral Particulate Exposure: Rele vance lo Exposure to Asbestos and Occur rence of Cancer. Amer. Ind. Hyg. Ass. J. .12: 651 (1971). 16. Warburg, O.: The Metabolism of Tumors. Constable, London (1930). 17. Aisenberg, A. C.. and H. P. Morris: Energy Pathways of Hepatoma No. 5121. Nature tLondon) 191.1314 (1961). IX. Racker. F..: Uutenergetics and Ihe Problems of Tumor Growth. Amer. Sci. 60 111:56 (1972). 19. Green, D. E.. and J. H. Young: Energy Trans duction in Membrane Systems. Amer. Sci. 59{ | ):92 (1971). 20. l.anger. A. M,, I. Rubin, and I. J. Selikoff: Electron Microprobe Analysis of Asbestos Bodies. Pneumoconiosis. Proceedings of the International Conference. Johannesburg, Cape town, Oxford University Pres* (1970). USOG600IS