Efficiency of pyoverdines in iron removal from flocking asbestos waste: An innovative bacterial bioremediation strategy.

The use of asbestos-containing products has been banned in many countries since the beginning of the 80's due to its carcinogenic properties. However, asbestos is widely present in private and public buildings, resulting in the need to process a vast amount of asbestos-containing waste. Among the current technologies for the destruction of asbestos fibers, biodegradation by fungi, lichens, and, more recently, bacteria has been described. We previously reported the involvement of the bacterial siderophore pyoverdine in the release of iron from the two asbestos groups, serpentines and amphiboles. Among the large diversity encountered in the pyoverdine family, we examined whether these siderophores can alter flocking asbestos waste as well. All the tested pyoverdines were efficient in chrysotile-gypsum and amosite-gypsum weathering, although some exhibited higher iron dissolution. Iron was solubilized by pyoverdines from Pseudomonas aeruginosa and mandelii in a time-dependent manner from chrysotile-gypsum within 24 h. Renewal of pyoverdine-containing supernatant every 24 or 96 h allowed iron removal from chrysotile-gypsum at each cycle, until a limit was reached after 42 days of total incubation. Moreover, the dissolution was concentration-dependent, as demonstrated for the pyoverdine of P. mandelii. Pyoverdine-asbestos weathering could therefore become an innovative method to reduce anthropogenic waste.

Iron removal from raw asbestos by siderophores-producing Pseudomonas.

Asbestos, mineral present in soil, are highly toxic due to the presence of iron. Microbes-mineral interactions occur naturally through various processes leading to their alteration. We examined the effect of siderophore-producing Pseudomonas with a particular focus on the role of pyoverdine and pyochelin on raw asbestos fibers such as amosite, crocidolite and chrysotile. We compared the efficiency of pyoverdine to the iron chelating agent EDTA in the release of iron from raw asbestos fibers. Pyoverdine was able to extract iron from all the tested raw asbestos with the higher efficiency observed for chrysotile and crocidolite. When asbestos were grinded, the iron removal was more important for all types. We monitored the effect of bacterial growth and siderophores containing bacterial supernatant on raw asbestos dissolution by solution chemistry analysis and transmission electron microscopy. The siderophore-containing supernatant allowed a higher iron solubilisation than the one obtained after bacterial growth. Moreover, the iron dissolution was faster with pyoverdine-containing supernatant than pyochelin-containing supernatant, with approximately the same iron level for the maximum extraction with a delay of 48 h. Our study clearly showed the involvement of bacterial siderophores, pyoverdine and pyochelin on chrysotile, crocidolite and amosite fibers weathering.

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.

The pathological response and fate in the lung and pleura of chrysotile in combination with fine particles compared to amosite asbestos following short-term inhalation exposure: interim results.

The pathological response and translocation of a commercial chrysotile product similar to that which was used through the mid-1970s in a joint compound intended for sealing the interface between adjacent wall boards was evaluated in comparison to amosite asbestos. This study was unique in that it presents a combined real-world exposure and was the first study to investigate whether there were differences between chrysotile and amosite asbestos fibers in time course, size distribution, and pathological response in the pleural cavity. Rats were exposed by inhalation 6 h/day for 5 days to either sanded joint compound consisting of both chrysotile fibers and sanded joint compound particles (CSP) or amosite asbestos. Subgroups were examined through 1-year postexposure. No pathological response was observed at any time point in the CSP-exposure group. The long chrysotile fibers (L > 20 microm) cleared rapidly (T(1/2) of 4.5 days) and were not observed in the pleural cavity. In contrast, a rapid inflammatory response occurred in the lung following exposure to amosite resulting in Wagner grade 4 interstitial fibrosis within 28 days. Long amosite fibers had a T(1/2) > 1000 days and were observed in the pleural cavity within 7 days postexposure. By 90 days the long amosite fibers were associated with a marked inflammatory response on the parietal pleural. This study provides support that CSP following inhalation would not initiate an inflammatory response in the lung, and that the chrysotile fibers present do not migrate to, or cause an inflammatory response in the pleural cavity, the site of mesothelioma formation.

Using in vitro iron deposition on asbestos to model asbestos bodies formed in human lung.

