Cytotoxic effects of quartz and chrysotile asbestos: in vitro interspecies comparison with alveolar macrophages

Cytotoxic effects of DQ12 quartz and chrysotile asbestos on alveolar macrophages of different animal species were compared in vitro. The type of cell reaction toward the cytotoxic dusts was always the same: a loss of cell viability (trypan blue dye exclusion test) was accompanied by the release of cytoplasmic and lysosomal enzymes. The extent of cellular destruction depended upon the amount of dust applied. In the range of 50-100 micrograms/ml quartz or chrysotile asbestos, species-specific variations were observed in the sensitivity of the cells. At this concentration alveolar macrophages of dogs, monkeys, and human patients were damaged to a greater extent than the cells from rats and cattle. Simultaneous incubation of the cells with quartz and L-alpha-dipalmitoyl lecithin resulted in a reduction of the cytotoxic quartz effect. The extent of the protective effect varied according to the species. In the case of chrysotile asbestos no reduction of the fibers cytotoxicity was observed in the presence of L-alpha-dipalmitoyl lecithin.     

Effect of poly-2-vinylpyridine-N-oxide and sucrose on silicate-induced hemolysis of erythrocytes

The biological activity of montmorillonite, palygorskite, kaolinite, chrysotile, and silica was examined using in vitro hemolysis of erythrocytes. The hemolytic potency was in the order montmorillonite greater than silica greater than palygorskite greater than chrysotile greater than kaolinite. The polymer poly-2-vinylpyridine-N-oxide inhibited hemolysis caused by montmorillonite, palygorskite, kaolinite, and silica, but it was less effective with chrysotile. The extent of polymer binding to the silicates and red blood cells was measured by UV spectroscopy. When sucrose was substituted for the saline solution as the incubating medium, hemolysis was eliminated in all systems except chrysotile-erythrocyte, where it was enhanced. The results indicate that both hydrogen bonding and ionic interactions between silicate surfaces and the erythrocyte membrane are important in the hemolytic process.     

The health effects of chrysotile: current perspective based upon recent data

This review substantiates kinetically and pathologically the differences between chrysotile and amphiboles. The serpentine chrysotile is a thin walled sheet silicate while the amphiboles are double-chain silicates. These different chemistries result in chrysotile clearing very rapidly from the lung (T(1/2)=0.3 to 11 days) while amphiboles are among the slowest clearing fibers known (T(1/2)=500 days to infinity). Across the range of mineral fiber solubilities chrysotile lies towards the soluble end of the scale. Chronic inhalation toxicity studies with chrysotile in animals have unfortunately been performed at very high exposure concentrations resulting in lung overload. Consequently their relevance to human exposures is extremely limited. Chrysotile following subchronic inhalation at a mean exposure of 76 fibers L>20 microm/cm(3) (3413 total fibers/cm(3)) resulted in no fibrosis (Wagner score 1.8-2.6), at any time point and no difference with controls in BrdU response or biochemical and cellular parameters. The long chrysotile fibers were observed to break apart into small particles and smaller fibers. Toxicologically, chrysotile which rapidly falls apart in the lung behaves more like non-fibrous mineral dusts while response to amphibole asbestos reflects its insoluble fibrous structure. Recent quantitative reviews of epidemiological studies of mineral fibers have determined the potency of chrysotile and amphibole asbestos for causing lung cancer and mesothelioma in relation to fiber type have also differentiated between these two minerals. The most recent analyses also concluded that it is the longer, thinner fibers that have the greatest potency as has been reported in animal inhalation toxicology studies. However, one of the major difficulties in interpreting these studies is that the original exposure estimates rarely differentiated between chrysotile and amphiboles. Not unlike some other respirable particulates, to which humans are, or have been heavily occupationally exposed, there is evidence that heavy and prolonged exposure to chrysotile can produce lung cancer. The value of the present and other similar studies is that they show that low exposures to pure chrysotile do not present a detectable risk to health. Since total dose over time decides the likelihood of disease occurrence and progression, they also suggest that the risk of an adverse outcome may be low if even any high exposures experienced were of short duration.     

