[CANCER RESEARCH 51, 4942-4947, September 15, 19911

Crocidolite Asbestos Fibers Undergo Size-dependent Microtubule-mediatedTransport after Endocytosis in Vertebrate Lung Epithelial Cells1

Richard W. Cole, Jeffrey G. Ault, John H. Hayden, and Conly L. Rieder2

Wadsworth Center for Laboratories and Research, Albany, New York ¡2201-0509[R. W. C., J. G. A., C. L. K.J; Department of Biology, Siena College, Loudenville, NewYork 12211 (3. H. H.J; and Department of BiomédicalSciences, State University of New York, Albany, New York 12222 [C. L. R.]

ABSTRACT

The large respiratory epithelial cells within primary cultures of newt(Tarichii granulosa) lung are uniquely suited for high resolution video-enhanced light-microscopic studies. We show here that these cells incor

porate crocidolite asbestos fibers within 18 h by endocytosis. Once insidethe cell, fibers less than 5 tim in length are seen by video light microscopyto undergo saltatory transport at a maximum velocity of 1.18 um/s. Bycontrast, fibers over 5 um long rarely exhibit saltatory motion. Over time,all of the fibers become preferentially located near the nucleus. Thisperinuclear accumulation is largely inhibited by disassembling the cyto-

plasmic microtubules with nocodazole. Same cell correlative light andelectron microscopy reveal that fibers exhibiting saltatory behavior areenclosed within a membrane. From these observations we conclude that,upon incorporation into epithelial cells, asbestos fibers undergo size-

dependent active transport along cytoplasmic microtubules. Our data arethe first to link the dimension-dependent transforming ability of asbestosfibers to a basic cellular function, i.e., the microtubule-dependent trans

port of cellular components.

INTRODUCTION

Once prized commercially for its high tensile strength, resistance to heat, flexibility, and durability, asbestos has becomea public health dilemma. A number of epidemiológica! andtoxicological studies have implicated asbestos fibers, especiallycrocidolite, in the etiology of various diseases including asbes-tosis, lung cancer, and mesotheliomas of the pleura, pericardium, and peritoneum (reviewed in Refs. 1-3). However, despite much research, the mechanism(s) by which these fiberspromote oncogenesis and cell injury remain obscure (4-6).Unlike most carcinogens, asbestos by itself does not appear tobe mutagenic (7-10).

Over the years, it has become clear that the transformingability of mineral fibers, including asbestos, is linked primarilyto the dimensions of the fiber [i.e., the length/width aspect ratio(11); reviewed in Refs. 1 and 12]. In general, long thin fibersare more tumorigenic than shorter, thicker ones. The importance of fiber dimensions to carcinogenicity is not relatedsimply to the selective clearance of certain sized fibers by thelungs, since the same relationship applies also to cultured cells(9, 13, 14). Thus, as noted by Pott (15), "A fiber has to be

regarded as a physical carcinogen that works by its elongatedshape." At present, it is unknown how carcinogenicity is related

to fiber dimensions. Perhaps the strongest hypothesis is thatlong thin fibers physically promote the genesis of aneuploidyduring mitosis, and that such a shift in chromosome complement is then responsible for immortilization and neoplastic

Received 4/23/91; accepted 7/8/91.The costs of publication of this article were defrayed in part by the payment

of page charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1This work was supported by NIH CMS Grants R01-40198 (C. L. R.) andR15-41871 (J. H. H.), and by RR 01219, awarded by the National Center forResearch Resources (Department of Health and Human Services/Public HealthService), to support the Wadsworth Center's Biological Microscopy and Image

Reconstruction Facility as a National Biotechnological Resource.2To whom requests for reprints should be addressed, at Wadsworth Center

for Labs and Research, P. O. Box 509, Albany, NY 12201-0509.

progression [(9, 14, 16, 17) reviewed in Refs. 5 and 18].If mineral fibers are physical carcinogens, then how they

behave within the cell, and what components they interact with,become critically important to understanding how they exerttheir deleterious effects. However, despite this obvious conclusion, virtually nothing is known regarding the behavior of suchfibers once incorporated into the living cell. The few previousLM3 studies remotely relevant to this topic were not concerned

with fiber behavior, but with their long term effects on cellmorphology and toxicity to cell populations (10, 19-21). Indeed, in these studies the fate of individual fibers within cellscould not be discerned due to the small size and poor opticalproperties of the mammalian cell lines utilized, and to limitations in the (phase-contrast) LM systems used.