Recent studies have shown that iron is an important factor in the chemical activity of asbestos and may play a key role in its biological effects. The most carcinogenic forms of asbestos, crocidolite and amosite, contain up to 27% iron by weight as part of their crystal structure. These minerals can acquire more iron after being inhaled, thereby forming asbestos bodies. Reported here is a method for depositing iron on asbestos fibers in vitro which produced iron deposits of the same form as observed on asbestos bodies removed from human lungs. Crocidolite and amosite were incubated in either FeCl(2) or FeCl(3) solutions for 2 h. To assess the effect of longer-term binding, crocidolite was incubated in FeCl(2) or FeCl(3) and amosite in FeCl(3) for 14 days. The amount of iron bound by the fibers was determined by measuring the amount remaining in the incubation solution using an iron assay with the chelator ferrozine. After iron loading had been carried out, the fibers were also examined for the presence of an increased amount of surface iron using X-ray photoelectron spectroscopy (XPS). XPS analysis showed an increased amount of surface iron on both Fe(II)- and Fe(III)-loaded crocidolite and only on Fe(III)-loaded amosite. In addition, atomic force microscopy revealed that the topography of amosite, incubated in 1 mM FeCl(3) solutions for 2 h, was very rough compared with that of the untreated fibers, further evidence of Fe(III) accumulation on the fiber surfaces. Analysis of long-term Fe(III)-loaded crocidolite and amosite using X-ray diffraction (XRD) suggested that ferrihydrite, a poorly crystallized hydrous ferric iron oxide, had formed. XRD also showed that ferrihydrite was present in amosite-core asbestos bodies taken from human lung. Auger electron spectroscopy (AES) confirmed that Fe and O were the only constituent elements present on the surface of the asbestos bodies, although H cannot be detected by AES and is presumably also present. Taken together for all samples, the data reported here suggest that Fe(II) binding may result from ion exchange, possibly with Na, on the fiber surfaces, whereas Fe(III) binding forms ferrihydrite on the fibers under the conditions used in this study. Therefore, fibers carefully loaded with Fe(III) in vitro may be a particularly appropriate and useful model for the study of chemical characteristics associated with asbestos bodies and their potential for interactions in a biosystem.

TNF-alpha increases tracheal epithelial asbestos and fiberglass binding via a NF-kappaB-dependent mechanism.

Tumor necrosis factor (TNF)-alpha is released from alveolar macrophages after phagocytosis of mineral fibers. To determine whether TNF-alpha affects the binding of fibers to epithelial cells, we exposed rat tracheal explants to TNF-alpha or to culture medium alone, followed by a suspension of amosite asbestos or fiberglass (MMVF10). Loosely adherent fibers were removed from the surface with a standardized washing technique, and the number of bound fibers was determined by scanning electron microscopy. Increasing doses of TNF-alpha produced increases in fiber binding. This effect was abolished by an anti-TNF-alpha antibody, the proteasome inhibitor MG-132, and the nuclear factor (NF)-kappaB inhibitor pyrrolidine dithiocarbamate. Gel shift and Western blot analyses confirmed that TNF-alpha activated NF-kappaB and depleted IkappaB in this system and that these effects were prevented by MG-132 and pyrrolidine dithiocarbamate. These observations indicate that TNF-alpha increases epithelial fiber binding by a NF-kappaB-dependent mechanism. They also suggest that mineral particles may cause pathological lesions via an autocrine-like process in which the response evoked by particles, for example, macrophage TNF-alpha production, acts to enhance subsequent interactions of particles with tissue.

Bromo-deoxyuridine (BRDU) uptake in the lungs of rats inhaling amosite asbestos or vitreous fibres at equal airborne fibre concentrations.

Rats were exposed, by inhalation, to target airborne fibre concentrations of 1000 f/ml (PCOM fibres by WHO criteria) of a long amosite asbestos sample and a vitreous fibre sample; the target was closely attained for both fibre samples. The size distributions of the two fibre samples was closely similar. Rats were placed in the chambers for 7 hours and then, following a further 16 hours in room air, were injected with bromo-deoxyuridine (BRDU). The presence of BRDU-positive cells in terminal bronchioles/alveolar ducts was assessed in blocks taken from various parts of the left lung, from apex to base. There were significant differences in the proliferative responses between animals but there were also significant differences between the treatments. Lungs from rats exposed to vitreous fibres showed no greater response than the controls, but there was a markedly greater proliferative response in the lungs of rats inhaling long amosite. There was a decreasing gradient of proliferative response from the apex of the lung to the base with all treatments. This could be explained by different degrees of deposition in different areas of the lung. Similar amounts of fibre accumulated in the lungs of rats exposed to the two fibre types and it is unlikely that dissolution could be important over the timescale used here. We conclude that, when amosite asbestos deposits in the lungs of rats it stimulates a proliferative response and that deposition of an equal number of similar-sized vitreous fibres has no effect.

Enhanced retention of asbestos fibers in the airways of human smokers.

To determine whether cigarette smoke increases the pulmonary retention of asbestos, we compared the asbestos-fiber burden in the airway mucosa of six cigarette smokers who had received heavy occupational asbestos exposure with that in a group of six subjects with similar exposure who were never smokers. The groups were matched in terms of age, sex, years of exposure, and mean parenchymal amosite burden. We found that the concentration of amosite in airway mucosa was significantly elevated (by approximately sixfold) in smokers (p < 0.02). Chrysotile parenchymal burdens were statistically similar in both groups, but the chrysotile airway burden was again higher (by approximately 50-fold) in smokers (p < 0.006). There were no differences in airway or parenchymal tremolite burdens between the two groups. Fibers of all three types of asbestos recovered from the airway mucosa or parenchyma of smokers were shorter than fibers recovered from nonsmokers, an observation in accord with experimental data suggesting that cigarette smoke leads to retention of shorter fibers. These findings indicate that cigarette smoking causes enhanced accumulation of both amosite and chrysotile in the airway mucosa. This process may play a role in potentiating the pathologic effects of asbestos.

