importance of biopersistence in the pathogenicity of asbestos
Draft 17 January 2011
A Review of Fiber Biopersistence
as a Potential Mechanism of Asbestos Tumorigenicity
David Bernstein
Thomas Hesterberg
Ken Donaldson
Gunther Oberdörster
Abstract:
It is well accepted that the dose, dimensions,durability of respirable fibers in the lung, and in some cases the surfacecharacteristics of the fibers,arethe critical determinants of their potential adverse health effects. This mechanistic understanding of fiber toxicology is based primarily on the results of well conducted chronic inhalation toxicology studies of synthetic vitreous fibers. More recently, studies have extended these concepts to the two major classes of asbestos;serpentine asbestos (chrysotile) and amphibole asbestos (e.g., crocidolite, amosite, etc.). Chrysotile asbestos is a very thin rolled sheet silicate,which can be dissolved and broken apart by the acidicenvironment insidethe lysosomes of macrophages. In contrast, amphibole asbestos is a chained silicate, which is formed in a solid cylindrical shape with a quartzlike external surface and is insoluble at all pHs. The existing database of fiber toxicity studies strongly suggests that human exposure to respirable fibers that are biopersistent in the lung and induce significant and persistent pulmonary inflammation, cell proliferation, and fibrosis should be viewed with concern.
Introduction:
‘Asbestos’ is not a mineral in itself. Instead, it is a collective term given to a group of minerals having crystals that occur in fibrous forms. The term ‘asbestos’ was adopted for commercial identification.
The six minerals commonly referred to as asbestos come from two distinct groups of minerals. One group is known as serpentines (chrysotile, white asbestos); while the other group is termed the amphiboles (amosite, brown asbestos; crocidolite, blue asbestos; anthophyllite; tremolite; and actinolite). While both are silicate minerals, the two groups are chemically and mineralogically distinct.
Recent studies have shown that these two classes of asbestos are very different in their toxicological potential. A greater knowledge of the differences in mineralogy and chemistry of these two classes of mineral fibers has provided a mechanistic basis for understanding the differences in their toxicity. Many of the earlier studies of these two classes of asbestos did not demonstrate such differences in toxicity and suggested that serpentine asbestos (chrysotile)was of similar potency to amphibole asbestos. This paper provides a review of not only the more recent studies, which demonstrate a marked difference between chrysotile and amphiboleasbestos, but also examines on a quantitative basis the earlier toxicological studies in order to determine whether there is coherence in the results.
THE DIFFERENCES IN SERPENTINE AND AMPHIBOLE ASBESTOS:
SERPENTINE ASBESTOS (CHRYSOTILE)
Chrysotile is a cylindrical fibrous silicate, which is formed as a very thin rolled sheet, as illustrated in Figure 1. The sheet, which is composed of a sandwich of magnesium and silica, is about 8 angstroms (0.8 nanometers) thick and the magnesium is on the outside of the role. Magnesium is an essential element in the body (the adult human body contains approximately 20-28 gm of magnesium, WHO, 2002) and is soluble in the lung surfactant (NAS, 1980). In the lung, the magnesium dissociates and the crystalline structure of this sheet silicate is readily attacked by acid, such as occurs upon contact with or when phagocytized by the macrophage (pH 4 - 4.5). This process causes the rolled sheet of the chrysotile fiber to break apart and decompose into small pieces (Figure 2). These pieces can then be readily cleared from the lung by macrophages through muco-cilliaryand lymphatic clearance. If the fiber is swallowed and ingested it is attacked by the even stronger acid environment (hydrochloric acid, pH 2) of the stomach.
Figure 1
Figure 2
AMPHIBOLE ASBESTOS (CROCIDOLITE, AMOSITE, TREMOLITE, ETC.):
This is in contrast to the amphibole asbestos class of fibers, which are formed as solid rods/fibers as illustrated in Figure 3. The structure of an amphibole is a double chain of silicate tetrahedral which makes it very strong and durable. The external surface of the crystal structures of the amphiboles is quartz-like, and has the chemical resistance of quartz. The amphibole fibers have negligible solubility at any pH that might be encountered in an organism.
Figure 3
The characteristics of these two classesof mineral fibers, serpentines and amphiboles, have been long described in the scientific literature. The differences in chemistry and solubility would imply that within the milieu of the pulmonary system that these two minerals would behave differently. Pundsack (1955) described the colloidal and surface chemistry of chrysotile and how it behaves in suspension. Hargreaves and Taylor (1946) indicated that “If fibrous chrysotile is treated with dilute acid the magnesia may be completely removed, and the hydrated silica remaining, though fibrous in form, has completely lost the elasticity characteristic of the original chrysotile fibre and gives an X-ray pattern of one or perhaps two diffuse broad bands indicating that the structure is 'amorphous' or 'glassy' in type.” The acid insolubility of amphiboles in comparison to chrysotile hasbeen confirmedexperimentally by Speil and Leineweber (1969).