The respiratory epithelial cells (i.e., pneumocytes) generatedfrom primary cultures of newt lungs are among the largest (250-íímdiameter) and flattest somatic cells in existence (22). Inaddition, they possess unique optical properties that allow forthe examination of the distribution, interaction, and dynamicbehavior of cellular components in the living cell, with unsurpassed clarity, by video-enhanced contrast LM methods [(23,24) reviewed in Ref. 22]. We reasoned that the behavior andinteraction of crocidolite asbestos fibers could be examined inconsiderable detail once incorporated into living newt lungepithelial cells. Our observations, which are reported here,confirm the utility of this model system for such studies. Moreover, our data clearly demonstrate that crocidolite fibersundergo size-dependent active transport within the interphasecell—afinding that may ultimately be relevant to understandinghow the carcinogenic properties of such fibers are related totheir dimensions.

MATERIALS AND METHODS

Cell Culture. Newt (Taricha granulosa) lung primary cultures wereprepared in Rose multipurpose chambers as detailed by Rieder andHard (22). Briefly, small lung fragments were cultured at 23*C on 25-mm2 coverslips in 0.5x L-15 medium supplemented with 10% fetal calf

serum and antibiotics. Under these conditions, extensive monolayersheets of ciliated and nonciliated respiratory epithelia, and individualmesothelia cells, migrate 1 to 2 mm from the lung fragments withinthe first week of culture. After the first week, the medium was removedand replaced with fresh medium containing either 0.5 mg/ml of International Union Against Cancer crocidolite asbestos (a kind gift fromDr. T. M. Fasy, Mount Sinai Medical Center, New York, NY), or acombination of crocidolite fibers and the microtubule-disrupting drugnocodazole (20 ^M; Aldrich Chemical Co., Milwaukee, WI). At varioustimes thereafter, the coverslip cultures were removed from the culturechambers and mounted on microperfusion chambers in fresh medium(25). These chambers allowed cells within the cultures to be examinedwith high resolution LM optics and to be rapidly perfused with fixativesduring observation.

Video-enhanced LM. The video-enhanced contrast LM system used,and the methods used to track fiber motion, have been described in

3The abbreviations used are: LM, light microscopic; DIC, differential interference contrast; PBS, phosphate-buffered saline; EM, electron microscopic; NA,numerical aperature.

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Alexander and Rieder (26). Briefly, DIG or polarized light images ofcells were obtained on a Nikon Microphot-FX LM using a HamamatsuC24000 Chalnicon camera. These analogue images were then digitizedand processed with a Hamamatsu DVS-3000 image processor beforestorage on optical memory discs. During data analysis, the optical discimages were reprocessed through the Hamamatsu DVS-3000, wherethey were expanded 2-8 times. Using the Hamamatsu's distance meas

uring function, calibrated to the 0.62-^m lattice spacing of the diatomPleurasigma angulatum, both cursors were superimposed on the edgeof a particle. The recording was then advanced one frame, and onecursor was moved to the particle edge. The distance separating them,and the time interval, were entered manually into Quatro-Pro wherethey were analyzed and manipulated. Under the optical conditions used(x40 objective; NA = 0.85) we could measure edge displacements onthe order of 0.1 ^m (26). Images of video recordings were photographedon Plus-X film (Eastman Kodak Co., Rochester, NY) using a freeze-frame video recorder (Polaroid Corp., Cambridge, England). This filmwas subsequently processed in Rodinal.