Interrelationship between hemolysis and lipid peroxidation of human erythrocytes induced by silicic acid and silicate dusts

Silicic acid and silicate dusts (slate dust and chrysotile asbestos) cause hemolysis of erythrocytes in vitro. The peroxidation of polyunsaturated fatty acids (PUFA) of erythrocyte membrane lipids is also enhanced by incubating the erythrocytes with silicic acid and silicate dusts in in vitro. Hemolysis of erythrocytes elicited by silicic acid and silicate dusts is inhibited significantly by polyvinyl-pyrrolidone and dipalmitoyl lecithin (DPL). These agents, however, have no effect on silicic acid and silicate dust induced peroxidation of erythrocyte membrane lipids. On the other hand, peroxidation of erythrocyte membrane lipids, induced by silicic acid and silicate dusts, is inhibited almost completely by adding superoxide dismutase and catalase to the incubation system, whilst the hemolysis of erythrocytes induced by silicic acid and silicate dusts is unaffected by these agents. Similarly the lysis of erythrocytes, induced by silicic acid and silicate dusts, proceeds at a much faster rate than silicic acid and silicate dust induced lipid peroxidation. These results indicate that silicic acid and silicate dust induced hemolysis and lipid peroxidation represent two independent processes.     

In Vitro Studies on the Cytotoxic Action of Different Varieties of Asbestos Dust on Macrophages

Abstract: The cytotoxic action of a variety of asbestos dusts viz. chrysotile, amosite, anthophyllite, tremolite and actionlite was tested in vitro using alveolar and peritoneal macrophage cell cultures. The release of acid phosphatase and the uptake of the dye erythrosin B were taken as parameters of cytotoxicity. Among the several varieties of asbestos tested for this purpose, chysotile proved to be most cytotoxic for both the alveolar and peritoneal macrophages, although the former were more sensitive. Amosite was only slightly toxic for alveolar macrophages. Anthophyllite, tremolite and actinolite behaved as control inert dusts. Carboxymethylcellulose exhibited an anticytotoxic effect when chrysotile was pretreated with the polymer or when the dust cell interaction was allowed to take place in its presence.     

in vitro Evaluation of the Cytotoxic Potential of a Novel Man-Made Fiber, Calcium Sodium Metaphosphate Fiber (Phosphate Fiber)

In Vitro Evaluation of the Cytotoxic Potential of a Novel Man-MadeFiber, Calcium Sodium Metaphosphate Fiber (Phosphate Fiber).LI, A. P., and Myers, C. A. (1988). Fundam. Appl. Toxicol 11,21-28. As part of a comprehensive effort to evaluate the toxicologicalpotential of calcium sodium metaphosphate fiber (Phosphate Fiber),the in vitro cytotoxicity of the fiber in cultured cells wasstudied. Two pulmonary-derived cell systems (rat alveolar macrophages,It RAM; rat lung epithelial cells, LEC) and an established cellline (Chinese hamster ovary, CHO) were used. Release of lactatedehydrogenase (LDH) was used as an endpoint for cytotoxicityfor all three cell types. In addition, inhibition of colonyformation was used for CHO cells. The cytotoxicity of PhosphateFiber was compared to a variety of mineral dusts and fibersincluding chrysotile asbestos, crocidolite asbestos, two glassfibers, calcium sulfate fiber, titanium dioxide, as well asthe nonfibrous raw material, calcium sodium metaphosphate glass.Results with all Three cell culture systems demonstrated thatthe Phosphate Fiber was less cytotoxic than the two asbestosfibers, similar in cytotoxicity to the glass fibers, and morecytotoxic than the calcium sulfate fiber and titanium dioxide.To further investigate the cytotoxicity of the Phosphate Fiber,it was fractionated by sedimentation into small and large fibers.The small Phosphate Fiber was found to be more cytotoxic andthe large Phosphate Fiber to be less cytotoxic than the UnfractionatedPhosphate Fiber. The in vitro data suggest that Phosphate Fiberis less cytotoxic than asbestos, but further determination ofsafety can only be made after the in vivo data havebeen obtained.     

On the cytotoxicity of chrysotile asbestos fibers toward pulmonary alveolar macrophages. II. Effects of nicotinamide on the cell metabolism

