Analytical transmission electron microscopy of amosite asbestos from South Africa.

Using the recognized amosite standard, we have performed transmission electron microscopy (TEM), scanning electron microscopy (SEM), and chemical analyses. We use high-resolution transmission electron microscopy (HRTEM) and zone-axis selected area electron diffraction (SAED) to describe the molecular structure of the fibers. We find that both microscopic observational evidence and statistical dimensional characteristics indicate that the amosite fibers are formed by longitudinal splitting, with surfaces produced by fine twinning and lateral boundaries formed by parting parallel to the planes of double and triple sheets of amphibole chain structures. Our findings indicate that amosite would not be regulated under current asbestos regulations, which define amphibole asbestos as whole crystals that are not split and that form fibril bundles, not found in our standard. However, it is fully documented that amosite causes mesothelioma, lung cancer, and asbestosis.

On the threshold conditions for electron beam damage of asbestos amosite fibers in the transmission electron microscope (TEM).

Asbestos amosite fibers were investigated to evaluate the damage caused by a transmission electron microscope (TEM) electron beam. Since elemental x-ray intensity ratios obtained by energy dispersive x-ray spectroscopy (EDS) are commonly used for asbestos identification, the impact of beam damage on these ratios was evaluated. It was determined that the magnesium/silicon ratio best represented the damage caused to the fiber. Various tests showed that most fibers have a current density threshold above which the chemical composition of the fiber is modified. The value of this threshold current density varied depending on the fiber, regardless of fiber diameter, and in some cases could not be determined. The existence of a threshold electron dose was also demonstrated. This value was dependent on the current density used and can be increased by providing a recovery period between exposures to the electron beam. This study also established that the electron beam current is directly related to the damage rate above a current density of 165 A/cm2. The large number of different results obtained suggest, that in order to ensure that the amosite fibers are not damaged, analysis should be conducted below a current density of 100 A/cm2.

Asbestos surface provides a niche for oxidative modification.

Asbestos is a potent carcinogen associated with increased risks of malignant mesothelioma and lung cancer in humans. Although the mechanism of carcinogenesis remains elusive, the physicochemical characteristics of asbestos play a role in the progression of asbestos-induced diseases. Among these characteristics, a high capacity to adsorb and accommodate biomolecules on its abundant surface area has been linked to cellular and genetic toxicity. Several previous studies identified asbestos-interacting proteins. Here, with the use of matrix-assisted laser desorption ionization-time of flight mass spectrometry, we systematically identified proteins from various lysates that adsorbed to the surface of commercially used asbestos and classified them into the following groups: chromatin/nucleotide/RNA-binding proteins, ribosomal proteins, cytoprotective proteins, cytoskeleton-associated proteins, histones and hemoglobin. The surfaces of crocidolite and amosite, two iron-rich types of asbestos, caused more protein scissions and oxidative modifications than that of chrysotile by in situ-generated 4-hydroxy-2-nonenal. In contrast, we confirmed the intense hemolytic activity of chrysotile and found that hemoglobin attached to chrysotile, but not silica, can work as a catalyst to induce oxidative DNA damage. This process generates 8-hydroxy-2'-deoxyguanosine and thus corroborates the involvement of iron in the carcinogenicity of chrysotile. This evidence demonstrates that all three types of asbestos adsorb DNA and specific proteins, providing a niche for oxidative modification via catalytic iron. Therefore, considering the affinity of asbestos for histones/DNA and the internalization of asbestos into mesothelial cells, our results suggest a novel hypothetical mechanism causing genetic alterations during asbestos-induced carcinogenesis.

Mineralogy of asbestos.

