Document ZBGk8XKbqwy4rmaJxvwMOkVEp

W- ><--^ ir^ . _/ /fr/ :fftf PLAINTIFF'S EXHIBIT s MON-296 :3 CHAPTER 3 ASBESTOS CARCINOGENESIS* Arthur M. Langer and Mary S. Wolff Environmental Sciences Laboratory Mount Sinai School of Medicine of The City University of New York New York, New York 10029 I. INTRODUCTION A. Cancer in Western Man In countries where vital statistics concerning causes of death have been kept and mortality and morbidity data are validated by clinical and autopsy information, cancer deaths have increased dramatically (e.g., the United States, Silverberg and Holleb, 1972.(1) Some forms of malignant tumors have increased at alarming rates; h such is the case for lung cancer, colon cancer, and bladder cancer, f ? As an example, lung cancer now accounts for one of every six male deaths in Glasgow, Scotland (Haddow, 1970).(2). Some investigators have attributed the increasing cancer incidence to the inadvertent introduction of carcinogenic agents into the environment as pollu tants (Boyland, 1969).(3) These agents, which represent low-level, long-term insult to the host, may represent one of the most impor tant factors in the etiology of malignant neoplasms in the general population (Saffioti, 1970).(4) The suggested correlation between exposure to an agent in the environment and occurrence of excess neoplasms in the general pop ulation has come about from the study of occupational cancers. The first such cancer was described over 200 years ago, and focused on the occurrence of scrotal cancers among chimney sweeps in London This work was NIEHS; Center* Post-doctoral* arted in part by the following grants from the it ES 00928; Career Award, ES 44812(AML), and towship, ES 02565(MSW) . 29 i it] il ECC-790 6113 18145 A.M. LANGER AND M.S. WOLFF (Pott, 1976).-(5) Since this time, many occupations have been scrut inized and studied, and agents found that have been correlated with the etio of specific malignant neoplasms (Hunter, 1969). (6) Some of tumors also have been observed in populations which are only, tly exposed to the agent, or which simply lived in the loc where^the agent was used. This has been defined in some de one"sucfHsubstance; asbestos (Selikofff and Hammond, 1968), (7 ich is shown to occur as an air pollutant (Selikoff et al., 1972)(8) and which commonly occurs as fibers in the lung tiss ues of individuals from the general population (e.g., Langer, et al., 1971;(9) Pooley et al., 1970, 1976).(10,11) B. Inorganic Particles and Disease Exposure to inorganic dusts in the workplace is capable of producing disease that may cause disability or premature death. These facts have been known since antiquity, and have been the sub ject of early extensive treatises (e.g.. Agricola, 1556).(12) Pneumoconiosis, a term that is recent in origin, was recognized first as a dust disease of the lungs (Zenker, 1867). (13) Indeed, it was not until the early 20th century that the most common of the dust diseases (silicosis) began to be understood on a sound basis (Collis, 1915).(j4) Since, this time, a number of other dust dis eases have been recognized' as related to exposure to inorganic agents. tHrpp have been reviewed extensively (Rosen, 1943;(15) Holt, 1957;(26) Hunter, 1969;(6> Aponte, 1970;(17) Lariqer and Mackler, 1972(18)1. Interestingly, not only have these diseases been reported in the areas where the materials are mined, but also in workplaces where milling, processing, and even use of the mater ials occur. Not all inorganic dusts produce only lung scarring; some dis seminate throughout the-host, producing reactions in extra-pulmonary organs, as is the case with silica (Holt, 1957).(16). In addition to scar-tissue development, a number of inorganic dusts have been implicated in the etiology of malignant neoplasms. Durinq the last 25 to 30 years, a number of inorganic particles have been implicated as the agent (or agents) responsible for malignant neoplasms in the workplace: chromium ores (Mackle and Greqorius, 1948); (19) heavy metals, especially lead, zinc, and copper (Breslow. 1954)? (20) hema tite ore, in the presence of free silica and other silicates (Lamy, et al., 1959;(21) talc (Kleinfeld et al., 1967);(22) uranium ore (Holaday, 1969).(23) Perhaps the best studied inorganic agent is asbestos. It is of particular importance because it is now used throughout the world, in thousands of products, so that it pervades all societ^(IARC, 1977) . (24\ To appreciate this great preoccupation with asbesSij^F-one must understand its disease potential. AS8ESTO: C, As: As) relativr potent ; with in* when Cot bestos i ports o; tosis O These ol have bet cancer. autopsy tos wor) among ir been exf epidemic cancer : In consider in the occurrer AOt only liduals" -mill In appearec among t> on the over to categor: (35) St\ some obs of asbes environc and Thor dividual where at holds or (Andersc BCC2731 6113 18146 *VOLFF -ch i in i in Tiond, 5 et -iss- al.# sub- /Iff ASBESTOS CARCINOGENESIS 31 C. Asbestos Exposure and Human Disease Asbestos, has been identified as a biologically active agent in relatively nflUfk-times. Asbestos fiber was first recognized as a potent fibrt|t^agent capable of producing a fatal lun.g scarring with intensi^H||pSJ.~severjLtyr~as observed in cases of silicosis, when Cooke, ||ig27 (25V noted-several cases of fibrosis among as bestos workaK^^Several years later, there were two separate re ports of primary bronchogenic carcinoma in individuals with asbestosis (Lynch and Smith, 1935, U. S. A.;(26) Gloyne, 1935, U. K.).(27) These observations were regarded as "curiosities" in that they may have been merely coincidental occurrences; i.e., asbestos and lung cancer. However, Wedler, in^L943(23,29) later reported several autopsy series in which the occurrence of lung cancer among asbes tos workers was far in excess of a similar matched autopsy series among individuals from the general population presumed to have not been exposed to asbestos. With the work of Doll in 1955(30), a firmrm*^ epidemiological basis was established for the occurrence of lung cancer in workmen exposed, to Asbestos fiber. 3, the *s iso # -S- 'nary -on to ast 'a ted the y hemasny, -S ies ation !%*> /iV In 1960, Wagner et al.(31) reported that a tumor, heretofore considered rare, frequently occurred among asbestos-exposed workers in the Northwest Cape Province of South Africa. They reported the occurrence of pleural nesothelLioma, which they suggested was a risk not only among occupationally exposed asbestos workers, but to indi viduals who merely lived in the area where asbestos was being mined and milled. Similar tumors were reported to have occurred intraabdominally in factory workmen in the United Kingdom (Keal, 1960).