Since it was recently shown that the addition of nicotinamide (NAM) to pulmonary alveolar macrophages (PAM) cell monolayers significantly altered their ATP pools (Nadeau and Lane, 1988), the effects of the vitamin on the metabolism of the cells, exposed or not to very short chrysotile asbestos fibers (VSF), were evaluated. First, it was found that the addition of NAM to the culture medium caused a dose-dependent (5-30 mM) decrease in the extracellular liberation of lactate and pyruvate by PAM. This is suggestive of a direct effect of NAM on the metabolism of glucose. A decrease in extracellular lactate was also observed when control PAM were exposed to 50 micrograms of VSF asbestos. This latter effect was however progressively abolished when the NAM was added to the asbestos-exposed cell monolayers. Second, contrary to the lactic acid production, the exposure to chrysotile caused an increase in the extracellular liberation of pyruvate by PAM. This cell response to the asbestos fibers could represent an antioxidative defense mechanism. Yet, interestingly enough, this effect of the VSF on PAM was not suppressed by the presence of the vitamin. The NAM also induced a dose-dependent decrease in the total lactate dehydrogenase content of PAM monolayers. By comparison, 3-aminobenzamide (up to 5 mM) did not appreciably modify these parameters. After an 18-hr incubation period with 20 mM NAM, the NAD+ pools of control PAM increased by approximately 300% comparatively to a approximately 40% increase for the NADP+ content. The exposure to the VSF asbestos caused a dose-dependent depletion of the cellular NAD+ and NADH pools. However, for the latter, the vitamin prevented the depletion effect of the asbestos fibers. Comparatively, the NADP(H) pools increased. This shift toward the phosphorylated pyridine nucleotide forms could also represent a defense of the cell against the oxygen radicals produced during the ingestion of the fibers. Overall, it is shown that changes in the energy metabolism could be implicated in the toxicity of chrysotile asbestos fibers toward PAM, and that the cells seem to be able to respond to an oxidant stress. Although not fully elucidated at the present time, these data tend nonetheless to point out that the protective effect of NAM could involve some modifications of the host defenses against prooxidants.     

In vitro fibrinolytic activity and viability of rat alveolar macrophages treated with inflammation generating mineral dusts

Rat alvolar macrophages demonstrated plasminogen dependent fibrinolysis in vitro which was inhibited to varying degrees by the addition of zymosan, the non-toxic particulate titanium dioxide, and the toxic dusts quartz and chrysotile asbestos. Assessment of viability suggested that the inhibition produced by zymosan and titanium dioxide could be accounted for by cytotoxic effects but in the case of quartz and chrysotile asbestos there was evidence that stimulation of fibrinolysis preceded cell death. Zymosan, which caused no observeable enhancement of alveolar macrophage fibrinolysis was found to markedly stimulate peritoneal macrophage fibrinolysis. The choice of assays of cell function to assess the action of toxic dusts are discussed.     

In vitro assays to predict the pathogenicity of mineral fibers

A number of mineral dusts are associated with the development of inflammation in lung and pulmonary interstitial fibrosis. In an effort to find alternative approaches to animal testing, cells (rat alveolar macrophages, hamster tracheal epithelial cells, rat lung fibroblasts) of the respiratory tract have been evaluated for cytotoxic and metabolic changes after exposure to fibers (defined as a greater than 3:1 length to diameter ratio) and particles. In all bioassays, fibrous materials provoked greater biological responses in cells in comparison to non-fibrous, chemically similar particulates at identical concentrations. For example, release of superoxide (O2-.) from alveolar macrophages (AMs) was increased (in comparison to that observed in untreated cells) after exposure to the fibers, crocidolite asbestos, erionite, Code 100 fiberglass and sepiolite. Riebeckite, mordenite and glass particles elicited minimal generation of O2-. at similar concentrations of dusts. In hamster tracheal epithelial (HTE) cells, fibers such as chrysotile asbestos, crocidolite asbestos, and Code 100 fiberglass caused increased release of 51Chromium in comparison to the particles antigorite, riebeckite and glass. Another area of exploration is the measurement of collagen and non-collagen protein in a cell line (RL-82) of rat lung fibroblasts as an indication of the fibrogenic potential of minerals. Crocidolite asbestos caused an increase in the ratio of cell-associated collagen to non-collagen protein in these cell types whereas glass beads did not affect biosynthesis of collagen. Results suggest that a battery of in vitro assays can be used to screen the capability of minerals to elicit cell damage and inflammatory changes in the respiratory tract.     