FACTORS WHICH INFLUENCE FIBER TOXICITY:
Mineral fiber toxicology has been associated with three key factors: dose, dimension and durability. One determinant of the dose to the lung is the fiber exposure level, which is determined by the fiber’s physical characteristics/dimensions, how the fibrous material is used, and the industrial hygiene control measures that are implemented. In addition, the thinner and shorter fibers will weigh less and thus can remain suspended in the air longer than thicker and longer fibers. Most asbestos fibers are thinner than commercial insulationfibers, and depending upon type are thicker or in the range of the new nano-fibers which are currently being developed.
The fiber dimensions (length and diameter)also determine whether a fiber isrespirable, i.e., whether it will be deposited in the alveolar region of the lung. Fiber length is also a factor in determining toxicity in the lung milieu once inhaled. Shorter fibres, of the size which can be fully engulfed by the macrophage, will be cleared by mechanisms similar to those for non-fibrous particles. These include clearance through the lymphatics and macrophage phagocytosis and clearance via the mucociliary escalator. It is only the longer fibers, which the macrophage cannot fully engulf, which if they are biopersistent can lead to disease.
This leads to the third factor, that of durability. Those fibers whose chemical structure renders them wholly or partially soluble once deposited in the lung are likely to either dissolve completely, or dissolve until they are sufficiently weakened locally to undergo breakage into shorter fibres. The remaining short fibres can then be removed though successful phagocytosis and mucociliary clearance.
Synthetic vitreous fibers (SVFs) such as fiber glass are amorphous silica structures. In the lung, these fibers have been found to dissolve by two principal mechanisms either through congruent or incongruent dissolution. Congruent dissolution of SVFs leads to complete dissolution of the fibers over time. In contrast, incongruent dissolution leads to leaching of metal oxides from the fiber, leaving a weakened silicon dioxide matrix, which can lead to fiber breakage. The shortening of fibers resulting from fiber breakage can enhance macrophage-mediated clearance from the lung, thus minimizing toxicity, even from long fibers, if they are biosoluble.
The association of longer fibers (20–50 µm) with pathological effects in the lung compared to the lack of toxicity of shorter fibers (3 µm or less) was reported as early as 1951 by Vorwald et al. (1951). Vorwald concluded that “The mode of action of the long asbestos fiber in the production of asbestosis is primarily mechanical rather than chemical in nature” and that “Long asbestos fibers are essential in the production of the peribronchiolarfibrosis; short fibers are incapable of producing this reaction.”
A fiber is unique among inhaled particles in that the fiber’s aerodynamic diameter is largely related to three times the fiber diameter (Timbrell, 1982). This is because fibers tend to line up with the air flow within the lung, making diameter the most important determinant of deposition(Bernstein, 2006). Thus long thin fibers can penetrate into the deep lung. Within the lung, shorter fibers, which can be fully engulfed by the macrophage, can be removed as with any other particle. However, those fibers, which are too long to be fully engulfed by the macrophage, cannot be cleared by this route. Zeidler-Erdely et al. (2006) have shown that 20 µm fibers can be fully engulfed by human alveolar macrophages. Longer fibers will remain in the lung and can be cleared from the lung only if they can dissolve or break apart.
In the lung, extensive work on modeling the dissolution of mineral fibers, using dynamic in-vitro dissolution techniques and inhalation biopersistence, has shown that the lung has a very large buffering capacity (Mattson, 1994). These studies have shown that an equivalent in-vitro flow rate of up to 1 ml/min is required to provide the same dissolution rate of SVFsat pH 7.4 as that which occurs in the lung.(at ph 4.5?) In humans, the normal amount of fluid entering the lung’s tissue which is removed through the lymphatic flow has been estimated as 0.2 ml/min in non-exposed lungs and can increase by more than 15 fold in response to injury (Taylor, 2006).