(32) In addition to mesotheliomas, Selikoff et al. in 1964(33) report ed the occurrence of excess gastrointestinal tumors among asbestos- ? exposed workmen as well. Less than 15 years ago, data accumulated suggesting that occupational exposure to asbestos fiber resulted in excess risk to asbestosis, pleural and peritoneal mesothelioma, lung cancer, and gastrointestinal cancer. ' In addition to workmen incurring increased neoplastic risk, it appeared that individualsTindirectlv exposed to asbestos were also among the "at risk" population. Several studies of shipyard workers cn the European continent demonstrated that asbestos disease carried over to all trades, rather than being confined to the insulation categories (or "laggers") (Harries, 1968;(34) Bohlig et al., 1970; (35) Stumphius, 1971;(36) Edge, 1976).(37) To add to these worri some observations, a number of papers also reported the occurrence l of asbestos-related tumors among individuals who merely lived in the*' environment of an asbestos plant (Wagner, et al.. I960:(Newhouse and Thompson, 1965;(38) Lieben and Pistawka, 1967).(39) Also, in dividuals wnc^Bjjrely lived within a short distance of the workplace where asbestc|g|4s.-being processed or used, and members of the house holds of asbe^aPworkers also incurred increased neoplastic risk (Anderson et jjpa-,1976). ( 40) B0C2732 32 A.M. LANGER AND M.S. WOLFF One can readily understand why asbestos has been investigated so intensively. The iiterature concerning this material has so in creased in the last decade that extensive review papers and bibliographigJB^Hf.-.been written (Harington, 1967; (41) IARC Monographs, 1972, 1 jpH|2^-24) Harington et al., 1975) . (43) Indeed, asbestos has beccflK^tt-'of thfi important 20th century carcinogens. D. Studies in Animals Utilizing a variety of animal species including substrains ancj colonies: rat, mouse, rabbit, guinea pig, gerbel, hamster; and diff erent routes of administration, such as oral, inhalation, intrapleu ral, intratracheal, intraperitoneal, subcutaneous, all asbestos fiber varieties have produced malignant tumors in animals (Tables 14-21, reference 24). All asbestos varieties have Induced mesoJdTeliomas, and most have produced other carcinomas.(43) Doses, ex posure intensities, fiber-types, and species of animals have been varied to establish carcinogenicity and dose-response curves for each fiber type.(24) E. In vitro Studies of Asbestos As the number of animal studies has grown, so have in vitro test systems. Most of these systems have dealt with relative tox icity of the fiber types and their ability to stimulate fibroblast growth and form scar tissue. Membrane systems (hemolysis) have pro vided a rough index of cytotoxicity, which appears to correlate well with relative fibrogenicity of the fibers.(43) However, little rel evance has been gained concerning carcinogenicity. Cell systems have concentrated on hemolysis of mature erythrocytes, alveolar macrophage systems (viability, lysosomal enzyme changes, phagocytotic properties, etc.), other cell lines, especially dividing cells? organ culture.involving specific tissues, e.g., tracheal transplant; and others; e^g., Davis, 1967;(44) Koshi et al., 1968;(45) Bey and Harington, 1971;(46) Allison, 1971, 1972;(47,48) Miller and H.arington, 1972).(49) Whereas the animal studies determined the fibroaenicity and carcinogenicity of the fiber types, so the in vitro test systems were oriented_toward determination of mechanisms. An extensive review of these papers may be found in Harington et al.(43) F. Status Concerning Asbestos Activity There continue to be many clinical and epidemiological studies concerning asbestos disease in workmen, an extensive literature concerning experimental carcinogenesis in animal models, and an ever growing literature in the area of mechanisms of interaction of asbestos fiber in cellular systems, all reflecting the importance of' asbestos as a diverse and complex agent in human disease, and a widespreaiBSSvironmentar contaminant. Significant quantitative and qualitati<HOgjie'stions remain including the relative hazards assoc iated witdfipF&h'"of the asbestos fiber types, and those mechanisms leading t ffltt'fease. ASBESTOS A. Ihe Asb silicate Normally asbestos used as commonly chrysoti bole sil amosite and trem viewed i 1977.(24 curled s has been X-ray cr ted area Jagodzin al., 195 Maser, e sue* p 64V- $tru P* composed and hydr sheet re the chry sheets a every 14 the stru the smal modation crystall the monc 1957). (r The minerals linked s The ampr. oxygens chain ax that is These ba BQCU793 6113 18148 so inbibliraphs, estos .ns and na diffcrapleu-os ables asoas, ex- been for : tro = toxcblast ave proate well ale relcems r.g cells; -.splant; :y and anng-,bro-tro a. An a al.(43) studies are an a ion of aance and a ave and assocnisms ASBESTOS CARCINOGENESIS 33 II. ASBESTOS AS A MATERIAL A. The Asbestos {isbe^MglglL.the name given to a group of naturally occuring silicate fibers in the serpentine and amphibole series, normally, B^nslx of these fibrous silicates are recognized as asbestos, although some rare forms of other mineral fibers may be used as well, e.g., fibrous clays (Whittaker, 1960).(50) The most commonly used fiber in the United States is the serpentine mineral chrysotile, sometimes referred to as white asbestos. Five amphi bole silicate fibers make up the other common varieties: actinolite, amosite (brown asbestos), anthophyllite, crocidolite (blue asbestos), and tremolite. The mineralThature of asbestos is extensively re viewed in Speil and Leineweber, 1969;(51) and in the IARC Monograph, 1977.(24) 1. Atomic Structure - Chrysotile has been shown to be a curled sheet silicate that spirals around a central capillary. This has been determined on the basis of X-ray diffraction, single-crystal X-ray crystallographic, transmission electron microscopic, and selec ted area electron diffraction studies (Bates et al., 1950; (52) Jagodzinski and Kunze, 1954; (53) Whittaker, 1956; (54) Zussman et al., 1957;(55) Kalousec and_Muttart, 1957;(56) Bates, 1959;(57) Maser, et al., 1960;(5S) Whittaker, 1960, 1963;(50,59,60) Huggins and Shell, 1965;(61) Clifton et al,, 1966; (62) Yada, 1967, 1971; (63,64) Langer et al., 1974).(65) These investigators determined the structure of chrysotile !to be the magnesium analog of kaolin, in which planar-linked silica tetra'nedra face an adjoining brucite sheet composed of magnesium ions coordinated octahedrally with oxygens and hydroxyl groups. Two of the three apical oxygens in the silica sheet replace the hydroxyl groups in the brucite sheet to complete the chrysotile structure. The distance between these adjacent sheets are on the order of 7.3 angstroms, with symmetry repetition every 14.6 angstroms. The curvature of chrysotile is a function of the structural mismatch of the larger brucite sheet "stretched over" the smaller silica sheet to structurally satisfy the cation accom modations. There are several structural types that characterize-the crystallography of chrysotile, the most important of which occurs in the monoclinic system and is"'called clinochrysotile (Zussman et al., 1957).