"The Mechanism of Asbestos-Induced Carcinogenesis: 
Calcium and Plasma Membrane Integrity" 
Joseph H. Guth, Ph.D. 
Interscience Research, Inc. 
2614 Wyoming Avenue 
Norfolk, VA 23513 
(Paper Presented to the Virginia Academy of Sciences, 59th Annual Meeting, Norfolk, VA), 
 May 14, 1981 
Abstract
   The major properties determining carcinogenic potential of asbestos fibers, are their size and shape.  The chemical composition has minimal relevance.  The initial cellular interaction is the partial or complete phagocytotic uptake of fibers of appropriate sizes.  Phagocytotically-active cell types, such as macrophages, appear to be the most sensitive.  The most carcinogenic fiber size is approximately 1.25 X 3.75 micron or longer.  Cell impalement, where the fiber is not completely taken into the cell, or has penetrated the membrane mechanically, seems to be the initial damaging event.  Within 36 hours after application of asbestos fibers, gross karyological abnormalities appear.  Polyploidy and chromosome breakage have been observed which must lead to mutagenesis and ultimately, carcinogenesis.  All of these results are predictable from what is known about the capabilities, compartmentation, and management of the calcium ion in mammalian cells.  Allowing excess Ca++ to leak into the cytosol is known to (1) cause abnormal chromosomal condensation, (2) activate the mitotic spindle contraction and mitotic cleavage off-schedule, and (3) modulate the phosphorylation of histones, and thus gene readout.  A leaky plasma membrane and a disturbance in Ca++ levels can bring together all major observations on asbestos carcinogenesis into a unified theory. 

   Carcinogens can be of various types.  Those which have been well studied include organic chemicals, mycotoxins, inorganic chemicals, ionizing radiation, tumor viruses and hereditary tumorigenesis.  Asbestos fibers, which are mineral silicates having crystallized in a fibrous habit, have been demonstrated to be a new class of carcinogen, namely particulate and fibrous.  This paper presents a more detailed proposal for the mechanism of fiber carcinogenesis. 
   Several different minerals form asbestos fibers under appropriate mineralogical conditions.  Chrysotile, amosite, crocidolite, tremolite, actinolite and anthophyllite, the six most common asbestiform minerals, fall into two broad mineralogical categories.  These two categories, the serpentines and amphiboles, are distinguished by the crystallographic lattices which form their basis.  From studies reported in the late 1960's and early 1970's, the chemical composition and crystallographic characteristics are not now considered to be relevant to the carcinogenic potential of asbestos fibers (Harington et al, 1975). 
   What has been implicated as the basis of the carcinogenic potential of asbestos fibers is the physical shape, size and insolubility of the fibers.  Specific, though indirect, evidence implicates fibers which are approximately 1.25 microns in diameter by 3.75 microns in length are the most carcinogenic size range (Stanton and Wrench, 1972).  The range of fiber sizes which proved carcinogenic ranged from 1 to 20 microns in length and from 0.05 to 3 microns in diameter.  The range of aspect ratios (ratio of fiber length to diameter) which had the highest carcinogenic efficacy was 3-to-1 or greater.  Other non-asbestos fibers with similar dimensional and durability properties were also demonstrated to have carcinogenic potential in this study. 
   Cell physiologists (Chambers and Chambers, 1961) experienced in animal cell micromanipulation techniques have found that cells can be routinely pierced with microelectrodes, microneedles and micropipettes roughly 0.2 to 0.5 micron in diameter without acutely injuring the cell.  Such impaled cells can be kept alive for many hours or even days.  If the micropipette is carefully removed, the cell can survive indefinitely.  Thus, cells can survive impalement under certain conditions. 