The relationship between chemical composition and dissolution and subsequent breakage was first reported by Hammad (1984). Synthetic mineral fibers <5 µm in length had the longest retention in the lung following short-term inhalation, with longer fibers clearing more rapidly and fibers >30 µm in length clearing very rapidly. He proposed that clearance of SVFs is the result of biological clearance and the elimination of fibers by dissolution and subsequent breakage. He proposed that the long fibers were leaching and breaking into shorter fibers, which explains the rapid disappearance of these fibers from the lung. The shorter fibers appear to have a longer retention time, because these fibers were added to the pool via breakage of longer fibers overtime. The importance of this relationship to fiber toxicity has been quantified for synthetic mineral fibers (Hesterberg et al. 1998a & b; Miller et al. 1999; Oberdörster, 2000; Bernstein et al 2001a & b) and more recently in differentiating the toxicity of serpentine asbestos, e.g., chrysotile, from amphibole asbestos, i.e., amosite and crocidolite (Bernstein & Hoskins, 2006).
Chronic inhalation toxicity studies of chrysotile
Early chronic inhalation studies of fibers were often performed without consideration of the respirability of the fibers in the rat lung and without preserving the length distribution of the fibers. In addition, they were often performed at very high total particle/fiber exposure concentrations. As asbestosfibers often occur in bundles of long strands, investigators would grind the fibers to produce a more respirable fraction instead of separating the long thin fibers from the bulk material. This process frequently pulverized the long respirable fiber fraction producing excessive particles and shorter fibers, sufficient to cause lung overload in the rats.
High concentrations of insoluble dusts [GO: Yes, but overload is defined for poorly soluble particles of low cytotoxicity: crystalline SiO2 is not part of this; are amphiboles? Are described earlier as having quartz-like surface], when administered by inhalation in the rat, have been shown to overload the lung by compromising the clearance mechanisms, which can then result in chronic inflammation, fibrosis and a tumor formation(Bolton et al., 1983; Muhle et al., 1988; Morrow, 19881992; Oberdorster, 1995a&b).
As illustrated in Figure 4, inhalation toxicology studies of asbestos wereperformed well above the levels towhich humans have been exposed. However, when the exposure level is elevated to more than 100,000 times human exposure, as occurred in mostolder inhalation studies usingchrysotile and amphibole asbestos, lung overload occurs. [GO: I did not associate asbestos-induced effects with overload in the PSP sense. If you do, you need to show that short asbestos is of low cytotoxicity, like carbon or TiO2. Thus, it should be presented differently: Effects seen in rats with inhaled high concentration PSPs occur with cytotoxic particles (e.g., crystalline SiO2) already at much lower concentrations, and obviously even more at PSP-like higher concentrations/doses. Fig. 4 shows the concept, for cytotoxic particles it is just shifted to the left. If text is left as is, will be cause for criticism, unless there are data shown that short amphibole is low cytotoxic.] [ DB: Davis et al., 1986, showed that short fiber amosite did not produce disease following chronic inhalation. (Davis, J.M.G., et al., "The Pathogenicity of Long Versus Short Fibre Samples of Amosite Asbestos Administered to Rats by Inhalation and Intraperitoneal Injection," British Journal of Experimental Patholog, 67:415-430, 1986.)
Figure 4
Problems in Interpretation of Past Inhalation Studies
In reviewing the scientific literature of animal studies, it is often difficult to evaluate the exposure or lung dose administered in comparison to human exposures, the bivariate size distribution of the fibers, whether dose-response was evaluated, and the route of exposure used in the study. The expression of fiber dose to the deep lung is often the weakest component in these earlier studies. Many studies reported only gravimetric concentration of the aerosol with no information provided on fiber number or bivariate fiber size distribution. This is of particular importance in differentiating the effects of various types of fibers, as fiber length is related to potency. Where fiber number is reported, it wasoften assessedusingphase contrast optical microscopy (PCOM) or scanning electron microscopy(SEM), which for asbestos fibers,can detectonly a fraction of the total fibers present. Only transmission electron microscopy (TEM) can accurately detect all of the fibers in the exposure aerosol or deposited in the lung. When conducting fiber toxicology studies, it should be standard practice to use TEM to determine the bivariate length and diameter of fibers in the exposure aerosol and lung. These data can then be used to determine exposure levels in fibers/cm3and lung deposition.