The Biopersistence of Canadian Chrysotile Asbestos Following Inhalation

Chrysotile asbestos is often included with other asbestos materials in evaluation and classification. However, chrysotile is a serpentine with markedly different physical and chemical characteristics in comparison to amphiboles (e.g., crocidolite, amosite, tremolite). In contrast to amphiboles, which are solid, rodlike fibers, chrysotile is composed like a rope of many fine fibrils, which tend to unwind. In order to quantify the dynamics and rate by which chrysotile is removed from the lung, the biopersistence of a sample of commercial chrysotile from the Eastern Townships area of Quebec, Canada, labeled QS Grade 3-F, which is the longest commercial grade intended for textile use, was studied. As the long fibers have been shown to have the greatest potential for pathogenicity, the chrysotile samples were specifically chosen to have more than 200 fibers/cm³ longer than 20 µm present in the exposure aerosol. This publication presents the results of this study through 3 mo postexposure. The study design included: (1) Fiber clearance (lung digestions): At 1 day, 2 days, 7 days, 14 days, 1 mo, 3 mo, and 12 mo (to be reported) following a 5-day (6 h/day) inhalation exposure, the lungs from groups of animals were digested by low-temperature plasma ashing and subsequently analyzed by transmission electron microscopy for total chrysotile fibers number in the lungs and chrysotile fiber size (length and diameter) distribution in the lungs. (2) Fiber distribution (confocal microscopy): This procedure was included in order to identify the location of the fibers in the lung. At 1 day, 2 days, 7 days, 14 days, 1 month, and 3 months (to be reported) postexposure, the lungs from groups of animals were analyzed by confocal microscopy to determine the anatomic fate, orientation, and distribution of the retained chrysotile fibrils deposited on airways and in the parenchymal region. Chrysotile was found to be rapidly removed from the lung. Fibers longer than 20 µm were cleared with T_1/2 = 16 days, most likely by dissolution and disintegration into shorter fibers. The shorter fibers were also rapidly cleared from the lung, with fibers 5–20 µm clearing even faster (T_1/2 = 29.4 days) than those <5 µm in length. The fibers <5 µm in length cleared at a rate (T_1/2 = 107 days) that is within the range of clearance for insoluble nuisance dusts. The breaking apart of the longer fibers would be expected to increase the short fiber pool and therefore could account for this difference in clearance rates. The short fibers were not found clumped together but appeared as separate, fine fibrils, occasionally unwound at one end. Short free fibers appeared in the corners of alveolar septa, and fibers or their fragments were found within alveolar macrophages. The same was true of fibers in lymphatics, as they appeared free or within phagocytic lymphocytes. Neutrophil-mediated inflammatory response did not occur in the presence of chrysotile fibers at the time points examined. Taken in context with the scientific literature to date, this report provides new robust data that clearly support the difference seen epidemiologically between chrysotile and amphibole asbestos.     

The biopersistence of Canadian chrysotile asbestos following inhalation

Chrysotile asbestos is often included with other asbestos materials in evaluation and classification. However, chrysotile is a serpentine with markedly different physical and chemical characteristics in comparison to amphiboles (e.g., crocidolite, amosite, tremolite). In contrast to amphiboles, which are solid, rodlike fibers, chrysotile is composed like a rope of many fine fibrils, which tend to unwind. In order to quantify the dynamics and rate by which chrysotile is removed from the lung, the biopersistence of a sample of commercial chrysotile from the Eastern Townships area of Quebec, Canada, labeled QS Grade 3-F, which is the longest commercial grade intended for textile use, was studied. As the long fibers have been shown to have the greatest potential for pathogenicity, the chrysotile samples were specifically chosen to have more than 200 fibers/cm3 longer than 20 micro m present in the exposure aerosol. This publication presents the results of this study through 3 mo postexposure. The study design included: (1) Fiber clearance (lung digestions): At 1 day, 2 days, 7 days, 14 days, 1 mo, 3 mo, and 12 mo (to be reported) following a 5-day (6 h/day) inhalation exposure, the lungs from groups of animals were digested by low-temperature plasma ashing and subsequently analyzed by transmission electron microscopy for total chrysotile fibers number in the lungs and chrysotile fiber size (length and diameter) distribution in the lungs. (2) Fiber distribution (confocal microscopy): This procedure was included in order to identify the location of the fibers in the lung. At 1 day, 2 days, 7 days, 14 days, 1 month, and 3 months (to be reported) postexposure, the lungs from groups of animals were analyzed by confocal microscopy to determine the anatomic fate, orientation, and distribution of the retained chrysotile fibrils deposited on airways and in the parenchymal region. Chrysotile was found to be rapidly removed from the lung. Fibers longer than 20 micro m were cleared with T(1/2) = 16 days, most likely by dissolution and disintegration into shorter fibers. The shorter fibers were also rapidly cleared from the lung, with fibers 5-20 micro m clearing even faster (T(1/2) = 29.4 days) than those <5 micro m in length. The fibers <5 micro m in length cleared at a rate (T(1/2) = 107 days) that is within the range of clearance for insoluble nuisance dusts. The breaking apart of the longer fibers would be expected to increase the short fiber pool and therefore could account for this difference in clearance rates. The short fibers were not found clumped together but appeared as separate, fine fibrils, occasionally unwound at one end. Short free fibers appeared in the corners of alveolar septa, and fibers or their fragments were found within alveolar macrophages. The same was true of fibers in lymphatics, as they appeared free or within phagocytic lymphocytes. Neutrophil-mediated inflammatory response did not occur in the presence of chrysotile fibers at the time points examined. Taken in context with the scientific literature to date, this report provides new robust data that clearly support the difference seen epidemiologically between chrysotile and amphibole asbestos.     

