Document pp3jye8xa5qZOaOrV07vjk5aB

JOURNAL OF THE AMERICAN COLLEGE QF TOXICOLOGY Volume 9. Number 5,1990 Mary Ann Liebert. Inc.. Publishers Mechanisms ol Asbestos Carcinogenicity PETER VOYTEK. MIRIAM 4-NVER. TODD THORSLUND, JILL CONLEY. and El IZABETH ANDERSON ABSTRACT We present a brief review of different potential mechanisms at the molecular and cellular levels that may be involved in asbestos-indufed carcinogenicity. The usefulness of considering such mechanisms in developing appropriate lioiogicaily based models to estimate carcinogenic risk at environmental levels of asbestos fibers discussed. INTRODUCTION sbestos is a general commercial name for a group of mineral fibers of hydrated silicates with a variety of Aphysical characteristics and metal compos itiions.' Occupational exposure to several types of asbestos fibers has been shown to have a causal rtlationswh;nth> the development of lung cancer and malignant pleural mesotheliomas.(:J' However, animal studies lave shown that morphological difference and sire can resuit in different carcinogenic potencies.14-6' Initially, vorkplace exposure to asbestos fibers was the primary concern for human health hazards: however, more recently attention has turned to potential risks to the general public exposed to asbestos used in building materials in the con imiction of schools and public and commercial buildings. There is a likely potential to release high fiber concernid itions into the environment during the removal of asbestos from these buildings in order to reduce long-term a bestos exposures. Costs for removal have been estimated to be greater than 53 billion dollars.1171 Therefore, it is mportant to have the most accurate estimates of carcinogenic risks in making decisions whether removal is recess:dry and would actually result in an adequate reduction in risk, Regulatory agencies are obligated by law to irotect the public from levels of carcinogens that would pose an unreasonable risk to people. This often invollies using epidemiological studies in which cancers have been observed to be statistically elevated in an exposi d population in comparison to an unexposed matched population and extrapolating to lower levels where the "aiicci ptable" risk of cancer would be in the order of 1 out of 10s or 106. Although most epidemiological studies are inadequate for precise quantitative extrapolations, without better alternatives they are used to set environment; 1 exposure limits for carcinogenic agents. In conducting such assessments, however, no information is used i onceming the mechanisms by which carcinogens cause normal cells to become carcinogenic. Conservative,. ant possibly inaccurate, assumptions are made by assuming that the induction of cancer can result from one: interact) ain or "hit" with DNA. that there is a linear relationship between external exposure and the number of interaction: with DNA. and that there is no "safe" exposure level where the probability of causing cancer is zero. In 1986 he EPA used epidemiological studies and these assumptions to estimate that a lifetime exposure to 0.0001! fiber of asbestos per ml of air would result in 2.4 asbestos-associated deaths from iung cancer and mesothelioma per 130.000 people exposed.'*1 However, the accuracy of using such linear models to extrapolate to lower levels of aslb stos exposures has not been verified in epidemiology studies, nor has there been any biological data that would i:nqicate that asbestos-induced carcinogenicity is a simple "one-hit mechanism. In ihe past two decades, many scientific scudi :s revealed the complex nature of interactions of asbestos with macromolecules and cellular responses to asbestd, exposures. These interactions can result in direct and/or indirect Clement imemauonai Corporation. Fairfax. Virginia 541 gpl-00955 \ OYTEK ET AL. damage to DNA. and may cause initiation event i mutations) which may cause cell transformation and eventually lead to cancer. Initiation is only one part of the c ircinogenesis process: however, agents such as asbestos'9 '"'also can act as cocarcinogens or promoters which r iduce the time necessary for an initiated or transformed cell to develop into a cancer cell. Here, we propose sev :rai biological mechanisms as to how asbestos fibers may inmate and promote cancer at the molecular level. How< ver. sufficient quantitative information is not yet available to use these mechanisms to develop more biologically b ised risk assessment models. Our