Immunofluorescence LM. For immunofluorescence LM, control andexperimentally treated newt lung coverslip cultures were simultaneouslyfixed and lysed in glutaraldehyde/detergent (Triton X-100), reduced,and blocked as described by Rieder and Alexander (25). They were thenincubated in a monoclonal antibody against ,f lubuliii (TU-27B; kindlyprovided by Dr. L. Binder, University of Alabama, Birmingham, AL)at 4°Cfor 18 h. After washing in PBS, the cultures were treated with a

fluorescein isothiocyanate-conjugated goat anti-mouse IgG (SigmaChemical Co., St. Louis, MO) at 37°Cfor 30 min. Subsequently, they

were washed in PBS and mounted on a slide in PBS/glycerol (pH 7.8)containing yV-phenylenediamine.

Cells processed for the indirect immunofluorescent localization ofmicrotubules were examined with a Nikon Optiphot microscopeequipped with a Nikon 60X Plan Apo objective (NA = 1.4). Fluorescentimages were photographed with an automatic exposure system on XP-1 film (Ilford Ltd., Basildon, Essex, England) using an ASA setting of1600. This film was then commercially developed by C-41 processing.

Electron Microscopy. Asbestos-treated newt lung coverslip cultureswere mounted on perfusion chambers as described above. Selected cellswere then followed by video LM until the desired time of fixation,during which the culture was perfused with 2% glutaraldehyde in 0.05

M phosphate buffer. After 20 min, the perfusion chamber was dismantled and the culture postfixed in 0.05% ruthenium tetroxide for 5 min.Ruthenium tetroxide was used during postfixation to enhance thecontrast of membranes. Once postfixed, the culture was washed indistilled H2O, dehydrated, and flat-embedded in Epon/Araldite asdescribed by Rieder el al. (27). Cells previously followed in vivo werethen relocated within the embedment, excised, mounted on Epon pegs,and serially thin (60 nm)-sectioned with a diamond knife using a SonaliMT-6000 ultramicrotome. The ribbons of serial sections were mountedon Formvar-coated slot grids and stained with uranyl acetate and leadcitrate. The sections were then viewed and photographed at 80 kV ona Philips 301 electrom microscope.

To facilitate thin (60-nm) serial sectioning, and to prevent tearingand distortion within the regions containing asbestos during the sectioning process, the asbestos fibers were largely removed from theembedded cells before sectioning by a brief treatment with hydrofluoricacid at 4°C(28). As a result of this treatment, conspicuous holes were

produced in the plastic where the asbestos had been located.

RESULTS

Newt Lung Epithelia Incorporate Crocidolite Fibers. After an18-h exposure to 0.5 mg/ml crocidolite fibers, many T. granulosa lung epithelial cells possessed a variable number of fiberswithin their cytoplasm. That these fibers were asbestos wassubsequently demonstrated by X-ray microanalysis (data notshown). They were easily detected in the living cell by through-focus DIC (cf. Fig. 1, A and D) or polarized (Figs. IE and 2)LM. Sixty-five h after exposure, 52% of the cells containingasbestos (n = 123 cells from 3 cultures) showed a strikingpreferential accumulation of fibers near the nucleus (Fig. 1, Dand E). It has previously been reported that Syrian hamsterembryo cells phagocytize crocidolite asbestos fibers within 24h of treatment, and that these fibers are concentrated aroundthe nucleus of fixed cells 24-48 h after exposure (5). Such aperinuclear accumulation could result simply from passive diffusion of the fibers within the cell that ultimately favors a net

•»' •La

'•it,

Fig. 1. Differential interference-contrast (A, D, F), antitubulin immunofluorescent (B, C), and polarized light (E) micrographs of newt lung epithelial cells before(A-C) and after (D-F) incorporating crocidolite asbestos fibers. The cell contains an extensive array of cytoplasmic microtubules (/?), which is completely disassembledby a 2-3-hr treatment with 20 ¿IMnocodazole (C). Pneumocytes with an intact cytoplasmic microtubule complex concentrate all of the asbestos fibers around thenucleus (D and E). By contrast, those lacking microtubules (F) incorporate fibers into the cytoplasm, but only some fibers are positioned immediately adjacent to thenucleus. The majority remain randomly dispersed throughout the cytoplasmic volume with the exception that they are excluded from the thin regions of the cellperiphery. A, D, E, and F are of living cells. Bar, 25 ^m.