The term asbestos collectively refers to a group of naturally occurring fibrous minerals which have been exploited in numerous commercial and industrial settings and applications dating to antiquity. Its myriad uses as a "miracle mineral" owe to its remarkable properties of extreme resistance to thermal and chemical breakdown, tensile strength, and fibrous habit which allows it to be spun and woven into textiles. Abundant in nature, it has been mined considerably, and in all continents save Antarctica. The nomenclature concerning asbestos and its related species is complex, owing to the interest held therein by scientific disciplines such as geology, mineralogy and medicine, as well as legal and regulatory authorities. As fibrous silicates, asbestos minerals are broadly classified into the serpentine (chrysotile) and amphibole (crocidolite, amosite, tremolite, anthophyllite, actinolite) groups, both of which may also contain allied but nonfibrous forms of similar or even identical chemical composition, nonpathogenic to humans. Recently, fibrous amphiboles, not historically classified or regulated as asbestos (winchite, richterite), have been implicated in the causation of serious disease due to their profusion as natural contaminants of vermiculite, a commercially useful and nonfibrous silicate mineral. Although generally grouped, classified, and regulated collectively as asbestos, the serpentine and amphibole groups have different geologic occurrences and, more importantly, significant differences in crystalline structures and chemical compositions. These in turn impart differences in fiber structure and dimension, as well as biopersistence, leading to marked differences in relative potency for causing disease in humans for the group of minerals known as asbestos.

High aspect ratio materials: role of surface chemistry vs. length in the historical "long and short amosite asbestos fibers".

In nanotoxicology the question arises whether high aspect ratio materials should be regarded as potentially pathogenic like asbestos, merely on the base of their biopersistence and length to diameter ratio. A higher pathogenicity of long asbestos fibers is associated to their slower clearance and frustrated phagocytosis. In the past decades, two amosite fibers were prepared and studied to confirm the role of fiber length in asbestos toxicity. Long fiber amosite (LFA) and short fiber amosite (SFA) have here been revisited, to check differences in their surface properties, known to modulate the biological responses elicited. We report: (i) micromorphology (abundance of exposed cylindrical vs. truncated surfaces; (ii) surface reactivity (oxidation and coordination state of surface iron, free radical generation and oxidizing potential); (iii) activation of nitric oxide (NO) synthase in lung epithelial cells, as representative of an inflammatory cell response. LFA shows a higher free radical yield, stimulates, more than SFA, NO production by cells and reacts with ascorbic acid, thus depriving the lung lining layer of its antioxidant defenses. The higher activity of LFA than SFA is ascribed to the presence of Fe2+ ions poorly coordinated to the surface. SFA shows only a large number of loosely bound Fe3+ ions, pristine Fe2+ ions having been oxidized during the grinding process converting LFA into SFA. Several factors determine a higher toxicity of LFA than SFA, beside length. The lesson from asbestos indicates that other features besides aspect ratio contribute to the pathogenic potential of a fiber type. All these aspects should be considered when predicting the possible hazard associated to any new fibrous material proposed to the market, let alone nanofibers.

An asbestos job exposure matrix to characterize fiber type, length, and relative exposure intensity.

The relationship between asbestos exposure and disease has been well documented, although questions persist as to variation in risk by the type and length of fiber. For a series of jobs with potential asbestos exposure, the primary fiber type (e.g., amosite, anthophylite, chrysotile, crocidolite, or tremolite) and fiber length were identified and the relative exposure intensity was estimated. The resulting job exposure matrix may be useful in epidemiological studies where asbestos is an exposure of interest.

Sorption of nonpolar aromatic contaminants by chlorosilane surface modified natural minerals.

The efficacy of the surface modification of natural diatomite and zeolite material by chlorosilanes is demonstrated. Chlorosilanes used were trimethylchlorosilane (TMSCI), tert-butyldimethylchlorosilane (TBDMSCI), dimethyloctadecylchlorosilane (DMODSCI), and diphenyldichlorosilane (DPDSCI) possessing different headgroups and chemical properties. Silanol groups of the diatomite and zeolite were modified by chemical reaction with the chlorosilanes resulting in a stable covalent attachment of the organosilanes to the mineral surface. The alteration of surface properties of the modified material was proved by measurements of water adsorption capacity, total organic carbon (TOC) content, and thermoanalytical data. The surface modified material showed great stability even when exposed to extremes in ionic strength, pH, and to pure organic solvents. Sorption of toluene, o-xylene, and naphthalene from water was greatly enhanced by the surface modification compared to the untreated materials which showed no measurable sorption of these compounds. The enhanced sorption was dependent on the organic carbon content as well as on chemical characteristics of the chlorosilanes used. Batch sorption experiments showed that the phenyl headgroups of DPDSCI have the best affinity for aromatic compounds. Removal from an aqueous solution of 10 mg/L of naphthalene, o-xylene, and toluene was 71%, 60%, and 30% for surface modified diatomite and 51%, 30%, and 16% for modified clinoptilolite, respectively. Sorption data were well described by the Freundlich isotherm equation, which indicated physical adsorption onto the lipophilic surface rather than partitioning into the surface organic phase. The chlorosilane modified materials have an apparent potential for application in environmental technologies such as permeable reactive barriers (PRB) or wastewater treatment.