intent is 10 identify and describe molecular mechanisms based on current studies < nd to provide stimulus to generate additional data useful for more accurate quantitative esnmate of asbestos carctn igenicitv. ' In addition to biological mechanisms, other actors are also important in assessing the carcinogenic nsk of exposure to asbestos fibers. These other factors nclude the pharmacokinetics of asbestos deposition in lung and pleural tissues, accumulation and elimination, target cell specificity, and physicochemical properties of the different asbestos fibers. Here we focus oniv on he potennal mechanisms at the molecular and cellular levels. EVIDENCE FOR DI SECT GENOTOXIC ACTIVITY Initiation events are the results of permanent all :irations of DNA expression caused by gene mutations (alteration of single bases in DNA or deletions within one ge I: chromosomal alterations, such as deletions of many genes or rearrangements of chromosomes where pan of one chromosome is translocated to another chromosome: and numencal alterations that occur dunng cell dii ision where daughter cells receive abnormal distributions of chromosomes. Depending upon what genes these events affect, the cell carrying the aberrations can die or survive as a normal cell, or if oncogenes or regulatory' gB' fliies are involved, the ceil can survive without the capability to differentiate, to control its growth, and to function normally. In vivo studies have shown that crocidolite stos fibers of mixed sizes can be engulfed by mice peritoneal macrophage cells after an interpentoneai injectio 1 and physically interact with skeletal cellular proteins1111 and. therefore, may have access to the genetic maiena; within the cell. Brody et al.1121 also have reported that inhaled chrysotile asbestos fibers are taken up by pulmona y epithelial cells of rats and then can associate with intracellular actin-contaming microfilaments, and therefore e the capacity to bind to protein polymers that are involved in chromosomal separation during cell division. Boti crocidolite and chrysotile asbestos fibers 1 the majority of fibers were less than 5 pirn in length and between 0. 1.0 qm in diameter) injected into the pleural cavity of guinea pigs have been shown to penetrate mesotheha cells within two weeks after exposure.1'3' These findings demonstrate that asbestos fibers can enter cells in vivo and have access to cytoplasmic macromolecules and potentially can interact with DNA. Many in vitro studies also have been conducted with asbestos fibers. Haugen et al.llJI reported that human bronchial epithelial cells were capable of engul ing amosite asbestos fibers and fibers within the cytoplasm appeared free or in membrane-bound vacuoles, 1 nev ialso reported that intranuclear amosite inclusions occur in human bronchial epithelial cells. Cultured rai miic sothelial ceils can phagocytize chrysotile A asbestos fibers113' having a mean fiber length of less than 4 qm aid both chrysotile A and crocidolite asbestos fibers ranging in lengths from more than 10 qm to I qm and from norethan I qm io less than 0.1 qm in diameter have been shown to adhere to chromosomes in metaphase and cause ' olyploidy .1161 In rat mesothelial cells, chrysotile asbestos fibers having a mean length of less than 4 qm with 8491 of the fiber having a diameter of less than 0.058 qm. caused chromosomal aberrations that were predominantly chromatid breaks as well as polyploidy .1171 Exposure of human pleural mesothelial cells to amosite asbestos; fibers induced aneupioid cells that exhibited altered growth char- acteristics."81 although isolated aneupioid clones were not found to be tumongenic when injected into athymic nude mice. Hesterberg et al."" reported that Syri in hamster embryo cells internalized chrysotile asbestos fibers and. in some instances, chromosomes appeared to >e wrapped around individual asbestos fibers. In another study, Syrian hamster embryo cells arrested at G,/S and e tposed to asbestos fibers caused a 22-fold increase in displaced chromosomes over control cultures. Hesterberg et .'191 postulated that the missegregation could have been caused by direct physical interference with chromosome: preventing or hindering their separation dunng cell division and/or interacting with proteins I tubulin) that form the spindle apparatus for chromosomal separation dunng cell division. Similar findings were reported for cultu ed Chinese hamster cells.120"221 However. Sincock et al.122' reported