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Fig. 2. Selected micrographs, from a time-lapse video-enhanced polarized LM sequence, of asbestos fibers (dark masses) undergoing saltatory transport within thecytoplasm of a newt lung epithelial cell. The cell nucleus, which is not within the field of view, occupied an area just below the bottom of each micrograph. Arrowheadin A and B, particle that moves over 12 urn within a selected 10-s period. Arrow in C-F, another particle that undergoes a lengthy excursion over a 70-s period.Elapsed time in min: s, relative to A (bottom right corners). Bar, 25 um.

FiberVelocityvs. TimeI.«K>-10.75-0.50.250-0.25-n

*\10J

KJ•1110

20 304Time(sec)

Fig. 3. Velocity versus time plot of the asbestos fiber noted by the arrowheadin Fig. 2, A and B. Within this 40-s plot the fiber exhibited a characteristic patternof saltatory motion including highly variable positive velocities followed by abruptstops, and even negative velocities (reversals of motion). Note that the fiberreached a maximum velocity of over 60 *im/min.

movement from the thin peripheral regions into the thickerperinuclear region. Alternatively, the accumulation of crocidol-ite fibers around the nucleus could also be mediated by a moreactive process that is analogous, for example, to the microtu-bule-mediated transport of endosomes and lysosomes that results in the accumulation of these organelles around the nucleus(e.g., Refs. 29-31). Indeed, when newt lung epithelia are treatedwith the microtubule disrupting drug nocodazole at the time ofasbestos exposure, only 7% (n = 111 cells from 3 cultures)exhibited a perinuclear accumulation of fibers at the end of 65h comparable to that of controls. Under these conditions, thecytoplasmic microtubule network was completely disassembled

within 1 to 2 h (cf. Fig. 1, B and C) but fiber incorporation wasnot inhibited (Fig. IF). Some of the fibers within the cytoplasmof nocodazole-treated cells were positioned near the nucleusbut, with the exception that they were largely excluded fromthe thin peripheral regions, most were distributed randomlythroughout the cytoplasmic volume (cf. Fig. l, D and F). Theseobservations on living cells support our hypothesis that croci-dolite fibers become concentrated in the thickest (perinuclear)region of the epithelial cell by movement along cytoplasmicmicrotubules.

Crocidolite Fibers Undergo Size-dependent Saltatory Motionswithin the Cell. The inhibitory effect of nocodazole on theperinuclear accumulation of crocidolite suggests that these fibers are actively transported within the cytoplasm along micro-tubules. To evaluate this hypothesis, we followed living croci-dolite-containing newt lung epithelial cells by time-lapse video-enhanced DIG or polarized LM. Under these conditions, manyof the fibers were seen to undergo rapid and conspicuous, buttransient, movements (Fig. 2). As a rule, these fibers ranged inlength from less than 1 urn to 5 /¿m,and in width from lessthan 0.4 jum (the resolution limit of our 0.85 NA objective lens)to 1.5 Mm.Fibers over 5 ßinlong rarely exhibited such saltatorymotions. The average rate of transport was found to be 0.48fim/s (25 particles undergoing a total of 32 translocations, SD= 0.25 Mm/s) with a maximum velocity of 1.18 ntn/s. The rateand characteristics of this transport (Fig. 3) were similar inevery respect to the saltatory movements recently described byAlexander and Rieder (26) for organelles within newt lungepithelial cells. Moreover, the saltatory movement of crocidolitefibers, like that of the organelles, was completely inhibited bynocodazole treatment.