Free radical activity of natural and heat treated amphibole asbestos.

The amphibole minerals amosite and crocidolite were subjected to calcination and to hydrothermal treatment in order to study the effect of these heat treatments on the ability of the minerals to trigger formation of free radicals, which is known to be a main factor causing asbestosis and other asbestos-induced diseases. Free radical activity of the natural and heat treated minerals was studied by using supercoiled DNA (pUC18 plasmid) as a target molecule, and also by means of EPR spectroscopy. It was shown that after calcination of the natural minerals at 1073 K their free radical activity was strongly decreased These results, which may have relevant consequences for asbestos technology, were correlated with concomitant alteration of the structure and surface chemistry of the minerals during calcination.

Estimation of dissolution rate from in vivo studies of synthetic vitreous fibers.

Although the dissolution rate of a fiber was originally defined by a measurement of dissolution in simulated lung fluid in vitro, it is feasible to determine it from animal studies as well. The dissolution rate constant for a fiber may be extracted from the decrease in long fiber diameter observed in certain intratracheal instillation experiments or from the observed long fiber retention in short-term biopersistence studies. These in vivo dissolution rates agree well with those measured in vitro for the same fibers. For those special types of fibers, the high-alumina rock wool fibers that could not be measured in vitro, the method provides a way of obtaining a chemical dissolution rate constant from an animal study. The inverse of the in vivo dissolution rate, the fiber dissolution time, correlates well with the weighted half life of long fibers in a biopersistence study, and the in vivo dissolution rate may be estimated accurately from this weighted half-life.

Biopersistence and durability of nine mineral fibre types in rat lungs over 12 months.

The study objectives were to assess the ability of intratracheal injection methods to discriminate between nine fibre types in respect of pulmonary biopersistence, and to provide approximate estimates of relative biopersistence and durability for a study of general relationships with biological and toxicological responses. The test fibres included six samples of size-selected fibre types specially prepared for research purposes, two commercially available fibres, and amosite. A 1 mg dose of each fibre type was administered to rats by intratracheal injection. The relative biopersistence of fibres in different size categories was assessed from the changes in mean lung burden, as determined by electron microscopy, at 3 days and 1, 6 and 12 months after injection. The ability of the test materials to resist dissolution was measured in a parallel series of simple in vitro acellular experiments at two pHs and in a continuous flow dissolution test. The observed differences in the persistence of fibres of differing length recovered from rat lungs were consistent with the current hypothesis that short fibres are cleared by cellular processes and long fibres by dissolution and disintegration. Differences in persistence of long (> 20 microns) fibres were correlated with measured rates of dissolution in vitro. Differences in persistence among those fibre types also studied by others workers were consistent with their findings after inhalation and intratracheal injection. Overall, the differences in the biopersistences of the test fibres following intratracheal injection were sufficient to enable an examination of the relationship of biopersistence with other biological and toxicological responses. Biopersistence was influenced by both fibre dimensions and solubility.

Chemical differences between long and short amosite asbestos: differences in oxidation state and coordination sites of iron, detected by infrared spectroscopy.