(55) The structure of the amphibole minerals is more complex. These minerals are termed inosilicates, occurring as double chains of linked silica tetrahedra that are cross-linked with bridging cations. The amphiMfca-.chain is formed by coplanar sharing of two of the three oxygens arfeig base of the silica tetrahedra to form an infinite chain axisflEp&e double chain is completed by the third basal oxygen that is shSgi lietween, two opposite-facing, single-chain structures. These basic units make up the asbestos fiber axis (Deer et al.. B0C2794 34 A.M. LANGER AND M.S. WOLFF 1967;(66) Whittaker;(50) Ernst, 1968;(67) Speil and Leineweber.(51) The different amphibole asbestos fibers possess the same basic structtuBM^with small modifications brought about by chemical vari- partions within this structure alter interplanar spac- _/angle at which the adjacent units are stacked within (referred to as the beta angle, forming the inclined onoclinic structure). 2. Fiber Unit - Electron micrographs demonstrate that single chrysotile fibers (called fibrils) are formed as hollow tubes (the internal capillaries surrounded by the spiraling sheet). These structures have been demonstrated many times. Studies of their dimensional characteristics indicate that the single fibril may ranqe considerably with diameters ranging from 100-600 Angstroms (e.g., Langer and Pooley^ 1973;(63) Langer et al., 1974). (65) Bundles of individual fibrils form the chrysotile fiher. These may ranqe in diameter and in lenqth, from millimeters to centimeters. Amphibole asbestos fibers appear to consist of single crystals, twinned crystals, and stack-faulted fibers, in which the width dis tribution tends to be different. It has been observed that the different amphibole varieties behave differently when comminuted to form width distributions that are unique and characteristic for each mineral species (Timbrell et al., 1970, 1971; (69,70) Timbrell, 1972).(71) These studies demonstrated that crocidolite forms the shortest and thinnest fibers, followed by amosite, followed by anthophyllite. The loq normal distribution of such width distri bution appears to center-at about O.liyand smaller for crocidolite, 0.16yfor amosite, and a polymodal distribution, greater than 0.20y for anthophyllite. The importance of such characteristics will be discussed in the section oh inhalation potential. However, all of the amphibole fibers appear generally as straight rods when exam ined by electron microscopical techniques, with diffraction contrast figures resulting from flexing of the structure under the electron beam; curved fibers are noted as well.(65) It has been suggested that some of the internal-structures of amphiboles, a fine, 20-50 Angstrom internal lamellar structure parallel to the fiber axis, may reflect twinning which imparts the unusual optical and tensile strength characteristic of the fiber (Seshan and Zoltai, 1976).(72) 3. Chemistry - The empirical chemical composition of chryso tile is Ma sSi205(OH)u. Although magnesium predominantly occupies the octahedral site within the structure, it is also common to have iron, nickel, and manganese substituting within this site up to sev eral tenths of a percent. Occasionally, aluminum may substitute for silicon ijastructure, and fluorine may occupy several of the hy droxyl pnBgrffons. In the Canadian chrysotile samples, originating from ulti^gEflc rock types^ the high iron content may be attribu table to _|r.growths_ of the mineral magnetite within the chrysotile ASBESTOS fiber b dolomit growth logical iaentif in cnry chrysot 1956;(7 1956;(7 1965;(7 Th complex by the origin; oral ff or hydi 1969, ' 1971) . impurit taminat 1952; (t BCC-735 6113 18150 !^COLFF (52 ) sic si vari- spac/ithin dined single (the "se eir siay roms 1) ese may ters. rvstals, th disihe ited to ,for each 11 s the ~y -tri.dolite, c- of xamontrast rtron sted 2-50 is, may A . (72) ASBESTOS CARCINOGENESIS fiber' bundle. Serpentine deposits that originate from silicified dolomites to have high calcium oxide content, reflecting inter- growth o te minerals. Other trace metals that may have bio- logical nce are nickel, chromium, and cobalt, which have been identifi ncerrtrations up to several thousand parts per million in chrys- originating from ultranraf ic rocks. The chemistry of chrysoti s been discussed at length in several papers: Pundsack, 1956; (73) Pundsack and Reimschussel, 1956;(74) Nagy and Faust, 1956; (75) Brindley and Zussman, 1957;(76) Gaze, 1965;(77) Harington, 1965;(78) Speil and Leineweber; (52) and Table 1.(24) The chemistry of the amphibole asbestos minerals is far more complex than the chrvsotile mineral. Generally, it may be described by the structural chemical formula:(67) w0. -1. x2, yo_ (Si40n)2 (0,0H,F)2 For anthophyllite, no cations occupy the W structural site, and the X and Y cation sites are theoretically filled with magnesium. There is limited substitution of iron for magnesium in the structure, and some limited substitution of aluminum for silicon. Amosite has no cations in the W site also, and contains almost total iron in the X and Y sites. Some magnesium does substitute for iron, as well as manganese. Actinolite and tremolite, both forming the end members of a solid solution series, may contain some calcium, sodium, or potassium in the W site, in limited amounts. The X site is filled with calcium and the Y sitejwith magnesium and iron (for actinolite the iron is greater than the magnesium, for tremolite the magnesium is greater than iron). Manganese may occur in these sites as well. Crociaolite may have traces of calcium and potassium in the W site, contain sodium in the X catign site, and iron in the Y site. Antho phyllite may contain traces::pf aluminum which substitutes for sili con in the basic chain structure. Crocidolite may contain substantial amounts of magnesium and occasionally traces of aluminum as well. Originating from different rock sources, there are a number of different trace metals that flay be associated with the amphibole asbestos varieties, again a function of provenance and geological origin. This is reviewed in_Whittaker;(50) Ernst;(67) Speil and Leineweber;(52) and in Tables- 5, 6, 7.