Crocidolite Fibers are Transported through the Cytoplasm asMembrane-bound inclusions. To distinguish whether asbestosfibers are transported as membrane-bound inclusions, or asdemembranated fibers, we followed crocidolite-containing newtlung epithelial cells in vivo by time-lapse video-enhanced polar-

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ized LM, and then fixed these cells during observation forsubsequent serial section EM analyses. Using this same-cellcorrelative LM/EM approach, we examined the ultrastructureof 8 asbestos fibers, over 3 cells, that we knew had undergonesaltatory motion before fixation (Fig. 4). In all cases, the movingfiber was enclosed within a membrane (e.g., Fig. 4, E to G).

DISCUSSION

The large diameter of the newt lung epithelial cell, its flatnature, and its optically clear cytoplasm provide a unique model

system for examining the behavior and interaction of mineralfibers within the living cell by high resolution video LM methods. Our results clearly demonstrate that once within the cell,crocidolite asbestos fibers exhibit a previously undescribed size-

dependent dynamic behavior. Such a behavior would be difficultto detect in the mammalian (e.g., hamster and human) cellscommonly used for asbestos studies because these cells areextremely small and round considerably after incorporatingfibers.

In an ultrastructural study, Brody et al. (32) found that manyof the chrysotile asbestos fibers ingested by rat aveolar epithelial

Fig. 4. Same-cell correlative light and electron microscopy of asbestos fibers undergoing saltatory motion. A and B are selected micrographs, from a time-lapsevideo-enhanced polarized light microscopic sequence, of an asbestos fiber (arrow ¡nA and B) undergoing a saltation. Time, in min:s. is at the bottom corner of eachmicrograph. The DIC micrograph in C is of the same cell after fixation. Bar, 20 ^m. An analysis of the complete video sequence revealed that 3 other particles (openarrowhead, asterisk, and dark arrowhead in A-C). in addition to that noted by the arrow in A and B, also underwent saltatory motions during the observational period.Three of these fibers (arrow, asterisk, and dark arrowhead) are included within the low-magnification electron micrograph in D, which is from one of the serialsections cut from the cell. Bar. 10 ^m. A high-magnification electron microscopic analysis of the series revealed that all 4 fibers that underwent translocations duringthe filming sequence were surrounded by endocytotic membranes. Three of these (open arrowhead, arrow, and asterisk in A-C) are shown in E-G, respectively. Bar, 1

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cells, after a 1-h inhalation exposure, were ultimately trans

ported across the cells to the interstitial compartments. Asubsequent ultrastructural examination revealed that actin filaments were associated with newly phagocytized fibers withinthe aveolar cells (33). This finding prompted these investigatorsto hypothesize that the transport of chrysotile across the pulmonary epithelium to the interstitial compartments was mediated by actin-containing microfilaments (33). However, although actin filaments are known to be necessary for endocy-tosis (34, 35), there is currently no evidence that they areinvolved in the saltatory transport of phagocytosed mineralfibers within cells.

The saltatory transport of cellular organelles is known tooccur along the surface of microtubules (36-38). Recent biochemical and in vitro data convincingly demonstrate that thisprocess is mediated by several mechanochemical proteins, including cytoplasmic dynein and kinesin, that appear to bind toboth the organelle membrane and the microtubule (reviewed inRefs. 39-41). The largely inhibitory effect of nocodazole on theperinuclear accumulation of asbestos suggests that these fibersare actively transported within the cytoplasm along microtubules. Indeed, when newt epithelial cells containing crocidolitefibers are viewed by video-enhanced DIC or polarized LM,many are seen to undergo size-dependent saltatory movementssimilar to those reported for other organelles (26) within thecytoplasm. Since this motion is inhibited by nocodazole, a drugthat disrupts cytoplasmic microtubules, we can conclude thatthe transport of crocidolite in interphase newt lung epithelialcells is mediated by cytoplasmic microtubules.

Upon endocytosis by macrophages, epithelial cells, and fibro-blasts, asbestos and other mineral fibers become membrane-bound endosomes (14, 42-44). Over time, such endosomeswould undoubtedly tend to move, by random diffusion from theexceedingly thin peripheral margins of the cell to the thickerperinuclear region that contains most of the cytoplasmic volume. Indeed, our time-lapse observations indicate that even thelargest crocidolite fibers undergo random diffusion (e.g., thelarge fibers in Fig. 2, bottom). However, when newt lung cellsare treated with nocodazole at the time of exposure to crocidolite, few fibers acquire a perinuclear location, and those thatdo are much less tightly packed than in controls not treatedwith nocodazole (cf. Fig. 1, D and F).