OBJECTIVES: Short fibres of amosite asbestos (SFA), obtained by ball milling of long fibres (LFA), have been shown to be less pathogenic than long fibres. Accumulating evidence suggests an important role for differences in surface chemistry between fibres. Iron has been implicated in the pathogenesis of asbestos fibres. In this study infrared (IR) spectroscopy was used to compare LFA and SFA in terms of the coordination and oxidation state of iron at the three cation sites (M1, M3, M1). METHODS: Infrared was used to examine LFA ad SFA, when dry and when hydrated in the presence and absence of the chelators desferroxamine and ferrozine. With appropriate software the proportions of iron and its oxidation states in the overlapping peaks were resolved and assigned, and the three coordination sites were identified. Data were obtained from 10 samples of both lengths of fibre for each of the four treatments. Iron release was also monitored. RESULTS: Iron was significantly more oxidised in LFA than SFA. Further oxidation of the dry fibres with water, ferrozine, or desferroxamine tended to abolish these differences. There were also significant differences between the proportions of iron held in the different coordination sites of the fibres. For LFA, a higher proportion of its iron was held in the cation sites coordinating less with iron and more with Mg. Interestingly, the sites coordinating single irons were significantly more oxidised than multiple sites. The single iron sites were more oxidised in LFA than SFA and were more readily oxidised by the treatments. CONCLUSIONS: Important chemical differences between LFA and SFA were found. There seemed to be some mobility of iron near the surface. Based on these data it is speculated that the 1 iron surface site may be important in pathogenesis.

Major and trace element compositions of the UICC standard asbestos samples.

Major and trace element compositions for chrysotile (2 samples), amosite, crocidolite, and anthophyllite UICC standard asbestos samples have been determined using UV-visible spectrophotometry, atomic absorption spectometry, flame photometry, volumetric analysis, and gravimetric analysis for major elements and x-ray and optical spectrometry for trace elements. The trace element data are for Li, S, Cl, Sc, V, Cr, Co, Ni, Cu, Zn, Ga, Rb, Sr, Zr, Nb, Ba, La, Ce, Pb, and Th and distribution in the various mineral phases is discussed.

Ferritin adsorption on amosite fibers: possible implications in the formation and toxicity of asbestos bodies.

In order to investigate how endogenous iron can be deposited in vivo on inhaled mineral fibers during early stages of formation of asbestos bodies, in vitro experiments were performed on the adsorption of ferritin onto amosite asbestos. The mineral dust was found to adsorb the protein from an aqueous solution containing 0.3 mg/ml horse spleen ferritin. In order to simulate physiological conditions the aqueous solution was adjusted with 150 mM saline. Polyacrylamide-SDS gel electrophoresis of the desorbed protein showed subunits of approximately 13 and 15 kD, aside from the 20-kD subunit present in the native protein. This suggests that as a result of interactions between ferritin molecules and the solid surface of the mineral fibers, the protein iron core may be released or partially exposed. Data indicate these interactions may have implications in the observed mineral fiber toxicities.

Mineral phases and some reexamined characteristics of the International Union Against Cancer standard asbestos samples.

Standard asbestos samples to be used for biomedical research were first prepared by the International Union Against Cancer (UICC) in 1966 in the United Kingdom and South Africa. Using modern techniques, X-ray diffractometry, analytical transmission electron microscopy, and thermal analysis, we have now analyzed these UICC samples to determine the mineral compositions (mineral phases) and their respective quantities. UICC chrysotile A (from Zimbabwe) contains 2% fibrous anthophyllite as impurity; chrysotile B (from Canada) does not contain any fibrous impurities, only non-fibrous minerals. UICC amosite and crocidolite are almost pure. UICC anthophyllite has 20-30% talc as impurity. The chemical compositions and fiber size distributions of the UICC asbestos samples have also been determined. The mean widths of the fibers of chrysotile A and B are smaller than those of the amphibole fibers. This agrees well with the earlier results which showed the two chrysotile samples to have a larger respirable fraction than the amphiboles.

The sizes, shapes, and mineralogy of asbestos structures that induce lung tumors or mesothelioma in AF/HAN rats following inhalation.