    This implies that when a cell plasma membrane is partially penetrated by a long and fine fiber, either mechanically or through phagocytosis-assistance, the membrane has the property of self-sealing itself around the fiber body.  Pathology studies of asbestos-induced tumors frequently involve cell types known to have an active, phagocytotic capability (e.g., mesothelial cells).  In in vitro studies, pairs of these types of cells are frequently found attached on both ends of asbestos fibers.  This picture was commonly found when the fiber length was too long for the cells to completely engulf the fiber phagocytotically.  Depending on the presence and geometry of submicroscopically separated asbestos fibrils in the membrane-sealing plane, one of the following can result from fiber impalement: 
(a) complete self-sealing, 
(b) partial self-sealing with fluid leakage pathways, or 
(c) gross membrane rupture and cell death. 
   If either (a) or (b) occurs, it will result in the survival of the cell membrane, and the cell (Figure 1).  Under these specific conditions, a slow rate of leakage of extracellular fluid into the cell's cytosol can be expected to occur.  Such occurrences are familiar to cell biologists who perform micromanipulations of cells and were described in detail to regularly occur during micromanipulation of various cell types (Chambers and Chambers, 1961).  Interaction of asbestos fibers with cultured cells has been reported to result in the release of lysosomal enzymes (presumably through stimulation of exocytosis).  This occurred without the release of cytosolic enzymes from mononuclear phagocytotic cells. This latter finding implies that the leakage pathway is not necessarily bi-directional (wide open) nor to result in a total loss of membrane semi-permeability (Davies et al, 1974).  The predictable results of this partial leakage condition form the proposed hypothesis for a mechanism for transformation of an initially normal cell into a cancerous cell. 
   Different carcinogens act on cells in different ways.  They all have certain aspects in common, however. 
1.  First, carcinogens all have the ability to damage the genetic composition of the cell, either directly through DNA modifications, or through inducing gross chromosomal abnormalities. 
2.  Second, the initial action of the carcinogen does not immediately cause the cell to lose its growth controls (contact inhibition and organized growth patterns with specific cell-cell adhesion).  Such a pre-cancerous cell must undergo at least one cell division in order to express the cancerous behavior condition.  This DNA and chromosome replication thus "sets up" or initiates the carcinogenic behavior. 
  When mammalian cells are exposed to asbestos fibers invitro, gross karyological abnormalities appear within 36 to 48 hours of fiber application.  Polyploidy, aneuploidy, chromosomal breakage, chromosomal inversions and fragmentation have been observed (Sincock and Seabright, 1975; Sincock, 1977) which must lead to mutagenesis, and then ultimately can proceed to tumorigenic or carcinogenic transformation. 
  What effects would impalement and fluid leakage cause in a normal, resting cell which might lead to genetic modification?  Guth (1975) extensively reviewed the relevant literature.  This literature described the central role which the cell membrane and intracellular/extracellular calcium ions play in the initiation of cell division, the switching of control points in the cell division cycle, and finally the ordered and equal separation of the new chromosomes into the daughter cells.  The relevant aspects of calcium control will now be described. 
  Cells are dynamic, ever-changing entities.  Virtually all dynamic activities which take place by cells and within cells are initiated and/or driven by calcium ion fluxes between different compartments of the animal cell (Guth, 1975).  These compartments and their range of calcium concentrations observed can be found in Figure 2.  The most important two compartments for our discussion are the following ones, which are separated by the cell's plasma membrane. 
   
No.	Compartment	Free Ca++ Concentration	
1	Extracellular Fluid	1 to 5 milliMolar	
2	Cytosolic Fluid	10-5   to  10-9  Molar	
  What should be noted is that there always exists a calcium conentration gradient, directed from the outside of the cell back into its interior.  This concentration gradient would immediately collapse to equal and uniform concentrations except for the presence of the semi-permiable, two molecule-thick plasma membrane.  This membrane defines the outermost limits of the living cell.  As long as this membrane is intact and operates in maintaining the inwardly-directed calcium gradient, the cell remains alive.  If the internal calcium concentration rises above approximately 10-5 molar, the cell loses its hold on life and dies (Guth, 1975). 