(24) It is of interest to note that both chrysotile and the amphi bole asbestos varieties may be contaminated with hydrocarbon traces, originating either from the geologic environment at the time of min eral formation (or migration of hydrocarbons after rock formation) or hydrocarbon qontamination^from processing and storage (Gibbs, 1969, 197Chfl5j?,f.80) Commins-and Gibbs, 1969; (81) Gibbs and Hui, 1971) . (82) fife addition to these sorbed hydrocarbons, occasionally impurities Sgit the metals encountered during processing might con taminate thJHfsamples "as well (Ayer and Lynch, 1967; (83) Baddolet, 1952;(84) Baddolet and Edgerton, 1961(65)). BCGLT3S 6113 18151 lil 36 A.M. DANGER AND M S. WOLFF 4. . Surface Charge - Chrysotile asbestos has a surface layer of magnesium hydroxide groups bonded to the silica layer through the sharing <flfejygens. The surface hydroxyl layer, having protons outermost, a net positive charge to the fibril surface. Zeta potent^gjffiSasurements show a surface charge of +40 to +100 mv from pH 4 tf^g|py 8.(73) The amphibole asbestos fibers tend to have surfacBfefs composed predominantly of silica, with oxygens on-the surface rendering a negative charqe. Surface charge measure ments of these fibers have been studied as a function of pH, ionic ' strengths, and differing cation concentrations of the media (Prasad and Pooley, 1973).(S6) The negative range of zeta potential for the amphiboles is similar to"~that of quartz, but smaller in magni tude. 5. Mineral Stability ^_It. is known that chrysotile, asbestos possesses a chemical stability only in liquid media with pH centered at 10.8. Contact with solutions below this pH value results in magnesium loss from the fiber. (Hargreaves and Taylor, 1946).(87) This instability has been noted in the mineralogical literature and was demonstrated to take place when the fiber was retained in resi dence in living tissues (Morgan and Holmes, 1970;(88) Danger et al., 1970, 1972;(89,90) Morgan et al., 1975).(91) Chemical degra dation of the amphibole minerals in vivo has also been observed, mostly by electron microprobe characterization of fibers in tissues. III. FACTORS TO BE CONSIDERED IN ASBESTOS CARCINOGENESIS A. Fiber Types and Malignant Disease in Humans Currently, all asbestos fiber types have been associated with malignant diseases: chrysotile exposure; e.g., McDonald and McDonald, 1976,(92) amosite exposure; (8,9) and anthophyllite ex posure (Meurman et al., 1974).(93) A detailed summary, reflecting the world literature concerning human populations studied, is pro vided in Table 22.(24) Exposure to, all asbestos fiber types, with the exception of anthophyllite, has been observed to be associated with mesothelioma (both pleural and peritoneal); all asbestos fiber types have been implicated as an'agent with bronchogenic carcinoma; chrysot:iile, ^ j crocidolite, and amosite have been associated with excess qraassttrro- J ' intestinal cancers. In addition to these tumors, chrysotile and/or the amphibole fibers are implicated!in excess malignancies of the trachea, kidney, and central nervous system among insulation workers 7 who use all fiber types. In addition to implication of all fibers with many tumor types, multiple primary tumors also have been found in single individuals exposed to asbestos. (94) MultiplfdjSshors in the same individual and in different indi viduals poin5b^the fact that asbestos fibers disseminate once in- ASBESTOS CAf haled and/c mation and exist as tt types (epit B. Fiber; A numi cf asbesto; workemn exj Most of th( fibers in i copy. Morf abundance . onment, as fended to 1 C. Retjpit The p: of exposure Hon rates animal mod1 exposure. idolite by left.3S aer et l_wprp sent IB of the ori tained. F cause extr amphibole grades in plicatinq D. Dose Util i loqical st nant tumor exposure t appears tr the miner; mesothelic fluenced 1 increases for factor holdinq a", also founc period im BQC2737 6113 18152 ^^LFF ^B^er ugh rotons a LOO mv -O Tygens easureionic Prasad for 'agni- estos 3ntered in 37) re and . resiet iegraed, issues. 3X- iting : pro of ^lioma ,seen -,roind/or the workers ibers found frtmt. *MI mumWTIltjaa HWjUwww --- T-nn^flWffliT I i - ' '* ASBESTOS CARCINOGENESIS 37 haled and/or ingested.(95,96,97,9?,99,100,101,102). Such infor mation andJata point to the fact that a number of different organs exist as for asbestos, as well as at least two major cell types (ep{Qgljp&l and mesenchymal cells). Tissues A number cf studies have been made demonstrating the presence of asbestos fibers in the lung and extra-pulmonary tissues, of vorkemn exposed to asbestos- fiber.(11,89,90,102,103,104,105,106) Most of these:studies demonstrated the prevalence of submicroscopic fibers in relation to those that may be observed by liaht micros copy. More importantly, asbestos fibers were found in greater abundance in individuals exposed to the material in the work envir onment, as compared with those in the general population (which tended to have fewer and smaller fibers). C. Retention of Fibers -- The presence of fibers in tissues, many years after cessation of exposure, points to retention for long periods of time. Reten tion rates experimentally determined.appear to be related to the animal model, route of administration, and fiber type involved in exposure. For example, rats exposed to~milligram amounts of crocidolite by inhalation were observed to retain about 35%, with about 9% left 30 days l-ater.(107) In inhalation studies in rats by Wagner et al., 1974(105) amphibole minerals (crocidol-ite and amosite) were retained, as much as 26% of the original amount being present 18 months after cessation of exposure; however, up to 59% of the oriqinal dosaae of the larqer anthophyllite fiber was re tained. Elimination rates for chrysotile were not. calculated be cause extremely low concentrations were found as compared to the amphibole fibers. Since iiflis known that the chrvsotilj? fiber dearades in vivo, it tends to become lost in this manner, thus com plicating rerention estimation. D. Dose Response and Latent Period Ftilizinq human studies in retrospective-prospective epidemioloaical studies, it has become apparent that the different maliqnant tumors possess different laihent^jggj^gds between first onset of exposure to dust and clinical appearance of disease. Lung cancer appears to peak after some.25-30 years from onset of exposure to the mineral dust; pleural mesot-hpl i nma. ^-in^vpars: peritoneal mesothelioma, 35-40 years. (7097 in, 771) Latent "fluenced by dose,in that the latent period for increases as the intensity of exposure decreases. for factoiSwprkers exposed to amosite. (5,9) In this latter study, holding alfi&pfeher factors equal, demonstrable neoplastib risk was also foundS5pi,Idecrease_ as the exposure decreased and the latency period incnrased; that is, those individuals exposed for short time 1 i: B0C273S 6113 18153 38 A.M. LANGER AND M.S. WOLFF periods had a significantly decreas'd attributable risk to neoplas tic disease which, when it occurred, appeared only after a qreatly extended time period. It is perimental dose-respon tion.