that no significant chromosome damage ' /as observed in pnmarv human fibroblast cells from skin and 542 SPI-00956 AS BEST )S CARCINOGENICITY lung or in human Ivmphoblastoid cell lines exp ised io either crocidolite or chrysotile asbestos tibers with mean particle lengths of I. I to 3.4 pm and mean diameters between 0.15 and 0.47 pm. Fomace'231 was unable to detect single-strand DNA breaks in human fibroblast c Ms that had been treated with amosite asbestos tibers at doses ot low cellular toxicitv. Mossman et have als > shown that crocidolite and chrvsoule asbestos libers at nontoxic levels did not cause single-strand breaks in ti e DNA of hamster tracheal epithelial cells. Libbus et al.'231 demonstrated that crocidolite asbestos libers ot a 'erase length and diameter of 14.8 pm and 0.9 pm. respectively, caused single-strand DNA breaks in cultured ra embryo cells. These investigators used a nick translation assay rather than alkaline elution to measure breaks. El ctron microscopic examinations of the treated cells indicated that strand breaks occurred both in cells that visibly :ontained fibers and in cells where no fibers were detected. The DNA damage appeared to be dose related but no ilinear These studies show that asbestos fibers can en er cells, bind to molecular components, and cause chromosomal damage and numerical chromosomal aberration in certain cell types by direct interaction or indirectly through some intermediate that can result in mutations \sbestos apparently causes severe chromosomal damage rather than alterations in just single genes since astx stos fibers have not been shown to cause gene mutations in mammalian cell**6 271 or in bacterial cell*281 :st systems or stimulate unscheduled DNA synthesis in rat hepatocytes.1291 However. Yang et al.l30> have < emonstrated that xeroderma pigmentosum fibroblast cell lines. deficient in excision repair of certain types ofr :hemical or ultraviolet light-induced DNA damage, are more sensitive to chrysotile. amosite. and crocidolite asbestos fibers than normal human fibroblasts. These findings suggest that some types of asbestos-induced DN/1 damage may involve small lesions that are reparable. Asbestos fibers (amosite. amhophvllite. crocit oiite. Rhodesian A chrysotile. and Canadian B chrysotile) have been shown to facilitate the transfection of viralI DNA into liver epithelioid cells from chimpanzee livers, rhesus monkey kidneys cells, human carcinoma, and N1 -1 3T3 mouse fibroblast cells.1311 However, the concentration of asbestos fibers needed to cause transfections wa s high (3-10 mg/ml). A linear dose-response relationship was observed at higher doses, but at 0.1 mg/ml. littli or no transfection was seen. Appel et al.1321 also were able to introduce plasmid DNA vectors into cos-7 monktly cells using Canadian chrvsotiie sample B asbestos fibers with an average diameter of 7 pm and lengths in the ange of 130-3 pm as the transfecting agent. Therefore, under experimental conditions using high concentration: of asbestos fibers, nucleic acids can be inserted inio the genome of cells. Ke et a!.1331 successfully transfected n irmal human mesotheiial cells with fragmented DNA from a transformed human mesotheiial cell line and obtai led transformed clones that were morphologically indistinguish able from the original transformed line. Hence, the inserted DNA can alter the expression of cellular oncogenes and antioncogenes, depending on the insertion sites, oi the transfected DNA could itself possess oncogenes that may be translated after insertion into genomic DNA. EVIDENCE FOR 1NDIRECT GENOTOXICITY There are two potential mechanisms where: astx sitos fibers can cause the formation of reactive oxygen species, These reactive oxygen species, in tum. can act: second messengers in causing genotoxicity. One mechanism involves the transfer of electrons from asbestos ibers (chrysotile with mean fiber length of 6 pm) to cellular molecules which, in tum. can interact with Dh A.1341 The second mechanism involves an asbestos-induced inflammatory response that causes the accumulationn of macrophages and polymorphonuclear leukocytes, which can release active oxygen species such as superox i(ie free radical and hydrogen peroxide, which then interact with DNA to cause mutations.