Data from inhalation studies in which AF/HAN rats were exposed to nine different types of asbestos dusts (in 13 separate experiments) are employed in a statistical analysis to determine if a measure of asbestos exposure (expressed as concentrations of structures with defined sizes, shapes and mineralogy) can be identified that satisfactorily predicts the observed lung tumor or mesothelioma incidence in the experiments. Due to limitations in the characterization of asbestos structures in the original studies, new exposure measures were developed from samples of the original dusts that were re-generated and analyzed by transmission electron microscopy using a direct transfer technique. This analysis provided detailed information on the mineralogy (i.e., chrysotile, amosite, crocidolite or tremolite), type (i.e., fiber, bundle, cluster, or matrix), size (length and width) and complexity (i.e., number of identifiable components of a cluster or matrix) of each individual structure. No univariate measure of exposure was found to provide an adequate description of the lung tumor responses observed among the inhalation studies, although the measure most highly correlated with tumor incidence is the concentration of structures > or = 20 microns in length. Multivariate measures of exposure were identified that do adequately describe the lung tumor responses. Structures contributing to lung tumor risk appear to be long (> or = 5 microns) thin (0.4 microns) fibers and bundles, with a possible contribution by long and very thick (> or = 5 microns) complex clusters and matrices. Potency appears to increase with increasing length, with structures longer than 40 microns being about 500 times more potent than structures between 5 and 40 microns in length. Structures < 5 microns in length do not appear to make any contribution to lung tumor risk. This analysis did not find a difference in the potency of chrysotile and amphibole toward the induction of lung tumors. However, mineralogy appears to be important in the induction of mesothelioma with chrysotile being less potent than amphibole.

Mediated, thin-layer cell, coulometric determination of redox-active iron on the surface of asbestos fibers.

Redox-active iron on the surface of asbestos fibers was detected and quantified using a thin-layer cell, coulometric method with soluble mediators to shuttle electrons between the mineral fibers and the solid electrode. The working and counter electrodes consisted of gold films on a glass slide with reference electrodes of silver. Asbestos fibers were entrapped in a thin-layer cell of 25 microns thickness. Hexaammineruthenium(II) or o-dianisidine (dication) was used as the reducing or oxidizing mediator, respectively. Hexaammineruthenium(III) undergoes a one-electron reduction, and protonated o-dianisidine undergoes a sequential two-electron oxidation. The measurement involved determination of the total charge for the oxidation or reduction of surface-immobilized Fe(II) or Fe(III) on the asbestos fibers. Analysis of the results showed that crocidolite and amosite have 4.3 +/- 0.7 and 3.3 +/- 0.7 nmol/mg of total redox-active iron that is accessible to the mediators, respectively. This corresponded to a surface coverage of accessible redox-active iron of approximately 4.3 x 10(-11) mol/cm2 for crocidolite and 9.5 x 10(-11) mol/cm2 for amosite. Furthermore, Fe(II) constituted 76% or 25% of the accessible redox-active iron on the surface of crocidolite or amosite, respectively. The method may be applied to other types of solid materials with redox-active species on their surfaces.

Relation between lung asbestos fibre burden and exposure indices based on job history.

Lung asbestos burden was compared with exposure indices derived from job history interviews in 42 male subjects originating from the Montréal Case-Control Study project, 12 of whom had documented asbestos exposed job histories. Job interview data consisting of a chronological timetable of job histories were translated into detailed exposure indices by an expert group of hygienists and chemists. Total and individual asbestos fibre type concentrations were quantified by transmission electron microscopy with fibre identification by energy dispersive chi ray spectrometry after deparaffinisation of tissue blocks and low temperature plasma ashing. Geometric mean or median asbestos content was higher in subjects with an asbestos exposed job history than those without for retained dose of amosite, total commercial amphiboles, and total asbestos fibre. Except for crocidolite fibre diameter, which was significantly less in the lungs of exposed workers, no consistent differences were found in measurements of fibre dimension for any fibre type. Subgroups of subjects exposed to silica, metals, or smokers and non-smokers without significant occupational exposure showed varying patterns of lung asbestos fibre type deficit compared with the asbestos exposed subgroup. There was an overall trend for higher lung asbestos content proportional to higher exposure indices for asbestos representing concentration, frequency, and reliability. These exposure indices as well as duration of exposure (in years) were independent predictors of total asbestos content in regression analyses when combined in a model with age. Stepwise regression indicated that exposure concentration was the most important variable, explaining 32% of the total variation in total asbestos content. Smoking, whether expressed in ever or never smoked dichotomy or in smoked-years, had no relation to lung asbestos content in this model.