Dose-response assessments arelacking in most in vivo and in vitro studies. The large majority of earlier inhalation exposure studies and many of the more recent studies have been performed at an apparently arbitrary dose of 10 mg/m3. As an example, the fiber number exposure attained at a 10 mg/m3 exposure to chrysotile has been reported as measured by phase contrast light microscopy (PCOM) to be approximately 2000 WHO fibers/cm3 (length greater than 5 µm). When a similar mass concentration of another chrysotile was measured by SEM, a total fiber count of 100,000 fibers/cm3was observed (Mast et al., 1995; Hesterberg et al. 1993). TEM analysis would show as much as 17 times more fibers than SEM analysis on the same aerosol samples (Breysse et al., 1989). Thus, TEM measurement of the 10 mg/m3 exposure to chrysotile would likely correspond to more than 1,000,000 fibers/cm3. This is 500 times the number of fibers/cm3measured using PCOM. Also, there are few quantitative data presented in past publications on nonfibrous particle content of test fiber preparations. Since nonfibrous particles can contribute to lung overload (Oberdörsteret al., 1995), particle/fiber numbers and dimensions should be comprehensively evaluated in materials used in future work. The historical chrysotile chronic inhalation studies are summarized in Table A1 (Appendix 1). The exposure concentration in all studies were based upon gravimetric determination. Of the 16 studies, 6 did not report the fiber concentration, 8 reported estimates by PCOM and 3 by SEM. The gravimetric exposure concentrations ranged from 2 to 86 mg/m3, which based upon the extrapolation described above (Mast et al., 1995; Breysse et al., 1989), corresponds to between 200,000 and 8,600,000 fibers/cm3. The large majority of these earlier studies targeted 10 mg/m3. The single study performed at the lowest concentration of 2 mg/m3 had a comparative concentration group of 10 mg/m3. In this study, the author’s reported “With a 2 mg/m3 cloud the percentage retention of chrysotile is almost double that for a 10 mg/m3 cloud,” which reflects the difficulty of evaluating dose response at these overload conditions.
Exposure of rats to high aerosol concentrations of fibers creates a very different dose profile in the lung in comparison to human exposures. The rat is considerably smaller than humans and correspondingly the rat’s lungs are more than 300 times smaller than the human lungs. While the rat inhales proportionally less air per minute, the doses administered in some toxicology studies can result in unrealistic fiber lung burdens as compared to human exposure. In addition, for the rat which is mandatory nasal breather, alveolar deposition is largely limited to fibers less than approximately 1 µm in diameter, while in humans, this limit is approximately 3 µm (Morgan, 1985). For most asbestos fibers, however, this difference is less important than for MMVF.
Bernstein (2007) has compared the respiratory and physiological parameters of the rat and human lung and their influence on fiber deposition (Table 3). It should be noted that 100 %[GO: why not using realistic deposition fraction?] deposition was assumed. Deposition is a function of fiber dimension and species. It is estimated that the actual deposition for such fibers is between 10 and 20 %.
Table 3: Respiratory and physiological parameters in the rat and human and their influence of fiber deposition(Reproduced from Table 4 in Bernstein (2007))
Parameter / Rat / Human / Rat/Human RatioMinute ventilation* (ml/min) / 100 [GO: default: 200] / 7,000 / 0.014
Volume inhaled/6 h day (ml) / 36,000 / 2,520,000 / 0.014
Lung Volume* (ml) / 13 / 4341 / 0.003
Alveolar Surface Area* (µm2) / 463,000,000,000 / 143,000,000,000,000 1 / 0.003
Number Alveoli/lung* / 40,000,000 / 1,000,000,000 / 0.04
Number Alveolar Cells/lung* / 130,000,000 / 56,000,000,000 / 0.002
Fiber Exposure Concentration (f/ml) / 100 / 0.2 / 500
Number Fibers Inhaled/day [WHO? Total?] / 3,600,000 / 504,000 / 7
Number fibers/alveoli/day / 0.09 ( see 2) / 0.0005 / 179
Number fibers/alveolar cell/day / 0.03 (see 2) / 0.000009 / 3,077 (see 3)
Number fibers/ µm2 of alveolar surface/day / 0.0000078 / 0.000000004 / 2,206
Fiber Exposure Concentration (f/ml) / 1,000 / 0.2 / 5,000
Number Fibers Inhaled/day / 36,000,000 / 504,000 / 71
Number fibers/alveoli/day / 0.9 / 0.0005 / 1,786
Number fibers/alveolar cell/day / 0.28 / 0.000009 / 30,769
Number fibers/ µm2 of alveolar surface/day / 0.000078 / 0.000000004 / 22,061
Fiber Exposure Concentration (f/ml) / 100,000 / 0.2 / 500,000
Number Fibers Inhaled/day / 3,600,000,000 / 504,000 / 7,142 (see 3)
Number fibers/alveoli/day / 90 / 0.0005 / 178,571
Number fibers/alveolar cell/day / 28 / 0.000009 / 3,076,923
Number fibers/ µm2 of alveolar surface/day / 0.0078 / 0.000000004 / 2,206,109
* Pinkerton et al. (1992)