Evaluation of cellular toxicity of TAFMAG, a natural substitute for asbestos from China

Asbestos is a very important material for industrial use. However, the need for a substitute for asbestos fiber is currently on the rise due to its high disease causing potential. This study evaluated the potential bio-hazardous effects of TAFMAG, a natural fibrous silicate produced in China, in comparison with chrysotile, a typical toxic asbestos. The physicochemical properties of TAFMAG were very similar to those of chrysotile when it was examined by a scanning electron microscope (SEM) and X-ray diffraction (XRD) analyses. Both of TAFMAG and chrysotile showed high content of magnetite and Fenton activity when compared with wollastonite, a non-asbestos fiber with a known low toxicity. When their cellular toxicity was assessed, TAFMAG showed no or less comparable to that of chrysotile in the hemolysis and lipid peroxidation of erythrocytes, and also on a MTT assay in RLE-6TN, a rat alveolar epithelial cell line. Pre-treatment of fibers with desferrioxamine, an iron chelator, showed that iron content of TAFMAG and chrysotile might be important in their cellular toxicity. These results suggest that TAFMAG is potentially toxic when inhaled into the lung and appropriate laws and regulations should be established for its use.     

Biological effects and comparative cytotoxicity of thermal transformed asbestos-containing materials in a human alveolar epithelial cell line

Asbestos fibres can be transformed into potentially non-hazardous silicates by high-temperature treatment via complete solid-state transformation.A549 cells were exposed to standard concentrations of raw cement asbestos (RCA), chrysotile and cement asbestos subjected to an industrial process at 1200 °C (Cry_1200 and KRY·AS, respectively), raw commercial grey cement (GC). Cell growth rate and viability (MTT test) were detected in vitro. RCA and KRY·AS subjected to comprehensive microstructural study by electron microscopy were further in vitro assayed to compare their cytotoxic potential by morphostructural studies, proliferation index (Ki-67 antigen), apoptosis induction (AO/EB staining) assays and detection of intracellular reactive oxygen species (ROS) with the fluorescent DCFA dye. More severe cytotoxic damage was induced by RCA than by KRY·AS after each incubation period. Exposure to KRY·AS and GC resulted in comparable cell growth rates and cytotoxic effects. Cells incubated with RCA showed greater apoptotic induction and ROS production and a lower cell proliferation index than those exposed to KRY·AS. Chrysotile asbestos and RCA subjected to heat treatment underwent complete microstructure transformation. The final product of heat treatment of cement asbestos, KRY·AS, was considerably more inert and had lower cytotoxic potential than the original asbestos material in all in vitro tests.     

Contrasting respirable quartz and kaolin retention of lecithin surfactant and expression of membranolytic activity following phospholipase A2 digestion.

Respirable-sized quartz, a well-established fibrogenic mineral dust, is compared with kaolin in erythrocyte hemolysis assays after treatment with saline dispersion of dipalmitoyl phosphatidylcholine, a primary phospholipid component of pulmonary surfactant. Both dusts are rendered inactive after treatment, but the membranolytic activity is partly to fully restored after treatment with phospholipase A2, an enzyme normally associated with cellular plasma membranes and lysosomes. Phospholipid-coated dusts were incubated for periods of 2-72 h at a series of applied enzyme concentrations, and the adsorbed lipid species and hemolytic activity were quantitated at each time for both dusts. Surfactant was lost more readily from quartz than from kaolin, with consequent more rapid restoration of mineral surface hemolytic activity for quartz. Interactions of surfactant and mineral surface functional groups responsible for the mineral-specific rate differences, and implications for determining the mineral surface bioavailability of silica and silicate dusts, are discussed.     