(35~381 Wong et al.,39) suggested that the ferrous iro l in certain asbestos fibers'may reduce oxygen to form the superoxide free radical. The superoxide anion ma / then result in the formation of other reactive oxygen species such as hydrogen peroxide and the hydroxyl free n dical. Eberhardt et al.1401 and Weitzman and Graceffa'411 have shown that asbestos fibers can catalyze the forma ;non of both the superoxide radical and hydroxyl radical from hydrogen peroxide. Mossman et al.,38> reported tha asbestosis in rats caused by inhaling crocidolite asbestos fibers could be inhibited if the animals also were giv :in catalase, which converts hydrogen peroxide into water, Cytotoxicity of hamster tracheal epithelial ceils induced by crocidolite and chrysotile asbestos fibers of lengths varying from > 10 pm to < 2 pm can be inhibited |bv superoxide dismutase. which converts superoxide radical to H:CN. and by hydroxyl radical scavengers.'421 Cbtalase was ineffective in protecting the cells from asbestos 543 SPI-00957 VOYTEK ET AL toxicity, but mannitol and dimethvlthiourea. both scavengers for the hydroxyl free radical, were very effective in reducing cell toxicity. Hydrogen peroxide is requltd to generate the hydroxyl radical: it is surprising therefore that catalase did not have a protective effect. Like supejnoxide dismutase. catalase is not likely to penetrate the cell wall, One explanation may be that hydrogen peroxide not formed at outer membrane surfaces but superoxide anion is. Unlike catalase and superoxide dismutase. mami itol and dimethvlthiourea are capable of entering the cell and interacting with hydroxyl radicals. When the e4posure time to asbestos was increased, superoxide dismutase activity was induced in the cells.'421 Fisher et aj.l43` reported that heating chrysotile tsbestos fibers decreased their binding to bovine serum albumin, but that ionizing radiation could restore the protein binding capability. The effects of untreated and treated asbestos on cell cytotoxicity and viability of human foresk un fibroblasts and bovine macrophage ceils were also analyzed, For both cell types, heat-treated asbestos fiber were less toxic than untreated, radiation-created, and heat/ radiation-treated asbestos. One possible mecham:tm consistent with these results involves the release or escape of metastable electrons within the mineral and the i|egaining of activated electrons upon radiauon. The toxicity of asbestos may be attributable to the fibers coma<(:ting cells and transferring electrons to cell surfaces or. upon entering the cell, electron transfer could occur f; om asbestos fibers to various biomolecules that cause geneuc damage leading to carcinogenesis. Valentine et al.'341 also demonstrated that heat pretreatment of chrysotile asbestos fibers with mean fiber lengths of 6 um educed cytotoxicity toward human fibroblast cells and bovine alveolar macrophages and that reactivation of cvt itoxic effects resulted when fibers were irradiated with x-rays, Leanderson et al.144' have demonstrated that the ncubaoon of chrysotile asbestos fibers with 2-deoxyguanosine produced 8-hydroxydeoxvguanosine. This showei that, under laboratory conditions, asbestos has the capability to modify DNA bases through a reaction mediated ty hydroxyl radicals. If this reacuon occurs in vivo, the altered base could result in genetic damage and mutauon Moalli et al.'45' have shown that crocidolite as t estos fibers injected into the peritoneal cavity of mice produced an inflammatory response and caused mesothelial cell injury and regeneration. Asbestos fibers were primarily found clustered near the stomata of the lymphatic at the peritoneal surface of the diaphragm. Fibers accumulated at these sites during the first three days after injectijon and some still remained after 6 months. Hemorrhaging on the peritoneal surface of the diaphragm occurred and here was accumulation of neutrophils and macrophages around the fiber clusters. The inflammatory response, a well as the presence of macrophages at the deposition sites. persisted even after 6 months. Other studies by Warheit et al.l37) and Brody i : al.141 have shown that inhaled chrysotile asbestos fibers can deposit on alveolar duct bifurcations in the lung; of rats and that pulmonary macrophages accumulate at these deposition sues. Additional studies by Warheit :t al.'47' have shown that asbestos fibers enhance pulmonary macrophage chemotactic responses that can resu|it in migration of additional macrophage cells to the asbestos deposition sues. Goodglick and Kane148' have onstrated that crocidolite asbestos fibers can induce peritoneal macrophages in mice to produce/release reactiv: oxygen species such as hydrogen peroxide. Weitzman and Stossel'4" have demonstrated that human phagocj t:ies can cause mutations in bacterial cells by producing reactive oxygen species. Therefore, the continual release if mutagenic reactive oxygen species in vivo and the continual regeneration of "target" cells fe.g.. mesothelial cel|:s) in the same vicinity could result in genetic alterations causing some of the mesothelial cells to be transformed. ASBESTOS AS A CANCER PROMOTER Topping and Nenesheim191 demonstrated that i hrvsotile asbestos fibers promote tumors in F344 rat tracheal transplants in the retroscapuiar region of 8-week old isogeneic recipients. Dimethylbenz|a)anthracene (DMBA) was used to initiate the transplanted cells which wdire then exposed to asbestos fibers at the transplantation sue. No carcinomas were found in animals given only DMIIIA or in animals exposed only to asbestos. However, using both initiator (25 p.g) and promoter (200 jig), the care noma incidence increased to 9/40 (23%). A previous study'501 showed that an asbestos concentration of 2000 p.j was required to get a 5% increase in the incidence of tracheal carcinomas without any initiation. Topping and Nenesheim1311 reported that tr <cheal carcinogenesis in rats was also enhanced by 12-0tetradecanoylphorbol-13-acetate (TPA). the well- tnown tumor promoter in skin, but when given alone was not 544 SPI-00958 ASBESTOS CARCINOGENICITY capable of initiating a cancer response. Like bestos fibers, phorbol esters stimulate the production of reactive oxygen species and induce DNA strand break 531 and as with asbestos, the toxic responses, as well as the promotional activity of TPA. can be inhibited by antioxidants.'34' Phorbol esters have been studied extensively with respect to their carcinogenic promotion aiqtivitv'33 34~561 and they have been shown to cause changes in cell membranes, generate reactive oxygen specie: increase lipid peroxidation, elevate certain enzyme activities, enhance phospholipid synthesis, alter methylat on of macromolecules, and cause chromosomal damage. Promotera may act initially through interactions with cell membranes and stimulate lipid peroxidation, which in turn, through various intermittent steps, produce fr :ie radical species that reach and damage genetic material.137 381 Yano'39' has demonstrated that chrysottle and rocidolite asbestos fibers can induce the formation of malondialdehyde. a product of free radical-induced lipid | eroxidation in human polymorphonuclear neutrophil cells, guinea pig peritoneal macrophages, and alveolar iavatje cells. Weinstein'361 has reviewed the current status of the effects of TPA on protein kinase C (PKC). PKC plays a key role in cell signal transduction involving acnva :iion of oncogenes, cellular growth, and tumor promotion. PKC is the primary receptor for TPA and the phorbol e ster binds at an allosteric site on the enzyme, enhancing its ability to phosphorvlate protein substrate!s). Cox et al l60' have reported that PKC phosphorvlates a NADPH oxidase on the plasma membrane that catalyzes the redu :tion of oxygen to the superoxide anion. PKC stimulation also increases the levels of ornithine decarboxylase (ODC). which is the rate-limiting enzyme in the biosynthesis of polyamines that are necessary for the initiation ct cell division. Marsh and Mossman'611 have shown that chrysotile and crocidolite asbestos fibers also induce ODC activity in cultured hamster tracheal epithelial cells, but that ODC activity is significantly reduced when calcium e|i try antagonists i verapamil or nifedipine) are added to the cultures, They found that the longer fibers were more efiective in stimulating enzymatic activity. Furthermore, palmuoyl carnitine and l-(5-isoquinolinylsulfonyl)-2-meijhvlpiperazine. inhibitors of PKC. also were effective in blocking ODC activity that was stimulated by asbestos ibets. It seems unlikely that asbestos fibers are promoting cells, creating active oxygen species, and stimulatin ; enzymes like ODC in a similar manner to TPA. TPA can bind directly to PKC and activate the enzyme. Howi rer. asbestos fibers are likely to interact at the plasma membrane, to cause an influx of calcium into the cell, which activates PKC and may stimulate membrane phospholipases.