It is known that lysosomes accumulate preferentially aroundthe nucleus, and that the translocation of endosomes, theireventual fusion with lysosomes, and the translocation of lysosomes are all mediated by cytoplasmic microtubules (29-31).Our finding that the expeditious perinuclear accumulation ofcrocidolite fibers is microtubule-dependent is consistent withthe hypothesis that after endocytosis, asbestos fibers are transported along microtubules as endosomes, and eventually accumulate within secondary lysosomes located near the nucleus.In this hypothesis, the perinuclear accumulation of asbestosfibers in part reflects the distribution of lysosomes, the perinuclear location of which is known to be maintained by thecytoplasmic microtubule network (29, 30).

We observed that the microtubule-dependent saltatory transport of endocytosed crocidolite fibers is primarily restricted tothose fibers equal to or less than 5 urn in length. There areseveral possible reason(s) why longer fibers are largely inhibitedfrom undergoing such transport. First, longer fibers may interact with many microtubules, all of which attempt to move themin different directions. Such fibers may also be sterically inhibited—e.g., once entangled within the anastomosing network of

intermediate (keratin and vimentin) filaments—from exhibitingrapid and extensive linear motions. Alternatively, longer fibersmay have a propensity to become demembranated. Indeed, it isnot uncommon to see mineral fibers within the cytoplasm ofcells containing endocytosed asbestos fibers that are not surrounded by an endosomal membrane (42-46). Whether thisdemembranation is selectively related to fiber length is unknown, and is an important consideration for ultimately understanding the mechanism(s) by which asbestos affects cellularprocesses. In all of the cases we examined by same cell correlative LM/EM, the (smaller) fibers undergoing saltatory transport were shown to be surrounded by a membrane. Althoughwe cannot conclude that demembranated fibers do not undergosaltatory transport, it seems highly unlikely that they do soconsidering what is currently known regarding the dependenceof such transport on a surrounding membrane (see above). It isalso notewothy that if fiber demembranation occurs after lysosomes fuse with the asbestos-containing endosóme, then asbestos cytotoxicity may be due, in part, to the resulting release ofdeleterious hydrolytic enzymes into the cytoplasm (47).

The perinuclear accumulation of crocidolite fibers positionsthem to interact with the forming mitotic spindle during celldivision. However, it has been shown that the robust radialarrays of astral microtubules generated by the separating spindle poles transport membrane-bound vesicles, lysosomes, andother organelles away from the region of the forming spindle,i.e., either towards the astral periphery or into the astral center(30, 48, 49). Therefore, the smaller membrane-bound asbestosfibers capable of saltatory motion would presumably be transported away from the region of the forming spindle along withother organelles. By contrast, larger membrane-bound asbestosfibers, and demembranated fibers, may not be as efficientlytranslocated by the astral microtubules. As a result, these fiberswould be preferentially positioned to interfere with spindleformation and chromosome movement. This pathway is consistent with the hypothesis that asbestos promotes transformation by interfering with the normal course of chromosomedistribution during mitosis to produce aneuploid cells, andprovides an explanation for why longer fibers possess greatertransforming ability than shorter ones. We are currently testingthese ideas by examining the interaction of asbestos fibers withthe mitotic spindle in newt lung epithelial cells.

ACKNOWLEDGMENTS

We thank A. DeMarco for technical assistance and Drs. J. Gierthy,C. A. Mannella, B. R. Brinkley, and S. S. Bowser for their discussionsand com incuts during the course of this study.

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1991;51:4942-4947. Cancer Res   Richard W. Cole, Jeffrey G. Ault, John H. Hayden, et al.   Lung Epithelial Cells

VertebrateMicrotubule-mediated Transport after Endocytosis in Crocidolite Asbestos Fibers Undergo Size-dependent

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