Biological activity of respirable industrial fibres treated to mimic residence in the lung.

The durability of fibres in the lung environment after deposition could be a key factor in determining whether they accumulate to a sufficient tissue dose to cause pathological change. There is a shortage of information on the relative durabilities of respirable industrial fibres of various types. We describe a strategy for assessing the ability of different fibre types to persist in the lung milieu and to retain their biological activity. This is particularly important for the development of mesothelioma, where the long latent time that characterises this disease would be expected to exclude, from culpability, fibres that are not durable. We have combined a pre-treatment step in pH 5.0 or 7.0 with an assay that relies on the ability of fibres to damage the mesothelium. The long-term aim is to assess the impact that treatment in various pH solutions has on (a) fibre size/number, (b) loss of key elements, (c) the ability to damage the mesothelium. Such information should enable us to better predict the potential of fibres to cause mesothelioma.

DNA strand breaks following in vitro exposure to asbestos increase with surface-complexed [Fe3+].

Surface functional groups on silicate dusts complex iron cations which can cycle through reduction and oxidation states to generate free radicals. These oxidants have a capacity to produce DNA strand breaks and mutations which are primary events in cancer induction. A differential in the capacity of fibrous silicates to produce carcinoma is recognized with the amphiboles demonstrating a greater biologic effect than the serpentine fiber chrysotile. We tested the hypothesis that the differences in genotoxicity of these fibrous silicates correspond to varying concentrations of iron complexed to the surface. Relative to chrysotile, the amphibole fibers complexed greater amounts of iron cations from both inorganic and in vivo sources. Increased concentrations of surface-complexed iron were associated with greater oxidant generation, measured as thiobarbituric acid-reactive products of deoxyribose, and more covalently closed, circular DNA strand scission. These results indicate that genotoxic effects of these fibers may correspond to their capacity to complex iron at the surface.

Effect of long-term removal of iron from asbestos by desferrioxamine B on subsequent mobilization by other chelators and induction of DNA single-strand breaks.

The long-term removal of iron from crocidolite or amosite by desferrioxamine B (DF) at pH 7.5 or 5.0 was studied. Crocidolite or amosite (1 mg/ml) was suspended in 50 mM NaCl at pH 7.5 or 5.0 with the addition of 1 mM DF for up to 90 days. Although the rate of iron mobilization decreased with time, iron was continuously mobilized from both forms of asbestos at pH 5.0 or 7.5. The amount of iron mobilized from crocidolite was at least twice that mobilized from amosite at either pH. Iron was mobilized more rapidly from crocidolite at pH 5.0 than at 7.5 for the first 15 days, but at later times the amount being mobilized at pH 7.5 became equal to or slightly greater than that at 5.0. For amosite, the mobilization at pH 5.0 was always greater than that at pH 7.5. Next, the effect of iron removal from asbestos by DF on subsequent iron mobilization by a second chelator (EDTA or citrate) and on induction of DNA single-strand breaks (SSBs) was studied. Asbestos, treated for up to 15 days with DF at pH 7.5, was washed to remove ferrioxamine and excess DF, then incubated with EDTA or citrate (1 mM). The rates of iron mobilization from both forms of asbestos by a second chelator decreased as more and more iron was removed by DF. Induction of DNA SSBs also decreased, reflecting the unavailability of iron to catalyze the damage. The results suggest three things. First, if long-term mobilization of iron from asbestos occurs in vivo as has been observed in vitro, it may play a role in the long-term biological effects of asbestos. Second, more rapid mobilization of iron from asbestos fibers may occur when the fibers are phagocytized by cells and maintained in phagosomes where the pH is 4.0-5.0. Third, treatment of asbestos by iron chelators, such as DF, prior to exposure to cultured cells or whole animals, may reduce the biological effects of asbestos resulting from iron, but may not completely eliminate them.