{bZ) The phospholipases hydrolyze membrane pol\ pihosphaudylinositols to form diacylglycerol and inositol 1.4.5triphosphate. Diacylglycerol is an activator of PKC'631 and inositol 1.4.5-tnphosphate causes release of intracellular stores of calcium.'54' Both calcium and diacylglycerol bind at the regulatory site of PKC and induce a conformational change that enhances the catalyt iic activity of the enzyme. Roney and Holian162' have examined the effects of the PKC inhibitors (fluphenazine ot staurosponnei on blocking the production of superoxide anion production by chrysotile asbestos fibers and he promoter, phorbol 12.13-dibutvrate. in guinea pig alveolar macrophages. These PKC inhibitors all redubied the amount of superoxide anion production. The authors postulated that asbestos is most likely stimuli titng membrane phospholipase C to produce diacylglycerol and inositol 1.4.5-triphosphate, which causes an ini irease in cell calcium levels, which in tum increase the activity of PKC. PKC then is responsible for inducing meiribrane NADPH oxidase to produce superoxide anion that leads to other reactive oxygen species and causes act! ration of oncogenes that affect cell growth and differentiation, However, crocidolite. anthophyllite. and amii isite did not stimulate the production of superoxide anion as effectively as chrysotile fibers. Gabrielson et al.163' exposed cultured human lung mesothelial cells to amosite asbestos fibers and were unable to detect any generation of free radicals, m r did free radical scavengers (glutathione, n-acetvl-cvsteine. D-alpha-tocopherol. or superoxtde dismutase) al ::ier the cytotoxic effects of asbestos. These findings are in contrast to other studies described above. The differen :es may be due in pan to different cell types and experimental conditions as well as to differences in asbestos iber characteristics. DISCUSSION Figure I summarizes six potential mechanist iis for the induction and promotion of cancer by asbestos tibers. Mechanism I involves asbestos fibers penetrat tng_ tne target cell, directly interacting with DNA. and causing chromosomal aberrations (mutations). Dependii g on the seventy and location of the mutation, some cells will not survive, other cells may survive and continue to iinrfinn nnrmnllv u/hil^ nrh^r* 545 SPI-00959 V3YTEK ET AL. 546 SPI-00960 FIG . I. Different mechanisms for asbestos-induced carcinogenicity. As,, = asbestos fibers outside the cell, -As, = asbestos fibers inside the cell; l',, = protein outside the cell; P, protein inside the cell; l)N A (, = genomic DNA, DNA,: = fragmented DNA; T = tubulin, M = membrane receptor. See tent for lunher details ASBEST )S CARCINOGENICITY DNA that would constitute the initiation step the carcinogenic process. Asbestos fibers readily interact with proteins both outside and inside cells. These tvp^s of interactions actually mav compete with genomic DNA for the asbestos fibers and consequently may serve to irotect the ceil from asbestos-induced mutations. Because of the many macromolecules available to bind to asb^st:ios fibers, it seems that at low fiber concentrations few. if any. fleers could penetrate the nucleus and interact v 'ith IDNA. Mechanism II illustrates the capability of asbfcst:ios fibers to interact with DNA fragments and to act as a earner for the fragments into the cell for insertion into g nomic DNA. In vitro expenments with asbestos fibers were done at concentrations of both DNA fragments anc asbestos fibers to maximize transformation; it is quesuonable whether appropriate conditions exist in vivo in i 'hich insertion mutagenicity can occur by this process. However, recent findings1641 have shown that DNA fragme its exist outside cell membranes and may constitute as much as 1 % of total cellular DNA. Furthermore, inflammaio v responses induced by asbestos fibers result in the destruction of macrophages and mesotheiial cells (MeChams n VI), which can release degraded genomic DNA fragments, Therefore, in vivo DNA fragments are available bind asbestos fibers that may be inserted into the DNA of normal cells. The likelihood of this mechanism occun|in g in vivo and contnbunng to asbestos carcinogenicity at low exposure levels is questionable as the concern rations of both earner and nucleic acid fragments at asbestos deposition sites may not be sufficient to cause trpnstection of fragmented DNA into genomic DNA. Asbestos fibers have been shown to interac: with chromosomes and have been proposed to interact with cvtoskeletaJ structural proteins and have beet shown to induce aneuploidy and polyploidy. Chromosomal imbalanced cells are abnormal and may be in'solved in the process of asbestos-induced carcinogenicity