  When the living cell is "at rest", it is not in the cell division cycle, is not phagocytosing, is not motile (with amoeboid or cilial movement), is not propagating electrical action potentials or secreting substances it has synthesized.  In the resting condition, the interior or cytosolic calcium concentration is approximately 10-9 molar.  Any normal stimulus which is applied to the cell, or which it encounters, causes a rapid influx of calcium into the cell.  The interior calcium concentration rises.  At certain concentrations, various dynamic processes are initiated or activated.  In cells which are capable of division (most types of mammalian somatic cells), calcium influxes trigger a resting cell to enter the cell division cycle (G0 to G1  transition).  After initiation, the internal concentration drops again through the combined action of internal cell organelles (i.e., the mitochondria and endoplasmic reticulum) and the active outward transport processes of the plasma membrane.  At various control points within this cycle, the division process will temporarily stop, awaiting another influx of calcium.  Each of these influxes is needed to allow the division cycle to proceed to completion. 
  Each influx has a different geometry and set of internal processes that it is linked to.  Should the cell be unable to control this up-and-down progression of its internal calcium ion concentration, it would suffer from many unplanned and uncoordinated events which would be triggered out of sequence.  Damage at the chromosomal level would be inevitable.  One of the post-initiation calcium increases occurs to initiate DNA synthesis (G1 - S control point).  Another occurs at the beginning of the mitotic phase (M phase).  That influx results in the condensation of the DNA into the form of newly synthesized chromosomes.  Next, another calcium influx is required to cause the mitotic spindle to contract, thus separating the new sets of chromosomes to the daughter cells.  Finally, at the end of the cell cycle, a band-like portion of the cell membrane surface surrounding the equator of the cell allows the calcium to flow in which triggers the pinching off of the cell membrane, which ultimately separates the two daughter cells (Guth, 1975). 
  The presence of an inward leakage pathway for calcium caused by the presence of an asbestos fiber penetrating the cell's membrane is capable of both initiating the cell division cycle in a resting cell, and severely interfering with subsequent,  low-calcium requiring reactions and programmed changes.  Interference with the calcium-mediated attachment of chromosomes to the mitotic spindle can result in aneuploidy.  DNA synthesis is initiated by calcium increase but does not continue to its completion unless the calcium levels are brought back to low levels.  Incomplete replication of chromosomes and chromosome breakage and fragmentation may therefore result from continuing calcium influxes caused by asbestos-induced leakage. 
  Thus, internal calcium mismanagement through a compromised membrane integrity is consistent with the observed karyological changes which attend asbestogenic cancer.  This mechanism lends itself to numerous predictions for testing its validity. 

References
Chambers, Robert and Edward L. Chambers,  "Explorations into the Nature of the Living Cell", (Commonwealth Fund, Harvard University Press, Cambridge, Mass., 1961) 
Davies, Phillip, Anthony C. Allison, Jill Ackerman, Ann Butterfield and Susan Williams, 
Nature, 251, 423  (1974) 
Guth, Joseph H.,  "Calcium Permeability Studies of Cultured Human Cells: Relation to Cell Proliferation and Aging", Ph.D. Dissertation, University of California, Berkeley, (1975), pp. 1-200 
Harington, J. S., A. C. Allison, and D. V. Badami,  "Mineral Fibers: Chemical, Physicochemical, and Biological Properties", in Advances in Pharmacology and Chemotherapy, vol. 12, 1975 (Academic Press, NY), pp. 291-406 
Sincock, A. M., "Preliminary Studies of the in Vitro Cellular Effects of Asbestos and Fine Glass Dusts", in Origins of Human Cancer, Book B, Mechanisms of Carcinogenesis, edited by H. H. Hiatt, J. D. Watson and J. A. Winsten, Cold Spring Harbor Conferences on Cell Proliferation, Vol. 4, pp. 941-54 (Cold Spring Harbor Laboratory, 1977) 
Sincock, Andrew and Marina Seabright,  Nature, 257, 56-8  (1975) 
Stanton, Mearl F. and Constance Wrench,  J. Nat. Cancer Inst., 48, 797-821  (1972) 

Figures 1 and 2 will be added to this file at a future date -- JHG