(112) erest to note that several investigators, in ex systems, have produced data suggesting that a ts for lunq scarrinq, but not for tumor produc es was also-suggested by Gold in 1970(112) who cor related the number of asbestos bodies in lunq tissue with severity and extent of fibrosis, but no such correlation existed in individ uals with tumors. Importantly, an "excess" dose which may produce asbestosis in animal or man,_may produce death in the orqanism be fore neoplastic rhange can occur. Thus, competitive risk between asbestosis and cancer suggests that high exposures may result in 7 asbestosis, whereas lower exposures-wm-id* produce an. increased life span, and a long enough latent period for the tumors to become manifest. On the basis of examination of the best cohort studies avail able, coupled with exposure data, Schneiderman, in 1974{114) con cluded that a zero cancer response may exist only at a zero exposure level. E. Cofactors in Disease On the basis of a number of epidemiological studies of asbes tos workers, it has been demonstrated that those individuals who smoke cigarettes and are exposed to asbestos incur a significantly elevated risk of developing lung cancer.(7,115,116,117) Indeed, the risk of bronchoqenic carcinoma has been demonstrated to be greater than 9.0 times more for the cjqarette-gmnkl.no a-thgetne unr<er than for a nonciqarette-smoking individual from the qeneral population who does not work with asbestos. Cofactors in the in duction of tumors in animals have been investigated in a dumber of laboratory studies as well. (115,119,120) As in the human studies, it has been demonstrated that polycyclic aromatic hydrocarbons, in addition to asbestos exposure, qreatly enhances tumor risk in liv ing orqanisms. The bronchoqenic carcinomas in cigarette-smoking asbestos workmen are positioned unlike the bronchoqenic carcinomas found in cigarette smokers; first, they tend to occur more peripherally and not in the main bronchus or bronchi of the individual; the tumor cell tvne tpnds to he more undifferentiated and of the more virulent "oat cell" variety. F. Size, Slype Characteristics Fiber sfife. includinq both diameter and length, influences the relative "toJB&ity" of the dust indirectly and directly. Particle ASBESTOS CAR size affect logical act potential; response; a. 1. A the work en because the meter.(121) in the tent spherical p fibers tend potential a 2. I work envirc ing upon th and the inc individual, be primaril 3. length) the greater ret ianissrS; a) that by,and ers: (<B> It is of i: reach the r ral surfac- 4. migrate am observatio gesting th 5. has demons rest in th of shorter fibers in greater air areas. Cc cytosis of "frustrate lead to collagen c human bas: B0C2799 6113 18154 .VO IFF s- y ex- 3 :uc:orrity ivid'riuce ` bepen in -ecome ailonposure ntly # :nr of ies, , in liv- a an and T rulent s the acle ASBESTOS CARCINOGENESIS 39 size affects many important properties to be considered in the bio logical activity of dusts: stabilitv of the aerosol; inhalation potential; s reaction; migration potential; type of cellular response; a ace area. 1. Ae^rejgg - Small asbestos fibers form stable aerosols in the work ern^PPSient. Asbestos fibers are aerodynamically stable because their falling speed is primarily determined by fiber dia meter. {121) Fiber diameters for the asbestos minerals tend to be In the tenth micron range, which compares with falling speeds of spherical particles in the submicron size range; therefore, asbestos fibers tend to form stable aerosols, thereby increasing inhalation potential and dosage. 2. Inhalation Potential - As asbestos dust persists in the work environment, the inhalation potential is increased. Depend ing upon the concentration of the dust, the duration of exposure, and the individuality factors-involved; e.g., physiology of the individual, etc.; the risk associated with each dust appears to be primarily related to particle size.(122) 3. Site of Reaction - With smaller particle size (primarily length) there is deeper penetration to the alveolar spaces and greater retention by means of - both diffusion and inertial|impaction mechanisms.(71) This latter study demonstrated that long fibers (>8pm) that come to rest in the trachea and tracheal bronchial tree are by and 'arge eliminated, or cause fibrosis, whereas the smaller fibers (<8im) penetrate to the alveolar spaces and are retained. It is of interest to note that the smallest fibers are those that reach the mesothelial lining Of both the visceral and parietal pleu ral surfaces. 4. Migration Potential - Small fibers tend to be those that miqrate and that are found ini extra-pulmonary organs.(102) These observations have been made by other investigators as well, suqgestinq that small fibers miqrate more efficiently than'large ones. 5. Type of Cellular Response - A number of investigations has demonstrated that the lonqer asbestos fibers tend to come to rest in the upper and middle bronchial tree, whereas the majority of shorter fibers lodge in the alveolar spaces. Long and short fibers in the tracheal bronchial tree tend to be eliminated in greater amounts than those that come to rest in the more peripheral areas. Complete phagocytosis of short fibers and incomplete phaqocytosis of long fibers, which Kuschner and Wright(123) have termed "frustrated g|gocytosis," occurs. This incomplete phagocytosis may lead to "leaKgjf" of lysosomal enzymes and cell death, followed by collagen dev^jEtunent. Observations, on both an experimental and a human basis,demonstrated that asbestosis tends to occur with tj *\ t t1 r Cl if \ 6113 18155 40 A.M. LANGER AND M.S. WOLFF long fibers.{124} Although peribronchiolar fibrosis is observed in humans (long fibers only?) the characteristic scar pattern developed involves sepdBBgiells as well. Long and short fibers are. impli cated , althotMBppme investigators consider long fibers more potent. Long fiber a^BSplty has carried over into the carcinogenesis area in that some Wlggstigators consider long fibers responsible for carcinogenes^Brire well.(125,126) Indeed, a number of experimental pathology studies utilizing short fiber have reported no responses in laboratory animals, thereby dismissing short fiber ( < 5u in length) as biologically innocuous.(124,127,128,129,130,131) It is of interest to note that each of these investigators produced short asbestos fiber by vigorous mechanical methods (e.g., ball-milling) which may have significantly altered the fiber surface and bio logical properties.(132) Indeed, a number of workers has demonstrated short fibers to be extremely active in a number of animal models.(133,134,135) These investigators produced short fibers using special techniques that prevented surface destruction. They remarked that most of the disease processes were intracedlular in nature and required small fibers at the onset. Pott et al., 1972(136) and Pott and Fried richs, 1972(112) noted the importance of short fibers in tterms of human carcinogenesis. Suzuki^andchurg, in 1969(137) noted cell ular response with chrysotile fibers less than one micron in length. These were also phagocvtized by attached epithelial cells and were observed to be present in alveolar septa where fibrosis and thick ened membranes indicated marked activity. Wagner, in 1968(138), and Wagner and Berry in 1969(139) produced more mesotheliomas with a superfine chrysotile fiber (92t <6u in length) than with any other fiber used experimentally. Indeed, this group reports that the smaller the fiber, the greater its carcinogenic potential.