The Regulation of Epidermal Hyperplastic Growth

Results of a meta-analysis indicate that the variation in potency factors observed across published epidemiology studies can be substantially reconciled (especially for mesothelioma) by considering the effects of fiber size and mineral type, but that better characterization of historical exposures is needed before improved exposure metrics potentially capable of fully reconciling the disparate potency factors can be evaluated. Therefore, an approach for better characterizing historical exposures, the Modified Elutriator Method (MEM), was evaluated to determine the degree that dusts elutriated using this method adequately mimic dusts generated by processing in a factory. To evaluate this approach, elutriated dusts from Grade 3 milled fiber (the predominant feedstock used at a South Carolina [SC] textile factory) were compared to factory dust collected at the same facility. Elutriated dusts from chrysotile ore were also compared to dusts collected in Quebec mines and mills. Results indicate that despite the substantial variation within each sample set, elutriated dusts from Grade 3 fiber compare favorably to textile dusts and elutriated ore dusts compare to dusts from mines and mills. Given this performance, the MEM was also applied to address the disparity in lung cancer mortality per unit of exposure observed, respectively, among chrysotile miners/millers in Quebec and SC textile workers. Thus, dusts generated by elutriation of stockpiled chrysotile ore (representing mine exposures) and Grade 3 milled fiber (representing textile exposures) were compared. Results indicate that dusts from each sample differ from one another. Despite such variation, however, the dusts are distinct and fibers in Grade 3 dusts are significantly longer than fibers in ore dusts. Moreover, phase-contrast microscopy (PCM) structures in Grade 3 dusts are 100% asbestos and counts of PCM-sized structures are identical, whether viewed by PCM or transmission electron microscope (TEM). In contrast, a third of PCM structures in ore dusts are not asbestos and only a third that are counted by PCM are also counted by TEM. These distinctions also mirror the characteristics of the bulk materials themselves. Perhaps most important, when the differences in size distributions and PCM/TEM distinctions in these dusts are combined, the combined difference is sufficient to completely explain the difference in exposure/response observed between the textile worker and miner/miller cohorts. Importantly, however, evidence that such an explanation is valid can only be derived from a meta-analysis (risk assessment) covering a diverse range of epidemiology study environments, which is beyond the scope of the current study. The above findings suggest that elutriator-generated dusts mimic factory dusts with sufficient reliability to support comparisons between historical exposures experienced by the various cohorts studied by epidemiologists. A simulation was also conducted to evaluate the relative degree that the characteristics of dust are driven by the properties of the bulk material processed versus the nature of the mechanical forces applied. That results indicate it is the properties of bulk materials reinforces the theoretical basis justifying use of the elutriator to reconstruct historical exposures. Thus, the elutriator may be a valuable tool for reconstructing historical exposures suitable for supporting continued refinements of the risk models being developed to predict asbestos-related cancer risk.     

Iron-loaded synthetic chrysotile: a new model solid for studying the role of iron in asbestos toxicity

The generation of reactive oxygen species and other radicals, catalyzed by iron ions at the fiber surface, is thought to play an important role in asbestos-induced cytotoxicity and genotoxicity, but a direct confirmation of this statement needs the availability of asbestos samples differing only for their iron content, without the interference of other physicochemical features. Synthetic stoichiometric chrysotile nanofibers, devoid of iron or any other contaminant, did not exert genotoxic and cytotoxic effects nor elicited oxidative stress in a murine alveolar macrophage cell line; on the contrary, the same nanofibers, loaded with 0.57% and 0.94% (w/w) iron, induced DNA strand breaks, lipoperoxidation, inhibition of redox metabolism and alterations of cell integrity, similarly to natural chrysotile. On the other hand, the incubation with ferric nitrilotriacetate, a cell-permeating iron complex, even if it caused an intracellular overloading of iron very similar to that elicited by iron-loaded synthetic chrysotile and by natural chrysotile, did not exert any of these effects. This suggests that chrysotile is not toxic by acting simply as a carrier of iron into the cell, but rather that the redox activity of iron is potentiated when organized at the fibers surface into specific crystallographic sites having coordination states able to activate free radical generation. Synthetic chrysotile fibers may be proposed as a standard reference sample and model solids for experimental studies on asbestos fibers aiming to clarify the mechanisms of its toxicity and to synthesize new fibers devoid of pathogenic effects.     