Gibas et al..147' Popescu et al..1681 and Tiainen et a 16,1 reported that high incidences of numencal chromosomal abnormalities are present in human mesotheliil cells, and the frequencies of certain abnormalities may be nonrandom. Oshimura et al.'70' reported that exp is'ure of Syrian hamster embryo cells to asbestos fibers resulted in a nonlinear dose-dependent increase in aneuploid tetraploid. and binucleated cells, and that the cvtogenic changes, primarily aneuploidy, correlated with cell trans ormation. Mechanism III in Figure I illustrates the binding of asbestos fibers to tubulin, a protein that makes ip the spindle apparatus necessary for chromosomal separation during cell division. It is still unclear if chrt imosomal abnormalities are part of the induction process of asbestos-induced carcinogenicity or whether the; may be nonspecific secondary alterations that evolve during the progression of malignant cell growth. Mechanism IV illustrates that asbestos fibers n transfer electrons to cellular molecules after entering the cell or by interacting at membrane surfaces: the cellt I;ar molecules in turn interact with genomic DNA and cause mutations. Mechanism V demonstratespromotio lal1 activity of asbestos in which asbestos binds to membrane sites and causes epigenetic effects that initially alter t eilular growth and function. These cellular alterations in DNA expression in an already initiated cell (transformed cell) may cause it to progress further toward a cancer cell and may. by stimulating reactive oxygen species, cai a second mutation (transformation) that may be necessary for the formauon of a cancer cell. The extent of the ptoimotional response depends upon the number of interactions of asbestos with "receptor" sites (if specific receptoi sites exist) on cell membrane surfaces. The kinetics of asbestos binding to membrane "receptors" and the extent if promotional activity are not available, but the kinetics would most likely be nonlinear, and a threshold level needed)to stimulate sufficient promotional activity may exist. The asbestos-induced inflammatory mechanisn i (VI) is multifaceted in that asbestos fibers attract macrophages, some of which are destroyed and release reactiv oxygen species and DNA fragments and cause the release of macrophage growth-stimulating factors that it in cell regeneration at the deposition sites. The release of reactive oxygen species may induce mutations in the neighboring dividing cells; the DNA fragments from destroyed macrophages may bind to asbestos rs. and become transplanted into nearby cells where the DNA fragments may be inserted into genomic DNA (Mechanism II). The dashed lines in Figure 1 represent the contributing effects that the inflammatory responsj: may have on the other postulated mechanisms by inducing cell division and making more target cells available :o be transformed and releasing DNA fragments that could be inserted into genomic DNA All six mechanisms presented here potentially could be involved in asbestos-induced mesothelioma and lung cancer and the mechanisms may vary with respect to cell type. Certain mechanisms may realistically occur only at very high concentrations of asbestos fibers and ire more likely to occur under in vitro laboratory conditions, Mechanisms V and VI. however, have been nstrated in vivo, but adequate dose-response relationships for use in quantitative nsk assessment are not yet ava_ table. Quanutative cancer nsk assessments have alwa' s suffered from lack of informanon on mechanisms of action of 547 gpi-00961 VOYTEKETAL. carcinogens, making n necessary 10 extrapolate acceptable environmental levels and to assume conservatively that one infraction with DNA will result in lie developmem ot a tumor and that tumor incidence is directly proportional to concentration. Enough data are t> comi ine available showing different molecular aspects of asbestos carcinogenicity to design experiments to detentme the penmen molecular mechanisms and dose-response relationships that occur in vivo at environmen exposure levels. This new information can then be used to determine asbestos cancer risks more accuratelv for regulatory purposes than the current mathematical extrapolanon models are capable ot doing. F EFERENCES 1. IARC. (1977). IARC Monographs on the Evaluatics ofthe Carcinogenic Risk ofChemicats to Man: Asbestos. Vol. 14 Lyon. France: World Health Orgamiauon. 2 CRAIGHEAD. 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