(135) These investigations are^supported in part by human data that suggests that the finer crocidolite fiber encountered in the North west Cape Province of South Africa produces more mesotheliomas as compared with similar fibers in the Transvaal of South Africa.(71) This feature seems to support the concept that small fibers are active in terms of carcinogenic^potential. G. Structure of the Fiber Bundle It has been proposed that the physical character of asbestos fiber is responsible for its biological acitvity; i.e., its polyfilamentous character.(140) Chrysotile asbestos was examined in various states and it was concluded that only when chrysotile was used in fiber bundle form did it possess biological activity. How ever, monofilljtentous fibrous glass has been demonstrated to pro duce tumors i'jnimals; (125) chrysotile reduced to single fibrils by vigorous m-fiEanical methods produced no response in animals;(127, 124) which maTFHS# explained by alteration of its surface activity; ASBESTOS CAI (132) "monc and Hanley duced by e> into an ent 1. i asbestos t* face areas the surfac* interactioi small part action. 2. ] asbestos i releases m. 142,143,14mented as bole fiber nificant r 3. ling such For exampi h carisa h net n pulling eral of th increase t far in exc osmotic fc cellular s might not magnesium have the a at a hight system, h type and i inma yielc 1973.(235; and carcii Macn; activitie; their maqt for magne: than untr< also grea- BGC2501 i WOLFF ;served in * developed impli:re potent, iis area _e for ;rimentai esponses S U in i) It is ced short -on 1 ling) i bio- -bers to .135) -chniques ;st of the =d small i Friedrarms of id cell- m length, and were d thick(138), and ^jih a ^^ither S^he .(135) .ata that -.he North.omas as "ica.(71) -s are -.sbestos :s poly.ned in ile was tty. How to pro fibrils .mals;(127, ictivity; % ASBESTOS CARCINOGENESIS (132) "monofilamentous" chrysotile asbestos fibers used by Gross and HadBhfein 1973(140) to demonstrate lack of activity was pro duced Mptreme heat, and thus dehydroxylated and recrystallized into a^gjlgirely different mineral phase. (132,51) urface Area - It has been estimated that several of the asbestos types, especially chrysotile, may be comminuted until sur face areas reach 90-100 square meters per gram of material. Since the surface of the alveolar spaces is about 100 square meters, vast interaction phenomenon can be possible. Large surface areas with small particle sizes provide great potential for cellular inter action. 2. Fiber Chemistry - It has been demonstrated that chrysotile asbestos is markedly unstable in an acid environment and readily releases maqnesium in aqueous media with pH less than 10.8.(141, 142,143,144) The In vivo degradation of asbestos has been docu mented as well, with magnesium loss from both chrysotile and amphibole fibers observed.(90) Thus, fiber chemistry may play a sig nificant role in carcinogenesis. 3. Magnesium - Magnesium may be an important ion in control ling such reactions as cell membrane lysis (especially the red cell). For example, sialic acid groups protrude on the surface of the cell, with carboxyl units outermost. These units are hydrophilic, with a hiqh net negative charge. It was postulated that magnesium ion, lost from asbestos fibers, interacts with these carboxyl groups and by pulling on the oppositely charged glycoproteins, cross-links sev eral of these groups at the surface, forming channels that tend to increase the passive permeability of both potassium and sodium ions far in excess of the normal transport system. (145) This induces an osmotic force within thfFcell, causing sodium and water accumulation, cellular swelling and bursting. Such an effect, it seems to us, might not necessitate removal of magnesium from the fiber. In fact, magnesium structurally confined by the fiber crystalline matrix would have the ability to stereochemically interact at membrane surfaces at a higher effective concentration. Experimentally, the membrane svstem, hemolysis, is used to relate to cytotoxicity.(45,146) Fiber type and hemolytic activity are apparently related to the mesothel ioma yield as observed in chrysotile-exposed rats by Wagner, in .1973 ,(135) The d irect .relationship between these membrane studies and carcinogenesis is presently unknown. Macnab and HaringtOn, in 1967(148) suggested that the relative activities observed for the asbestos fiber types may be related to rheir^Bgriesium concentrations. Using chelating acfcnts specifically for mJj^sium, treated fibers were observed to be fhr less hemolytic chan jfijreared fibers. The leachinq of .magnesium from the fiber also greatly decreased tfhe hemolytic potency. The use of several t t ;l 1 l i! J: i .. * 1 ii BCC2S02 A2 AM. LANGER AND M.S. WOLFF varieties of chelating agentcalcium specific versus magnesium specific, demonstrated again the imcortance of magnesium in mem brane intera ,, i48) A number of adsorbed chemical aqents have been used to [g.onize the hemolytic response. (146,148,149,150) Again, these1 %stigations suggested the importance of magnesium, However, th ihelation and adsorption studies do not preclude more cotnple face mechanists such as those involving silicate or-hydroxyl groups. 4. Iron - A number of -investigators have considered iron and several of its compounds as possibly being involved in carcinogen esis. Some have suggested that iron plays a role in enzyme blockage, in interference with the immune system, and in upsetting ironsensitive cellular energy dynamics and enzymatic transformations through interference with iron+2: iron+3 equilibrium.(151,152,1511 As is the case with magnesium, the iron contents of the vari-us as bestos fibers range greatly, jand those iron-rich fibers tend to keep this cation structurally bound when in biological residence. The low iron content of certain biologically quite active fibers speaks against its being the only factor in carcinogenesis. 5. Silicon - The question may be posed whether silicon as a silicate entity is a chemical carcinogen. Several investigators consider this unlikely in that silicon is normally present in ban at concentrations that are detectable.(154) However, exposed silica surfaces (whic can exist in amphibole asbestos minerals, and in cnrysotile that has been degraded by organic acids in vivo) may act as powerful hydrogen-bonding agents.