Interactions of asbestos fibers with hepatocytes in vitro: an ultrastructural study

Cultures of rat hepatocytes have been used to study the interaction of chrysotile asbestos fibers with epithelial cells. The hepatocytes were incubated for 20 h with chrysotile asbestos (UICC B and a preparation of short fibers: 90% less than 2 micron) at concentrations of 1 or 10 micrograms/ml. Morphologic studies were then performed by means of transmission electron microscopy. Fibers were incorporated into plasma membrane invaginations, cytoplasmic vacuoles and phagolysosomes. These observations demonstrate that rat hepatocytes can engage in phagocytosis of chrysotile fibers. The results are compared to those previously obtained with mesothelial cells and tracheobronchial epithelium, exposed to asbestos in vitro. The advantages of the hepatocyte model to investigate the effects on cells of the association of asbestos fibers with genotoxic agents in relation to carcinogenesis are discussed.     

Ultrastructural study of mineral fiber uptake by hepatocytes in vitro

Cellular interactions of a series of fibrous materials were examined by the use of a well established in vitro system. Primary cultures of hepatocytes were exposed to natural attapulgite, synthetic xonotlite and natural sepiolite. Ultrastructural analyses revealed that hepatocytes can engage in the phagocytosis of all 3 types of fibers over an exposure period of 20 h. Attapulgite fibers were found in plasma membrane invaginations, and deeper in the cytoplasm, in vesicles exhibiting various shapes. Xonotlite was also incorporated in plasma membrane invaginations; furthermore, these fibers were present in large vacuoles where they were circumscribed by membranes and appeared somewhat isolated from the cytoplasm. Sepiolite fibers were also taken up by the cells and could likewise be identified in the previously described structures. These observations point to the relevance of the hepatocyte model for investigating the effects of fibrous materials at the cellular level.     

The biopersistence of brazilian chrysotile asbestos following inhalation

With the initial understanding of the relationship of asbestos to disease, little information was available on whether the two different groups of minerals that are called asbestos were of similar or different potency in causing disease. Asbestos was often described as a durable fiber that if inhaled would remain in the lung and cause disease. It has been only more recently, with the development of a standardized protocol for evaluating the biopersistence of mineral fibers in the lung, that the clearance kinetics of the serpentine chrysotile have been shown to be dramatically different from those of amphibole asbestos, with chrysotile clearing rapidly from the lung. In addition, recent epidemiology studies also differentiate chrysotile from amphibole asbestos. The biopersistence studies mentioned have indicated that chrysotile from Canada and California clear rapidly from the lung once inhaled. However, variations in chrysotile mineralogy have been reported depending upon the region. This is most likely associated with variations in the forces which created the chrysotile fibers centuries ago. In the present study, the dynamics and rate of clearance of chrysotile from the Cana Brava mine in central Brazil was evaluated in a comparable inhalation biopersistence study in the rat. For synthetic vitreous fibers, the biopersistence of the fibers longer than 20 microm has been found to be directly related to their potential to cause disease. This study was designed to determine lung clearance (biopersistence) and translocation and distribution within the lung. As the long fibers have been shown to have the greatest potential for pathogenicity, the chrysotile samples were specifically chosen to have more than 450 fibers/cm(3) longer than 20 microm in length present in the exposure aerosol. For the fiber clearance study (lung digestions), at 1 day, 2 days, 7 days, 2 wk, 1 mo, 3 mo, 6 mo, and 12 mo following a 5-day (6 h/day) inhalation exposure, the lungs from groups of animals were digested by low-temperature plasma ashing and subsequently analyzed by transmission electron microscopy (at the GSA Corp.) for total chrysotile fiber number in the lungs and chrysotile fiber size (length and diameter) distribution in the lungs. This lung digestion procedure digests the entire lung with no possibility of identifying where in the lung the fibers are located. A fiber distribution study (with confocal microscopy) was included in order to identify where in the lung the fibers were located. At 2 days, 2 wk, 3 mo, 6 mo, and 12 mo postexposure, the lungs from groups of animals were analyzed by confocal microscopy to determine the anatomic fate, orientation, and distribution of the retained chrysotile fibrils deposited on airways and those fibers translocated to the broncho-associated lymphoid tissue (BALT) subjacent to bronchioles in rat lungs. While the translocation of fibers to the BALT and lymphatic tissue is considered important as in cases of human's with asbestos-related disease, there has been no report in the literature of pathological changes in the BALT and lymphatic tissue stemming from asbestos. Thus, if the fibers are removed to these tissues, they are effectively neutralized in the lung. Chrysotile was found to be rapidly removed from the lung. Fibers longer than 20 microm were cleared with a half-time of 1.3 days, most likely by dissolution and breakage into shorter fibers. Shorter fibers were also rapidly cleared from the lung with fibers 5-20 microm clearing even more rapidly (T1/2 = 2.4 days) than those < 5 microm in length (T1/2 weighted = 23. days). Breaking of the longer fibers would be expected to increase the short fiber pool and therefore could account for this difference in clearance rates. The short fibers were never found clumped together but appeared as separate, fine fibrils, occasionally unwound at one end. Short free fibers appeared in the corners of alveolar septa, and fibers or their fragments were found within alveolar macrophages. The same was true of fibers in lymphatics, as they appeared free or within phagocytic lymphocytes. These results support the evidence presented by McDonald and McDonald (1997) that the chrysotile fibers are rapidly cleared from the lung in marked contrast to amphibole fibers which persist.     