Fiber levels and disease in workers from a factory predominantly using amosite.

The Cape Boards Plant at Uxbridge produced insulation board containing amosite asbestos between 1947 and 1973 with only small amounts of chrysotile. After 1973 only amosite was used. In this study we examined lung samples from 48 workers who had been employed at the plant and who had come to autopsy. The study investigated the fiber levels against the lung pathology including amount of interstitial fibrosis and numbers of ferruginous bodies. The degree of interstitial fibrosis and number of asbestos bodies were graded and the tissues were analyzed by transmission electron microscopy and energy dispersive X-ray analysis and the fibers counted and typed. The 48 cases included 5 mesotheliomas and 14 lung cancers. The mineral analysis results were dominated by the amosite fiber levels. The amounts of chrysotile were relatively small. There were higher levels in lung cancer cases than mesotheliomas and higher levels in mesothelioma cases than those who had died from nonasbestos related diseases. Analysis of the lung tissues showed a consistent pattern of high amosite levels, which confirms the impression that amosite was the predominant form of asbestos used and also indicates that the factory had been a very dusty one.

Deposition and clearance of chrysotile asbestos.

Studies of human lungs indicate that, for virtually all types of exposure, the relative proportion of amphibole asbestos retained in the lung far exceeds the proportion in the original dust and, conversely, the relative proportion of chrysotile is far less than that in the original dust. Although amphiboles appear to accumulate in lung in proportion to exposure and chrysotile does not, failure of chrysotile deposition is probably not the reason for the disproportionate retention of amphibole fibres. The available data suggest that chrysotile is deposited in the parenchyma but is cleared extremely rapidly, with the vast bulk of fibres removed from human lungs within weeks to months after inhalation; by comparison, amphibole clearance half-lives are of the order of years to decades. The mechanisms of preferential chrysotile clearance remain uncertain, but fragmentation of chrysotile into short fibres, possibly accompanied by extremely rapid dissolution of such fibres, appears to be important in this process. Chrysotile fibres do penetrate to the periphery of the lung, so that differences in mesothelial pathogenicity of chrysotile and amphiboles in regard to mesothelioma are not caused by failure of chrysotile to reach the pleura. The theory that the tremolite contaminant rather than the chrysotile itself is the cause of 'chrysotile-induced' disease (especially mesothelioma) is consistent with the available human data, but the contrary ideas that disease is caused either by the total transient burden of inhaled chrysotile fibres or by a small, sequestered, long-retained fraction of chrysotile fibres still need to be excluded.

Iron associated with asbestos bodies is responsible for the formation of single strand breaks in phi X174 RFI DNA.

The ability of amosite cored asbestos bodies isolated from human lungs to catalyse damage to phi X174 RFI DNA in vitro was measured and compared with that of uncoated amosite fibres with a similar distribution of length. Asbestos bodies (5000 bodies) suspended for 30 minutes in 50 mM NaCl containing 0.5 micrograms phi X174 RFI DNA, pH 7.5, did not catalyse detectable amounts of DNA single strand breaks. Addition of the reducing agent ascorbate (1 mM), however, resulted in single strand breaks in 10% of the DNA. Asbestos bodies in the presence of a low molecular weight chelator (1 mM) and ascorbate catalysed the formation of single strand breaks in 21% of the DNA with citrate or 77% with ethylenediamine tetra-acetic acid (EDTA), suggesting that mobilisation of iron may increase damage to DNA. Preincubation for 24 hours with desferrioxamine B, which binds iron (Fe (III)) and renders it redox inactive, completely inhibited the reactivity of asbestos bodies with DNA, strongly suggesting that iron was responsible. Amosite fibres (5000 fibres/reaction), with a similar length distribution to that of the asbestos bodies, did not catalyse detectable amounts of single strand breaks in DNA under identical reaction conditions. The results of the present study strongly suggest that iron deposits on the amosite core asbestos bodies were responsible for the formation of DNA single strand breaks in vitro. Mobilisation of iron by chelators seemed to enhance the reactivity of asbestos bodies with DNA. It has been postulated that the in vivo deposition of the coat material on to fibres may be an attempt by the lung defenses to isolate the fibre from the lung surface and thus offer a protective mechanism from physical irritation. These results suggest, however, that the iron that is deposited on asbestos fibres in vivo may be reactive, potentially increasing the damage to biomolecules, such as DNA, above that of the uncoated fibres.