(255J Nash et al. sug gested that hydrogen donors, especially those of the phenol con figuration, can be extremely^damaging to living cells. Silicic acid, as well as other weak acids, may act as hydrogen donors. 6. Hydroxyl Groups - The concept of hydrogen donors in the form of "phenolic-like" groups as damaging to living cells as dis cussed by Nash et al. may be the fundamental, or at least initial approach to understanding biological interaction of asbestos, which inevitably contains active surface or interlayer hydroxyl groups in the crystalline structure. Pundsack(73) estimated that 7% of the total hydroxyl groups of chrysotile were on the surface of the fiber. Allison(145) described such hydroxyls as phenolic-like in activity, and we have found that these surface hydroxyls interact With a number of organic compounds to reduce or oxidize them.(132) We have observed chemisorption, free radical and acid-base interactions at the surface of the fiber which, suggest that the surface of chry sotile is amphoteric (as befits magnesium hydroxide). SUch re actions arefc\ributed to surface activity related to the phenolic groups. Eophenomena vary greatly as a function of the pretreat ment of the^fcber (especially heating and other manipulations which alters the flftface configuration of the phenolic groups). These ASBESTOS CAR reactions c usually to least quali tation of t ly reactive / i bestos fibc ing up to ; the concept early in a; of action, cause of e: duced in ai of trace m< important, 8. that the aers are re theory can bestos car izing fibe However, t >ny exper ^dch para incide nical r he benzof actions w and enaogt asbestos ' of the accinogen; < on asbest pounds, perylene, surface c has been specific (through others pr Those pol or magnes BCC2303 6113 18158 m .m n,ave '0) xum. n and ?enockage, ns 153) 3 C.S -- D ~e. rs is a "S nan silica n :n e ,is, al nich s in -.he fiber. ity. Cions hry- eatnich ASBESTOS CARCINOGENESIS . 43 reactions oc usually to least quali tation of t ly reactive r with other magnesium silicates and amphiboles, smaller degree, which can be accounted for at ely by the stereochemical and electronic presenOH groups. Such reactions have been seen at high- fclytic surfaces, e.g., for zeolites. 7. Trace Metals - As indicated previously, most of the as bestos fibers contain significant quantities of trace metals (rang ing up,to several thousand ppm). It is of interest to note that the concept of nickel, chromium, and cobalt activity, implicated early in asbestos carcinogenesis research as one of the mechanisms of action, is now less important. The idea lost favor mainly be cause of experiments wherein the same number of tumors were pro duced in animals utilizing fibers with and without detectable amounts of trace metals.(139,135) However, the role of trace metals may be important, and can be considered in the category of iron. 8. Adsorbed Hydrocarbons - A number of studies has Suggested that the adsorbed hydrocarbons on the surfaces of the asbestos fib ers are responsible for their carcinogenic properties. Again, this theory cannot account entirely""'for the general phenomenon Of as bestos carcinogenesis, since tumors were produced in animals util izing fiber with and without adsorbed hydrocarbons.(139,147,156) However, this concept should not be discarded since it represents many experimentally substantiated synergistic carcinogenesis studies, which parallels strikingly the! asbestos-cigarette smoking lung can cer incidence in man. Furthermore, in the light of the very active chemical redox reactions observed by us and others, it is clear that the benzopyrene-asbestos synergism may be less important than inter actions with more potent electron donors and acceptors, exogenous and endogenous. It is also of interest to consider the interference of asbestos with repair mechanisms in this regard, so that the presence of the active fiber would (a) prolong the insult of a proximate car cinogen; or (b) inhibit repair processes. The potential carcinSgenicity of the hydrocarbons adsorbed on asbestos has led many investigators to study these surface com pounds. Polycyclic aromatic hydrocarbons such as phenanthrene, perylene, anthracene, and the carcinogen benzpyrene, adsorb on the surface of chrysotile.(156' Adsorption of various organic polymers has been studied extensively by Schnitzer et al.(150) Indeed, specific polymers adsorb more readily to the surface of chrysotile (through intwScation with the -"phenolic" surface groups) whereas, others prefe4(B3*e oxide surface of the amphibole asbestos fibers. Those polymes^fchat theoretically inactivate the surface hydroxyls or magnesiumdjt their adsorption also reduce hemolytic potency. 1 B0C2304 6113 18159 8! 44 A.M. LANGER AND M.S. WOLFF 9. Surface Sorption Properties - It has been nbted that as bestos fibers; e.g., chrysotile, can adsorb five times more protein from human serum than many other mineral species; e.g,, quartz.(147) The ad S' ionof specific substances at the surface of asbestos, is well kn The physisorption of benzene, ethanol, hexane, and other organi .ids has been reported, with polar molecules possessing a great inity for chrysotile as compared to the amphiboles. This S' has been investigated by Gorski and Stettler.(157) This aspect can be independent of those discussed here; for example, by causing an imbalance in cellular components. One cannot readily distinguish between true surface phen omenon, or physical-chemical interaction. It is known, for example, that proteins may be denatured at the surface of quartz particles by interacting with the surface oxide groups with the : hydrogens on the amine groups of the proteins.(158) This mechanism of inter action may take place at the surface of the amphibole asbestos fibers, or at the surface of chemically degraded chrysotile. It has been reported that silicon may be chelated directly from the surface by serum proteins, causing possible changes in the organic materials.(159) Indeed, these investigators consider the surface of silica compounds as the key to biological interaction. In add ition, such oxide surfaces provide secondary amide-hydrogen bonding to amino acids.(160) The extent of this interaction in terms of carcinogenesis is presently unknown. In addition, direct adsorp tion of substances including "tumor inhibitors" has been proposed by Oppenheimer et al.(161) and by Bischoff and Bryson.(154) Asbestos fibers adsorb a number of organic compounds. These organic compounds are both endogenous to the organism and exo genous, derived from pollutants. These materials tend to be non polar for the amphibole asbestos types and polar for the serpentine asbestos variety. The mineral surfaces can act as solid-state cata lysts' and may reduce and/or oxidize these compounds, creating al tered forms. It has been suggested that the denaturation of pro teins may produce materials that are antigenic to the host, iniatir.g an immune response. Protein or polysaccharide adsorption may simply cause an undesirable concentration gradient or stereochemical effect (e.g., membrane configuration). Adsorption of other bio chemical substrates such as those leading to hemosiderin deposition may interfere with iron-dependent energetics (redox). It has been reported that silicotic nodules contain substances rich in gamma globulin.