Comparison of Calidria chrysotile asbestos to pure tremolite: final results of the inhalation biopersistence and histopathology examination following short-term exposure

Calidria chrysotile asbestos, which is a serpentine mineral, has been shown to be considerably less biopersistent than the durable amphibole mineral tremolite asbestos, which persists once deposited in the lung. The initial results of this inhalation biopersistence study in rats that demonstrates this difference were reported in Bernstein et al. (2003). This article presents the full results through 1 yr after cessation of the 5-day exposure. This study was based upon the recommendations of the European Commission (EC) Interim Protocol for the Inhalation Biopersistence of synthetic mineral fibers (Bernstein & Riego-Sintes, 1999). In addition, the histopathological response in the lung was evaluated following exposure. In order to quantify the dynamics and rate by which these fibers are removed from the lung, the biopersistence of a sample of commercial-grade chrysotile from the Coalinga mine in New Idria, CA, of the type Calidria RG144 and that of a long-fiber tremolite were studied. For synthetic vitreous fibers, the biopersistence of the fibers longer than 20 microm has been found to be directly related to their potential to cause disease. This study was designed to determine lung clearance (biopersistence) and the histopathological response. As the long fibers have been shown to have the greatest potential for pathogenicity, the aerosol generation technique was designed to maximize the number of long respirable fibers. The chrysotile samples were specifically chosen to have 200 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. These longer fibers were found to be largely composed of multiple shorter fibrils. The tremolite samples were chosen to have 100 fibers/cm3 longer than 20 microm in length present in the exposure aerosol. Calidria chrysotile has been found to be one of the most rapidly cleared mineral fibers from the lung. The fibers longer than 20 microm in length are cleared with a half-time of 7 h. By 2 days postexposure all long fibers have dissolved/disintegrated into shorter pieces. The fibers between 5 and 20 microm in length were cleared with a half-time of 7 days. This length range represents a transition zone between those fibers that can be fully phagocytosed and cleared as particles and the longer fibers that cannot be fully engulfed by the macrophage. The fibers/objects shorter than 5 microm in length were cleared with a half-time of 64 days, which is faster than that reported for insoluble nuisance dusts such as TiO2. By 12 months postexposure, 99.92% of all the remaining chrysotile was less than 5 microm in length. Following the 5 days of repeated exposure to more than 48,000 chrysotile fibers/cm3 (190 fibers L > 20 microm), histopathological examination revealed no evidence of any inflammatory reaction either after the cessation of the last exposure or at any time during the subsequent 12-mo period. This is in marked contrast to the amphibole tremolite, which was also investigated using the same inhalation biopersistence protocol. The long tremolite fibers, once deposited in the lung, remain over the rat's lifetime with essentially an infinite half-time. Even the shorter fibers, following early clearance, also remain with no dissolution or further removal. At 365 days postexposure, there was a mean lung burden was of 0.5 million fibers L > 20 microm and 7 million fibers 5-20 microm in length with a total mean lung burden of 19.6 million fibers. The tremolite exposed rats, even with exposure to 16 times fewer total fibers than chrysotile, showed a pronounced inflammatory response with the rapid development of granulomas as seen at day 1 postexposure, followed by the development of fibrosis characterized by collagen deposition within these granulomas and by 90 days even mild interstitial fibrosis. With the short exposure, this study was not designed specifically to evaluate pathological response; however, it is quite interesting that even so there was such a marked response with tremolite. These findings provide an important basis for substantiating both kinetically and pathologically the differences between chrysotile and the amphibole tremolite. As Calidria chrysotile has been certified to have no tremolite fiber, the results of the current study together with the results from toxicological and epidemiological studies indicate that this fiber is not associated with lung disease.