(162) As in the present instance, no specific antigen or immunological factor has been observed for the asbestos minerals. In addition to the surface-controlled chemical phenomena, ihere agtoars to be chemisorption which may involve specific com ponents ffi^.the organic compounds, producing alteration to specific sites o(3S^e organic molecule. Again, these reactions may involve ASBESTOS either < amphotiu the suri Chi hundred length, durable halatioi produce of the short d the par act ion nma cha once in other o spleen, publish Sm phagocy of chry perimen liar an . activit phagocj cant lc T1 ever, n Once pi sotile present like h1 geometi macromi in imm the mi the st (espec C its ch ox id at metals molecu eCC-305 6113 18160 LFF pie, or .na. mu ASBESTOS CARCINOGENESIS 45 either oxidation or reduction reaction. Some surfaces may behave ampnoterically and may change with time as chemical degradation of the surface proceeds, as is the case for chrysotile. (132) IV. S ACTIVITY: CHRYSOTILE AS A MODEL Chrysotile^Bfe' comminute to particles that are only several hundred angstreifty in diameter and a few thousand angstroms in length. The aerosol formed by such particles is extremely stable, durable in the environment, and has associated with it a high in halation potential. The inhalation of such small fibers would produce deposition in the alveolar spaces and the peripheral areas of the respiratory tract. Such fibers may have only to miqrate a short distance to reach the mesothelial surfaces of the lung and the parietal pleura. The fibers could be easily available to re action to form both mesotheliomas and the peripheral lung carcin oma characteristic Of asbestos exposure. In addition to its pres ence in the deep regions of the lung, chrysotile may migrate to other organs for interaction. We have observed fibers in liver, spleen, testicle, brain, and intestinal tissue (Langer et al., un published results). Small particle sizes suggest that these fibers are easily phagocytized and removed from the lung tissue; hence, the levels of chrysotile observed in lung tfissues (even in highly dosed^ ex perimental animals! are often very much less than observed in sim ilar animals exposed to the amphibole fibers. Also, the chemical activity of chrysotile is such that one would suspect that both phagocytosis and chemical degradation would account for a signifi cant loss of fiber from the lung. This very process of chemical degradation of chrysotile, how ever, may provide several major mechanisms for cellular interaction. Cnee pnaqocytizpd by cells within the lung, the surface of chrysotile structure may readily oxidize and/or reduce organic bompounds present within the cell. Release of magnesium may follow pfienoliclike hydroxyl group interaction, causing alteration of membrane geometry, and cause lysis. Denaturing of proteins, alteration of macromolecules of various kinds, or adsorption of proteins involved in immunological processes also may take place on the surface of the mineral. Trace metals may be released, or trace .iron within the structure be present, causing interference with enzyme systems (especially those that are governed by iron equilibrium). Chrysotile asbestos offers'continuous interaction throughout its chemical degradation ("phenolic" interaction at the surface, oxidation-redij^fion reaction, magnesium release, iron and trace metals releas^fcsilicic acid release) or even a reaction with macro molecules, meiggiy providing a "passive" mechanism for alteration. i. K' 7i BGCL306 6113 18161 46 A.M. LANGER AND M.S. WOLFF Ind^^^all these processes may take part in its biological activity. greater membrane activity of chrysotile versus amphibole asD4K'Os is, we hypothesize, a more complex event than any single potential interaction. With chrysotile, interaction may proceed through sequential layers: proton or hydrogen radical: hydroxyl ion or radical,- oxide (Mg-O) ion or radical: magnesium ion: sili con-oxygen or hydroxyl group: silicon ion or radical: cation or anion substitutions (Fe, F-) . The various reactions through such layer stripping have extreme potential biological ramifications, especially when combined with the stereochemical control that can occur at its well-defined crystalline surfaces. Amphibole fibers possess all of these molecular possibilities, in an entirely different sequence and chemical range. These factors, coupled with factors that are related to the host conditions of work and the presence of other agents that might act synergistically with the fiber, underscore the complexity of the problem. Investigators in this field cannot help to amplify the observations of Eryson and Bischoff(159) who said: "Considering the extent to which silicates comprise man's environment on this planet, and bearing in mind the silicate-induced diseases (including cancer) had been recognized since antiquity, the sparsity of animal experimentation concerned with silicate carcinogenesis is striking and deplorable." Asbestos research has gone beyond the "sparse" stage and has been increasingly detailed and focused. The problem is not so much the lack of experimentation as the complexity of mechanisms of car cinogenesis. REFERENCES 1. Silverberg, E. and Holleb, A., Cancer Statistics, American Cancer Society, New York, 1972, 1971. 2. Haddow, H. A., Health Page 23, summer 1970. %i Boyland, E., Prog. Exp. Tumor Research 11, Page 222, 1969. 4Test Saffioti, U., Evaluation of Environmental Carcinogens: Ad Hoc Committee on,fhe Evaluation of Low Levels of Chemical Carcin ogens, Report to the Surgeon General, USPHS, April 1970. ASBEST 10. BGC~807 6113 18162 Vi.S. WOLFF [ivity. '.hibole single 'oceed :roxyl sili'n or ;n such ions, -.at can oilities. :o the -.at might ity of .the :y the -se T 1 Q #rs much 5 of car- ASBESTOS CARCINOGENESIS 47 Pott, ?.,' Chirurgical Observations, Hawkes, Clarke and Collings, London, U.K., 1976. ,, D., The Diseases of Occupations, English University London, 1969. 7. SeliSPEf, X. J., Hammond, E. C. and Churg, J., J. American Medical Association 204 , Page 106, 1968. 3. Selikoff, I. J., Nicholson, W. J. and Langer, A. M., Arch. Environmental Health 25, Page 1; Selikoff, I. J., Hammond, E. C. and Churg, J., Ibid, Page 183, 1972. 9. Langer, A. M., Selikoff, I. J. and Sastre, A., Arch. Environ. Health 22., Page 348, 1971. 10. Pooley, F. D., Oldham, P., Chang-Hyum, U. 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